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Predictive Value of Diminished Serum PDGF-BB after Curative Resection of Hepatocellular Cancer

Predictive Value of Diminished Serum PDGF-BB after Curative Resection of Hepatocellular Cancer Hindawi Journal of Oncology Volume 2019, Article ID 1925315, 8 pages https://doi.org/10.1155/2019/1925315 Research Article Predictive Value of Diminished Serum PDGF-BB after Curative Resection of Hepatocellular Cancer 1 2 2 3 Bibek Aryal , Munekazu Yamakuchi , Toshiaki Shimizu, Jun Kadono, 3 1 4 5 Akira Furoi, Kentaro Gejima, Teruo Komokata, Chihaya Koriyama, 2 1 Teruto Hashiguchi , and Yutaka Imoto Cardiovascular and Gastroenterological Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima -, Japan Department of Laboratory and Vascular Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima -, Japan Department of Surgery, Kirishima Medical Center, Kirishima -, Japan Department of Surgery, Kagoshima Medical Center, National Hospital Organization, Kagoshima -, Japan Department of Epidemiology and Preventive Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima -, Japan Correspondence should be addressed to Bibek Aryal; drbibekaryal@gmail.com and Teruto Hashiguchi; terutoha@m3.kufm.kagoshima-u.ac.jp Received 24 June 2018; Accepted 13 December 2018; Published 6 January 2019 Academic Editor: Ozkan Kanat Copyright © 2019 Bibek Aryal et al. is Th is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose. Platelet derived growth factor-BB (PDGF-BB) has emerged as one of the key cytokines in malignant transformation of dieff rent cells. PDGF-BB also exhibits a potent mitogenic effect on liver cells; studies have advocated clinical implications of monitoring serum PDGF-BB (sPDGF-BB) in patients with liver disease. We thus investigated the predictive relevance of perioperative sPDGF-BB aeft r curative resection of hepatocellular carcinoma (HCC). Methods. We evaluated perioperative sPDGF- BB in a prospective homogenous cohort of 40 patients diagnosed with HCC. During the first two-year follow-up, patients were evaluated every three months for postresection HCC recurrence. Results. Patients who developed recurrence during two-year follow-up were found to have lower concentration of sPDGF-BB than those without recurrence in both pre- and postoperative settings (P< 0.05 and P< 0.001, resp.). We validated that the reduced postoperative sPDGF-BB (< 2133.29 pg/mL) was associated with an increased incidence of postresection HCC recurrence [area under curve (AUC)> 0.8, 95% confidence interval (CI) = 0.68 -0.94, P< 0.001]; furthermore, we were able to demonstrate that postoperative sPDGF-BB was an independent predictor of HCC recurrence (hazard ratio = 5.64, 95% CI = 1.56 - 20.30, P< 0.01). Conclusions. es Th e findings provide a new insight into an association between diminished perioperative sPDGF-BB and HCC recurrence. Patients with low perioperative sPDGF-BB progressed early HCC recurrence. Therefore, evaluating perioperative sPDGF-BB may provide useful clinical information to characterize patients with postresection HCC recurrence. 1. Introduction patients, ideally before the recurrence sets in, by integrating both clinical and molecular information into a synergistic Curative resection (partial hepatectomy) is a safe, effective, understanding of the tumor recurrence. and preferred therapy for selected patients with hepatocel- Platelet-related biomarkers, detected in the circulation lular carcinoma (HCC)[1]. However, the tumor recurrence (whole blood, serum, or plasma), have long been considered particularly in the remnant liver is exceedingly frequent after as a potential diagnostic tool in cancer[3–7]. These growth the surgical resection of HCC[2]. The high incidence of factors have demonstrated several implications in the eld fi HCC recurrence intensifies the need for identifying high-risk of oncology including screening, diagnostic, and prognostic 2 Journal of Oncology relevance of diseases[7]. Platelet-related growth factors reflect centrifuged at 2810× g to isolate platelets. The supernatant, paradoxical association with different physiological and platelet-poor plasma (PPP), was collected precisely and pathological events[8]. For instance, the platelet-sequestered removed completely by decantation method. Platelet pellets isolated from each 200𝜇 l of PRP were suspended in 220𝜇 lof vascular endothelial growth factor (VEGF), which is linked to various pathological conditions[9] including carcinogenesis, lysis buffer (RIPA) and were vortexed after incubating for 20 has also been found to be crucial in physiological events like minutes. CBC was performed in 3 preparations: whole blood, liver regeneration after partial hepatectomy[10, 11]. PRP, and PPP. Growing lines of evidence recognize the role of platelet All samples were stored in aliquots at -80 C immediately derived growth factor-BB (PDGF-BB) at every stage during after preparation. the continuum of liver injury, repair, and fibrosis[12–14]. Despite a wealth of literature on PDGF-BB in other tumor .. Quantification of Cytokines. Platelet derived growth types, there are relatively a few studies on the PDGF-BB and factor-BB and P-selectin were measured using enzyme-linked its predictive relevance in HCC. Also, to date, no evidence immunosorbent assay (ELISA) tests (Quantikine; R&D Sys- examining the feature of serum PDGF-BB (sPDGF-BB) on tems,Minneapolis, MN, USA).Intraplatelet (IP) PDGF-BB postresection HCC recurrence has been published in English was calculated from isolated platelets using the equation literature. In this study, we aimed to determine whether to calculate cytokines inside each platelet, as described the perioperative sPDGF-BB could reflect the oncological before[10]. Concisely, 220 𝜇 lof lysis solution (RIPA) was outcome aeft r the curative resection of HCC. added to the platelets isolated from each 200𝜇 lof PRP. The concentration of the cytokine was adjusted to the platelet 2. Materials and Methods count obtained from the PRP and IP PDGF-BB was expressed per 10 platelets. .. Study Cohort. The study cohort consisted of forty patients diagnosed with primary HCC who went on to have liver . . Statistics. Statistical analyses were performed using SPSS resection. The trial is registered in UMIN Clinical Trial Reg- 25.0 software (SPSS, Inc., Chicago, IL, USA) and Graph- istry (UMIN000026380). The institutional ethics committee Pad Prism (version 6.0d for MacOS X, USA, GraphPad (Kagoshima University # 24-155/ 26-77, Kirishima Medical Sow ft are, San Diego, California, USA) and were mainly Center # 2505 and Kagoshima Medical Center # 25-30) based on nonparametric tests (Mann-Whitney U test and approved analyses of blood samples and patient data; all Wilcoxon test). Receiver operating characteristic (ROC) patients gave signed, informed consent. analysis was performed to assess the specificity and sensitivity of PDGF-BB levels to predict recurrence. Youden’s J index .. Follow-Up. Disease-free interval (DFI) was defined was applied to determine cut-off points in ROC analyses. according to the Guidance for Industry Clinical Trial End- Cox's proportional hazards regression model was used for points for the Approval of Cancer Drugs and Biologics of the the univariable analyses (UVA) and multivariable analyses US Food and Drug Administration (FDA). DFI represents the (MVA) to determine the variables independently associated time period between liver resection and tumor recurrence. with recurrence. Two-tailed P values of less than 0.05 were Follow-up period was standardized to two years; patients considered statistically significant. IP PDGF-BB and IP P- were followed up every three months aeft r surgery. Follow-up selectin concentrations were expressed per 10 platelets. was routinely scheduled and comprised of ultrasonography (USG) as well as evaluation of tumor markers; if USG showed any evidence of tumor, further assessment of thoracic 3. Results and abdominal CAT scans or MRI was performed. Tumor recurrence was diagnosed based on analysis of radiological .. Patient Demographics. We included forty patients with findings and comprised of local and distant recurrence. HCC who were selected for liver resection. Baseline char- acteristics of the study cohort are summarized in Table 1. None of the patients received platelet transfusion during the .. Sample Preparation. Venous blood was collected preop- sample preparations. During two-year follow- up, 15 patients eratively, immediately before surgery (PRE OP), and four developedHCC recurrence andone patient diedwith a cause weeks after surgery (POST OP). Complete blood count other than the cancer recurrence. (CBC) was performed with an automated hematology ana- lyzer Sysmex XE-5000 (Sysmex Corporation, Kobe, Japan). Whole blood was collected in the serum-separating .. Perioperative sPDGF-BB Concentrations Were Lower in tube and an EDTA-2k. Serum tube was incubated at room Patients with HCC Recurrence. We first compared perioper- temperature for 30 minutes before centrifuging at 1710× gfor ative sPDGF-BB concentrations in patients with postresec- 10 minutes. tion HCC recurrence. Patients with two-year postresection HCC recurrence had significantly lower concentrations of . . Platelet Extract. Venous blood in citrate tubes was sPDGF-BB prior to liver resection (PRE OP median sPDGF: centrifuged at 90× g for 15 minutes. To avoid contaminations nonrecurrent, 2444 pg/mL; recurrent, 1592 pg/mL, P = 0.02; with other cells, upper two-thirds of the resultant platelet- Figure 1(a)) and aer ft four weeks of liver resection compared rich plasma (PRP) were gently pipetted. The PRP was then to the patients with no cancer recurrence (POST OP median Journal of Oncology 3 Table 1: Demographics and clinical and pathological data of the cohort. Variables Data Age, median (min, max) 72.52 (51, 83) Sex, n(%) Male, 30 (75 %) Female 10 (25 %) Child Pugh Class, n(%) A 40 (100 %) B 0 C 0 Etiology, n(%) HBV 12 (30 %) HCV 13 (32.5 %) Nonviral 15 (37.5 %) Fibrosis grade, n(%) 0-2 22 (55 %) 3-4 18 (45 %) Tumor staging, n(%) I-II 28 (70 %) III-IV 12 (30 %) Tumor size, n(%) < 5cm 27 (67.5 %) ≥ 5cm 13 (32.5 %) Tumor number, n(%) Single 31 (77.5 %) Multiple 09 (22.5 %) Vascular invasion, n(%) Present 08 (20 %) Absent 32 (80 %) Tumor histology (differentiation), n(%) Well-to-moderate 32 (80 %) Poor 08 (20 %) Type of hepatectomy, n(%) Major hepatectomy 14 (35 %) Minor hepatectomy 26 (65 %) Prior treatment, n(%) RFA/TACE 19 (47.5 %) None 21 (52. 5 %) Total bilirubin before resection, n(%) < 1mg/dl 28 (70 %) ≥ 1mg/dl 12 (30 %) INR before resection, n(%) > 1 30 (75 %) < 1 10 (25 %) AFP before resection, n(%) < 20 ng/ml 25 (62.5 %) ≥ 20 ng/ml 15 (37.5 %) Clavien-Dindo postoperative (severe) morbidity, n(%) Absent 35 (87.5 %) Present 05 (12.5 %) Min., minimum; max., maximum; n/N, numbers; HBV, hepatitis B virus; HCV, hepatitis C virus; INR, international normalized ratio, AFP, alpha-fetoprotein; PIVKA II, protein induced by vitamin K absence/antagonist-II; TACE, transarterial chemoembolization; RFA, radiofrequency ablation; INR, international normalized ratio. 4 Journal of Oncology PRE OP POST OP ∗∗∗ 0 0 Recurrence (-) Recurrence (+) Recurrence (-) Recurrence (+) (a) (b) Figure 1: Serum PDGF-BB (sPDGF-BB) concentration in recurrent and nonrecurrent cases (a) before (PRE OP) and (b) 4 weeks aeft r liver ∗ ∗∗∗ resection (POST OP). < 0.05 and < 0.001. P P sPDGF: nonrecurrent, 2852 pg/mL; recurrent, 1592 pg/mL, P low-risk patients in terms of HCC recurrence. This translates < 0.001; Figure 1(b)). to the positive predictive value (PPV) of 54.99% and negative Furthermore, we compared separately isolated, predictive value (NPV) of 79.98% for preoperative sPDGF- intraplatelet (IP) concentrations of PDGF-BB in patients BB and similarly PPV of 63.16% and NPV of 85.71% for with and without recurrence. We observed similar but a postoperative sPDGF-BB. weak trend; patients with HCC recurrence had lower IP PDGF-BB concentrations preoperatively (PRE OP median . . Postoperative sPDGF-BB Independently Predicts Postre- IP PDGF-BB: nonrecurrent, 12.38 pg/10 platelets; recurrent, section HCC Recurrence. We observed distinctly depleted 8.94 pg/10 platelets, P = 0.709; Fig S1A) and after four perioperative concentrations of sPDGF-BB levels in patients weeks of liver resection (POST OP median IP PDGF-BB: with postresection HCC recurrence; furthermore, it was of 6 6 nonrecurrent, 12.43 pg/10 platelets; recurrent, 9.24 pg/10 interest to investigate if sPDGF-BB could independently pre- platelets, P = 0.43; Fig S1B). The difference in IP PDGF-BB dict postresection recurrence. Therefore, we rfi st performed between recurrent and nonrecurrent cases did not yield a univariable (UVA) and multivariable survival analysis (MVA) statistically significant result. using Cox’s proportional hazard model. Among the 4 sig- We also examined the possibility of increased platelet nificant variables in UVA, we excluded the postoperative activation in patients with or without recurrence by evalu- platelet count in MVA because of its collinearity to all other 3 ating the total platelet P-selectin in patients with or without variables. recurrence and found no substantial difference in the concen- As seen in Table 2, postoperative sPDGF-BB was able to trations of total platelet P-selectin between the groups (PRE independently predict the postresection recurrence (hazard OP median IP P-selectin: nonrecurrent, 520.7 pg/10 platelets; ratio = 5.64, CI = 1.56 - 20.30, P = 0.008). recurrent, 507 pg/10 platelets, P = 0.709; Fig S2A, and POST Furthermore, a log rank test was run to determine OP Median IP P-selectin: nonrecurrent, 477 pg/10 platelets; the differences in disease-free interval (DFI) distribution recurrent, 455 pg/10 platelets, P = 0.61; Fig S2B). between groups with high and low perioperative serum sPDGF-BB. The Kaplan-Meier curves generated distinct DFI pattern between the patients with high and low perioperative .. Perioperative sPDGF-BB Values Specifically Characterize PDGF-BB; patients with lower preoperative [𝜒 2(2) = 5.97, P Patients with Postresection HCC Recurrence. Given the sub- = 0.01] or postoperative [𝜒 2 (2) = 11.04, P = 0.001] sPDGF-BB stantial difference in concentrations of sPDGF-BB in patients demonstrate shorter DFI (Figures 3(a) and 3(b)). with and without postresection HCC recurrence, we further focused on its clinical relevance. First, we determined a cut-off point for both pre- and postoperative sPDGF-BB by 4. Discussion using the receiver operating characteristics (ROC) curves (Figures 2(a) and 2(b)). We then applied Youden’s J index The overexpression or mutations of PDGF and its prognostic and identiefi d a cut-off value of 2062.34 pg/ml for PRE relevance observed in different cancers[15, 16] prompted us OP sPDGF-BB [area under curve (AUC): 0.709, sensitivity: to investigate if soluble (serum) PDGF-BB could provide 73.3%, specificity: 64.0%, 95% cond fi ence interval (CI) = 0.53 useful information to diagnose HCC recurrence. We detected – 0.88, P = 0.029] and 2133.29 pg/ml for POST OP sPDGF-BB a substantially lower concentration of perioperative PDGF- (AUC: 0.816, sensitivity: 80.0%, specificity: 72.0%, 95% CI = BB in the serum of patients with early postresection HCC 0.68 – 0.94, P = 0.001) to distinguish between the high- and recurrence. sPDGF-BB (pg/ml) sPDGF-BB (pg/ml) Journal of Oncology 5 POST OP PRE OP 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 00 0.0 02 0.2 04 0.4 06 0.6 08 0.8 10 1.0 0.0 0.2 0.4 0.6 0.8 1.0 1- Specificity 1- Specificity (a) (b) PRE OP sPDGF-BB POST OP sPDGF-BB Cut-off 2062.34 pg/mL 2133.29 pg/mL AUC 0.70 0.81 Sensitivity 73.3 % 80.0 % Specificity 64.0 % 72.0 % PPV 54.99 % 63.16 % NPV 79.98 % 85.71 % P value 0.029 0.001 Figure 2: Receiver operating characteristic curve for the preoperative (a) and postoperative (b) serum PDGF-BB (sPDGF-BB). In this study, we evaluated perioperative PDGF-BB con- We believe that this is the first report on depleted serum centration in serum, as serum concentrations also represent concentrations of PDGF-BB in patients with cancer (HCC) PDGF-BB pool stored in platelets. Platelet-stored PDGF-BB recurrence. us, Th the query rises about the molecular mecha- is easily available for the tumor and surrounding stroma[14]; nism involved in the exhaustion of sPDGF-BB concentrations thus sPDGF-BB reflects the magnitude of proangiogenic in patients with recurrence. Based on our finding, we can influence related to the growth factor. Platelet dysfunction speculate that decreased synthesis or increased degradation is already a known phenomenon in cancer progression; of PDGF-BB (or both) may occur during the process of HCC platelets hyperactivity and platelet exhaustion are frequently recurrence. Some other functional studies have stressed on seen in cancer patients[17–20]. We previously observed the response of tumor vessels pericyte to PDGF-BB[21–23]. exhaustion of IP serotonin in patients with early cancer Frequently irregular and disorganized vascular networks in recurrence[17]; likewise, we speculated that the depleted tumor largely influence the tumor progression and outcome. sPDGF-BB might attribute to the platelet exhaustion; how- Intriguingly, endothelial, and not tumor, cells production of ever, we observed a weak trend with total platelet (IP) PDGF- PDGF-BB is essential for proper pericytes installation and BB. Furthermore, no remarkable difference in total platelet coverage and formation of stable pericyte-endothelial cell P-selectin, observed in the study, rules out the possibility contacts[21]. In a previous study, ablation of pericytes by of a varied pattern of platelet activation in patients with anti-PDGF agents augmented vascular tortuosity and tumor or without recurrence. It is clearly known that PDGF-BB growth, suggesting a negative role of anti-PDGF-BB-induced in serum is not a soul reefl ction of PDGF-BB content pericyte loss in tumor angiogenesis and growth[22]. The same in platelets; a variety of cells secrete PDGF-BB [13–15]. study revealed a dose-dependent effect of PDGF-BB in tumor u Th s, the exhaustive behavior of PDGF-BB in serum is vessels pericyte loss and demonstrated a detrimental effect not fully attributed to the platelet kinetics in HCC recur- of substantially lower PDGF-BB in tumor progression [22]. rence. Stabilization of tumor blood vessel by pericyte is suggested to Sensitivity Sensitivity 6 Journal of Oncology Table 2: Univariable and multivariable analyses with Cox proportional hazard model. Univariable Multivariable BExp(B) 95%CI P value B Exp(B) 95% CI P value Variables 0.006 1.00 0.94-1.06 0.83 Age at resection -0.03 0.97 0.30-3.03 0.95 Sex 0.34 1.40 0.55-3.58 0.47 Etiology (viral/nonviral) Fibrosis grade (0-2/3-4) -0.68 0.50 0.17-1.41 0.19 -0.82 0.92 0.75-1.12 0.42 Tumor size, cm -0.38 0.96 0.30-3.02 0.94 Tumor stage (I-II/III-IV) 0.48 1.61 0.51-5.09 0.41 Tumor Multiplicity -0.50 0.60 0.13-2.69 0.51 Microvascular invasion Histological dieff rentiation 0.48 1.63 0.36-7.22 0.52 (well-to-moderate/poor) 1.36 3.91 1.24-12.32 0.02 1.16 3.18 0.99-10.22 0.05 Prior TACE/RFA Type of hepatectomy -0.38 0.682 0.21-2.14 0.51 (major/minor) 1.77 5.90 0.77-44.94 0.09 INR≥ 1.0 0.60 1.83 0.66-5.06 0.24 AFP> 20 ng/mL PIVKA II> 40, mAU/L -0.25 0.77 0.28-2.14 0.62 0.20 1.01 0.99-1.04 0.09 AST, U/L -0.005 0.99 0.97-1.01 0.61 ALT, U/L Albumin, g/L 0.20 1.23 0.46-3.24 0.67 0.14 1.15 0.34-3.92 0.81 TB, mg/dL -0.08 0.92 0.83-1.01 0.10 Preop. platelet count× 10 /𝜇 L -0.15 0.85 0.74-0.99 0.04 Postop. platelet count× 10 /𝜇 L Preop. sPDGF-BB , 1.33 3.78 1.20-11.94 0.02 0.84 2.32 0.71-7.54 0.16 pg/mL 1.87 6.52 1.82-23.24 0.004 1.73 5.64 1.56-20.30 0.008 Postop. sPDGF-BB ,pg/mL -0.77 0.46 0.06-35.0 0.45 Clavien-Dindo severe morbidity ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; TB, total bilirubin; AFP, alpha-fetoprotein; PIVKA II, protein induced by vitamin K absence/antagonist-II; TACE, transarterial chemoembolization; RFA, radiofrequency ablation; INR, international normalized ratio;Preop., preoperative; Postop., postoperative; sPDGF-BB, serum platelet derived growth factor-BB; , categorical variable. be a desirable therapeutic goal where new vessel may inhibit injury[24, 25]. The dimeric form of PDGF including at least formation and tumor growth thereby arrested[21]. Similarly, one B chain (PDGF-AB and PDGF-BB) was found to be more PDGF-BB-induced pericyte detachment was observed in two potent for HSC with predominant expression of PDGF-R different cancer cell lines[22]. In another study by McCarty 𝛽 subunits[26]. The “good” prohealing action of PDGF-BB et al., overexpression of PDGF-BB decreased colorectal and observed earlier has now been translated for clinical appli- pancreatic cancer growth by increasing the tumor pericyte cations, establishing an inverse association between sPDGF- content [23]. The authors observed that the tumor with BB concentrations and advanced br fi osis[14]. Takayema et al. highest PDGF-BB expression in vitro had the slowest tumor observed an association between decreased concentration of growth rate in vivo. Another compelling evidence on oppos- sPDGF-BB and poor outcomes in patients with fulminant ing effects of PDGF-BB levels in tumor microenvironment hepatic failure (FHF) suggesting a protective role of PDGF- warrants a caution while selecting the patients for anti-PDGF BB in liver repair[27]. This observation, to some extent, drugs[22]. These mechanisms somehow justify the n fi dings rationalizes the peculiar results we obtained in our cohort; of our study; a similar phenomenon such as the increased however, we did not observe a signicfi ant association between proportion of cancer cells leaking through the less stabilized fibrosis stage and sPDGF-BB concentrations. Nevertheless, tumor vasculature in patients with low PDGF-BB could have the ubiquitous mechanism of fibroproliferative response led to early postresection HCC recurrence. interlaced with PDGF-BB and the resultant phenotypic Investigations on liver b fi rogenesis, particularly with the change that may occur during the process of cancer recur- identification of the role played by PDGF on hepatic stellate rence cannot be completely ignored. cells (HSC), have clearly suggested PDGF as the most potent Our results provide rfi st in-human evidence for an mitogen in the series of tissue repair process aer ft liver inverse association between perioperative sPDGF-BB and Journal of Oncology 7 PRE OP Ethical Approval 1.0 sPDGF-BB > 2062.34 pg/ml All procedures performed in studies involving human par- 0.8 ticipants were in accordance with the ethical standards of P < 0.01 the institutional and/or national research committee and 0.6 with the 1964 Helsinki Declaration and its later amendments sPDGF-BB < 2062.34 pg/ml or comparable ethical standards. The institutional ethics 0.4 committee (Kagoshima University # 24-155/26-77, Kirishima Medical Center # 2505, and Kagoshima Medical Center # 25- 0.2 30) approved analyses of blood samples and patient data. 0.0 0.0 5.0 10.0 15.0 20.0 25.0 Consent Time ( months ) 0 All patients gave signed, informed consent. 0-censored Disclosure 1-censored (a) Teruto Hashiguchi and Yutaka Imoto are joint senior authors. POST OP 1.0 sPDGF-BB > 2133.29 pg/ml Conflicts of Interest + + 0.8 All the authors have no conflicts of interest to disclose. P < 0.001 0.6 sPDGF-BB < 2133.29 pg/ml Acknowledgments 0.4 + Dr. Bibek Aryal (https://orcid.org/0000-0003-0257-7439) is 0.2 a fellow of Japan Society for the Promotion of Science (JSPS). The authors thank Nobue Uto, Ryoko Narimatsu 0.0 (Department of Laboratory and Vascular Medicine), and Miyuki Sugo (Kirishima Medical Center) for their techni- 0.0 5.0 10.0 15.0 20.0 25.0 cal support. This study was funded by Grants-in-Aid for Time ( months ) Scientific Research, JSPS KAKENHI (Grant nos. 16F16420, 18F16420, and 16H05229). 0-censored 1-censored Supplementary Materials (b) Supporting Figure 1: intraplatelet (IP) PDGF-BB concentra- Figure 3: Kaplan-Meier disease-free survival curves based on tions in recurrent and nonrecurrence cases (A) before (PRE preoperative (PRE OP) (a) and postoperative (POST OP) (b) serum OP) and (B) 4 weeks after liver resection (POST OP). IP PDGF-BB (sPDGF-BB) concentration. concentrations are expressed per 10 platelets. Supporting Figure 2: intraplatelet (IP) P-selectin concentrations in recur- rent and nonrecurrence cases (A) before (PRE OP) and (B) 4 postresection HCC recurrence. Given the relatively small weeks after liver resection (POST OP). IP concentrations are sample size of this pilot study and with no direct mechanistic expressed per 10 platelets. 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Predictive Value of Diminished Serum PDGF-BB after Curative Resection of Hepatocellular Cancer

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Copyright © 2019 Bibek Aryal et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Oncology Volume 2019, Article ID 1925315, 8 pages https://doi.org/10.1155/2019/1925315 Research Article Predictive Value of Diminished Serum PDGF-BB after Curative Resection of Hepatocellular Cancer 1 2 2 3 Bibek Aryal , Munekazu Yamakuchi , Toshiaki Shimizu, Jun Kadono, 3 1 4 5 Akira Furoi, Kentaro Gejima, Teruo Komokata, Chihaya Koriyama, 2 1 Teruto Hashiguchi , and Yutaka Imoto Cardiovascular and Gastroenterological Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima -, Japan Department of Laboratory and Vascular Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima -, Japan Department of Surgery, Kirishima Medical Center, Kirishima -, Japan Department of Surgery, Kagoshima Medical Center, National Hospital Organization, Kagoshima -, Japan Department of Epidemiology and Preventive Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima -, Japan Correspondence should be addressed to Bibek Aryal; drbibekaryal@gmail.com and Teruto Hashiguchi; terutoha@m3.kufm.kagoshima-u.ac.jp Received 24 June 2018; Accepted 13 December 2018; Published 6 January 2019 Academic Editor: Ozkan Kanat Copyright © 2019 Bibek Aryal et al. is Th is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose. Platelet derived growth factor-BB (PDGF-BB) has emerged as one of the key cytokines in malignant transformation of dieff rent cells. PDGF-BB also exhibits a potent mitogenic effect on liver cells; studies have advocated clinical implications of monitoring serum PDGF-BB (sPDGF-BB) in patients with liver disease. We thus investigated the predictive relevance of perioperative sPDGF-BB aeft r curative resection of hepatocellular carcinoma (HCC). Methods. We evaluated perioperative sPDGF- BB in a prospective homogenous cohort of 40 patients diagnosed with HCC. During the first two-year follow-up, patients were evaluated every three months for postresection HCC recurrence. Results. Patients who developed recurrence during two-year follow-up were found to have lower concentration of sPDGF-BB than those without recurrence in both pre- and postoperative settings (P< 0.05 and P< 0.001, resp.). We validated that the reduced postoperative sPDGF-BB (< 2133.29 pg/mL) was associated with an increased incidence of postresection HCC recurrence [area under curve (AUC)> 0.8, 95% confidence interval (CI) = 0.68 -0.94, P< 0.001]; furthermore, we were able to demonstrate that postoperative sPDGF-BB was an independent predictor of HCC recurrence (hazard ratio = 5.64, 95% CI = 1.56 - 20.30, P< 0.01). Conclusions. es Th e findings provide a new insight into an association between diminished perioperative sPDGF-BB and HCC recurrence. Patients with low perioperative sPDGF-BB progressed early HCC recurrence. Therefore, evaluating perioperative sPDGF-BB may provide useful clinical information to characterize patients with postresection HCC recurrence. 1. Introduction patients, ideally before the recurrence sets in, by integrating both clinical and molecular information into a synergistic Curative resection (partial hepatectomy) is a safe, effective, understanding of the tumor recurrence. and preferred therapy for selected patients with hepatocel- Platelet-related biomarkers, detected in the circulation lular carcinoma (HCC)[1]. However, the tumor recurrence (whole blood, serum, or plasma), have long been considered particularly in the remnant liver is exceedingly frequent after as a potential diagnostic tool in cancer[3–7]. These growth the surgical resection of HCC[2]. The high incidence of factors have demonstrated several implications in the eld fi HCC recurrence intensifies the need for identifying high-risk of oncology including screening, diagnostic, and prognostic 2 Journal of Oncology relevance of diseases[7]. Platelet-related growth factors reflect centrifuged at 2810× g to isolate platelets. The supernatant, paradoxical association with different physiological and platelet-poor plasma (PPP), was collected precisely and pathological events[8]. For instance, the platelet-sequestered removed completely by decantation method. Platelet pellets isolated from each 200𝜇 l of PRP were suspended in 220𝜇 lof vascular endothelial growth factor (VEGF), which is linked to various pathological conditions[9] including carcinogenesis, lysis buffer (RIPA) and were vortexed after incubating for 20 has also been found to be crucial in physiological events like minutes. CBC was performed in 3 preparations: whole blood, liver regeneration after partial hepatectomy[10, 11]. PRP, and PPP. Growing lines of evidence recognize the role of platelet All samples were stored in aliquots at -80 C immediately derived growth factor-BB (PDGF-BB) at every stage during after preparation. the continuum of liver injury, repair, and fibrosis[12–14]. Despite a wealth of literature on PDGF-BB in other tumor .. Quantification of Cytokines. Platelet derived growth types, there are relatively a few studies on the PDGF-BB and factor-BB and P-selectin were measured using enzyme-linked its predictive relevance in HCC. Also, to date, no evidence immunosorbent assay (ELISA) tests (Quantikine; R&D Sys- examining the feature of serum PDGF-BB (sPDGF-BB) on tems,Minneapolis, MN, USA).Intraplatelet (IP) PDGF-BB postresection HCC recurrence has been published in English was calculated from isolated platelets using the equation literature. In this study, we aimed to determine whether to calculate cytokines inside each platelet, as described the perioperative sPDGF-BB could reflect the oncological before[10]. Concisely, 220 𝜇 lof lysis solution (RIPA) was outcome aeft r the curative resection of HCC. added to the platelets isolated from each 200𝜇 lof PRP. The concentration of the cytokine was adjusted to the platelet 2. Materials and Methods count obtained from the PRP and IP PDGF-BB was expressed per 10 platelets. .. Study Cohort. The study cohort consisted of forty patients diagnosed with primary HCC who went on to have liver . . Statistics. Statistical analyses were performed using SPSS resection. The trial is registered in UMIN Clinical Trial Reg- 25.0 software (SPSS, Inc., Chicago, IL, USA) and Graph- istry (UMIN000026380). The institutional ethics committee Pad Prism (version 6.0d for MacOS X, USA, GraphPad (Kagoshima University # 24-155/ 26-77, Kirishima Medical Sow ft are, San Diego, California, USA) and were mainly Center # 2505 and Kagoshima Medical Center # 25-30) based on nonparametric tests (Mann-Whitney U test and approved analyses of blood samples and patient data; all Wilcoxon test). Receiver operating characteristic (ROC) patients gave signed, informed consent. analysis was performed to assess the specificity and sensitivity of PDGF-BB levels to predict recurrence. Youden’s J index .. Follow-Up. Disease-free interval (DFI) was defined was applied to determine cut-off points in ROC analyses. according to the Guidance for Industry Clinical Trial End- Cox's proportional hazards regression model was used for points for the Approval of Cancer Drugs and Biologics of the the univariable analyses (UVA) and multivariable analyses US Food and Drug Administration (FDA). DFI represents the (MVA) to determine the variables independently associated time period between liver resection and tumor recurrence. with recurrence. Two-tailed P values of less than 0.05 were Follow-up period was standardized to two years; patients considered statistically significant. IP PDGF-BB and IP P- were followed up every three months aeft r surgery. Follow-up selectin concentrations were expressed per 10 platelets. was routinely scheduled and comprised of ultrasonography (USG) as well as evaluation of tumor markers; if USG showed any evidence of tumor, further assessment of thoracic 3. Results and abdominal CAT scans or MRI was performed. Tumor recurrence was diagnosed based on analysis of radiological .. Patient Demographics. We included forty patients with findings and comprised of local and distant recurrence. HCC who were selected for liver resection. Baseline char- acteristics of the study cohort are summarized in Table 1. None of the patients received platelet transfusion during the .. Sample Preparation. Venous blood was collected preop- sample preparations. During two-year follow- up, 15 patients eratively, immediately before surgery (PRE OP), and four developedHCC recurrence andone patient diedwith a cause weeks after surgery (POST OP). Complete blood count other than the cancer recurrence. (CBC) was performed with an automated hematology ana- lyzer Sysmex XE-5000 (Sysmex Corporation, Kobe, Japan). Whole blood was collected in the serum-separating .. Perioperative sPDGF-BB Concentrations Were Lower in tube and an EDTA-2k. Serum tube was incubated at room Patients with HCC Recurrence. We first compared perioper- temperature for 30 minutes before centrifuging at 1710× gfor ative sPDGF-BB concentrations in patients with postresec- 10 minutes. tion HCC recurrence. Patients with two-year postresection HCC recurrence had significantly lower concentrations of . . Platelet Extract. Venous blood in citrate tubes was sPDGF-BB prior to liver resection (PRE OP median sPDGF: centrifuged at 90× g for 15 minutes. To avoid contaminations nonrecurrent, 2444 pg/mL; recurrent, 1592 pg/mL, P = 0.02; with other cells, upper two-thirds of the resultant platelet- Figure 1(a)) and aer ft four weeks of liver resection compared rich plasma (PRP) were gently pipetted. The PRP was then to the patients with no cancer recurrence (POST OP median Journal of Oncology 3 Table 1: Demographics and clinical and pathological data of the cohort. Variables Data Age, median (min, max) 72.52 (51, 83) Sex, n(%) Male, 30 (75 %) Female 10 (25 %) Child Pugh Class, n(%) A 40 (100 %) B 0 C 0 Etiology, n(%) HBV 12 (30 %) HCV 13 (32.5 %) Nonviral 15 (37.5 %) Fibrosis grade, n(%) 0-2 22 (55 %) 3-4 18 (45 %) Tumor staging, n(%) I-II 28 (70 %) III-IV 12 (30 %) Tumor size, n(%) < 5cm 27 (67.5 %) ≥ 5cm 13 (32.5 %) Tumor number, n(%) Single 31 (77.5 %) Multiple 09 (22.5 %) Vascular invasion, n(%) Present 08 (20 %) Absent 32 (80 %) Tumor histology (differentiation), n(%) Well-to-moderate 32 (80 %) Poor 08 (20 %) Type of hepatectomy, n(%) Major hepatectomy 14 (35 %) Minor hepatectomy 26 (65 %) Prior treatment, n(%) RFA/TACE 19 (47.5 %) None 21 (52. 5 %) Total bilirubin before resection, n(%) < 1mg/dl 28 (70 %) ≥ 1mg/dl 12 (30 %) INR before resection, n(%) > 1 30 (75 %) < 1 10 (25 %) AFP before resection, n(%) < 20 ng/ml 25 (62.5 %) ≥ 20 ng/ml 15 (37.5 %) Clavien-Dindo postoperative (severe) morbidity, n(%) Absent 35 (87.5 %) Present 05 (12.5 %) Min., minimum; max., maximum; n/N, numbers; HBV, hepatitis B virus; HCV, hepatitis C virus; INR, international normalized ratio, AFP, alpha-fetoprotein; PIVKA II, protein induced by vitamin K absence/antagonist-II; TACE, transarterial chemoembolization; RFA, radiofrequency ablation; INR, international normalized ratio. 4 Journal of Oncology PRE OP POST OP ∗∗∗ 0 0 Recurrence (-) Recurrence (+) Recurrence (-) Recurrence (+) (a) (b) Figure 1: Serum PDGF-BB (sPDGF-BB) concentration in recurrent and nonrecurrent cases (a) before (PRE OP) and (b) 4 weeks aeft r liver ∗ ∗∗∗ resection (POST OP). < 0.05 and < 0.001. P P sPDGF: nonrecurrent, 2852 pg/mL; recurrent, 1592 pg/mL, P low-risk patients in terms of HCC recurrence. This translates < 0.001; Figure 1(b)). to the positive predictive value (PPV) of 54.99% and negative Furthermore, we compared separately isolated, predictive value (NPV) of 79.98% for preoperative sPDGF- intraplatelet (IP) concentrations of PDGF-BB in patients BB and similarly PPV of 63.16% and NPV of 85.71% for with and without recurrence. We observed similar but a postoperative sPDGF-BB. weak trend; patients with HCC recurrence had lower IP PDGF-BB concentrations preoperatively (PRE OP median . . Postoperative sPDGF-BB Independently Predicts Postre- IP PDGF-BB: nonrecurrent, 12.38 pg/10 platelets; recurrent, section HCC Recurrence. We observed distinctly depleted 8.94 pg/10 platelets, P = 0.709; Fig S1A) and after four perioperative concentrations of sPDGF-BB levels in patients weeks of liver resection (POST OP median IP PDGF-BB: with postresection HCC recurrence; furthermore, it was of 6 6 nonrecurrent, 12.43 pg/10 platelets; recurrent, 9.24 pg/10 interest to investigate if sPDGF-BB could independently pre- platelets, P = 0.43; Fig S1B). The difference in IP PDGF-BB dict postresection recurrence. Therefore, we rfi st performed between recurrent and nonrecurrent cases did not yield a univariable (UVA) and multivariable survival analysis (MVA) statistically significant result. using Cox’s proportional hazard model. Among the 4 sig- We also examined the possibility of increased platelet nificant variables in UVA, we excluded the postoperative activation in patients with or without recurrence by evalu- platelet count in MVA because of its collinearity to all other 3 ating the total platelet P-selectin in patients with or without variables. recurrence and found no substantial difference in the concen- As seen in Table 2, postoperative sPDGF-BB was able to trations of total platelet P-selectin between the groups (PRE independently predict the postresection recurrence (hazard OP median IP P-selectin: nonrecurrent, 520.7 pg/10 platelets; ratio = 5.64, CI = 1.56 - 20.30, P = 0.008). recurrent, 507 pg/10 platelets, P = 0.709; Fig S2A, and POST Furthermore, a log rank test was run to determine OP Median IP P-selectin: nonrecurrent, 477 pg/10 platelets; the differences in disease-free interval (DFI) distribution recurrent, 455 pg/10 platelets, P = 0.61; Fig S2B). between groups with high and low perioperative serum sPDGF-BB. The Kaplan-Meier curves generated distinct DFI pattern between the patients with high and low perioperative .. Perioperative sPDGF-BB Values Specifically Characterize PDGF-BB; patients with lower preoperative [𝜒 2(2) = 5.97, P Patients with Postresection HCC Recurrence. Given the sub- = 0.01] or postoperative [𝜒 2 (2) = 11.04, P = 0.001] sPDGF-BB stantial difference in concentrations of sPDGF-BB in patients demonstrate shorter DFI (Figures 3(a) and 3(b)). with and without postresection HCC recurrence, we further focused on its clinical relevance. First, we determined a cut-off point for both pre- and postoperative sPDGF-BB by 4. Discussion using the receiver operating characteristics (ROC) curves (Figures 2(a) and 2(b)). We then applied Youden’s J index The overexpression or mutations of PDGF and its prognostic and identiefi d a cut-off value of 2062.34 pg/ml for PRE relevance observed in different cancers[15, 16] prompted us OP sPDGF-BB [area under curve (AUC): 0.709, sensitivity: to investigate if soluble (serum) PDGF-BB could provide 73.3%, specificity: 64.0%, 95% cond fi ence interval (CI) = 0.53 useful information to diagnose HCC recurrence. We detected – 0.88, P = 0.029] and 2133.29 pg/ml for POST OP sPDGF-BB a substantially lower concentration of perioperative PDGF- (AUC: 0.816, sensitivity: 80.0%, specificity: 72.0%, 95% CI = BB in the serum of patients with early postresection HCC 0.68 – 0.94, P = 0.001) to distinguish between the high- and recurrence. sPDGF-BB (pg/ml) sPDGF-BB (pg/ml) Journal of Oncology 5 POST OP PRE OP 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 00 0.0 02 0.2 04 0.4 06 0.6 08 0.8 10 1.0 0.0 0.2 0.4 0.6 0.8 1.0 1- Specificity 1- Specificity (a) (b) PRE OP sPDGF-BB POST OP sPDGF-BB Cut-off 2062.34 pg/mL 2133.29 pg/mL AUC 0.70 0.81 Sensitivity 73.3 % 80.0 % Specificity 64.0 % 72.0 % PPV 54.99 % 63.16 % NPV 79.98 % 85.71 % P value 0.029 0.001 Figure 2: Receiver operating characteristic curve for the preoperative (a) and postoperative (b) serum PDGF-BB (sPDGF-BB). In this study, we evaluated perioperative PDGF-BB con- We believe that this is the first report on depleted serum centration in serum, as serum concentrations also represent concentrations of PDGF-BB in patients with cancer (HCC) PDGF-BB pool stored in platelets. Platelet-stored PDGF-BB recurrence. us, Th the query rises about the molecular mecha- is easily available for the tumor and surrounding stroma[14]; nism involved in the exhaustion of sPDGF-BB concentrations thus sPDGF-BB reflects the magnitude of proangiogenic in patients with recurrence. Based on our finding, we can influence related to the growth factor. Platelet dysfunction speculate that decreased synthesis or increased degradation is already a known phenomenon in cancer progression; of PDGF-BB (or both) may occur during the process of HCC platelets hyperactivity and platelet exhaustion are frequently recurrence. Some other functional studies have stressed on seen in cancer patients[17–20]. We previously observed the response of tumor vessels pericyte to PDGF-BB[21–23]. exhaustion of IP serotonin in patients with early cancer Frequently irregular and disorganized vascular networks in recurrence[17]; likewise, we speculated that the depleted tumor largely influence the tumor progression and outcome. sPDGF-BB might attribute to the platelet exhaustion; how- Intriguingly, endothelial, and not tumor, cells production of ever, we observed a weak trend with total platelet (IP) PDGF- PDGF-BB is essential for proper pericytes installation and BB. Furthermore, no remarkable difference in total platelet coverage and formation of stable pericyte-endothelial cell P-selectin, observed in the study, rules out the possibility contacts[21]. In a previous study, ablation of pericytes by of a varied pattern of platelet activation in patients with anti-PDGF agents augmented vascular tortuosity and tumor or without recurrence. It is clearly known that PDGF-BB growth, suggesting a negative role of anti-PDGF-BB-induced in serum is not a soul reefl ction of PDGF-BB content pericyte loss in tumor angiogenesis and growth[22]. The same in platelets; a variety of cells secrete PDGF-BB [13–15]. study revealed a dose-dependent effect of PDGF-BB in tumor u Th s, the exhaustive behavior of PDGF-BB in serum is vessels pericyte loss and demonstrated a detrimental effect not fully attributed to the platelet kinetics in HCC recur- of substantially lower PDGF-BB in tumor progression [22]. rence. Stabilization of tumor blood vessel by pericyte is suggested to Sensitivity Sensitivity 6 Journal of Oncology Table 2: Univariable and multivariable analyses with Cox proportional hazard model. Univariable Multivariable BExp(B) 95%CI P value B Exp(B) 95% CI P value Variables 0.006 1.00 0.94-1.06 0.83 Age at resection -0.03 0.97 0.30-3.03 0.95 Sex 0.34 1.40 0.55-3.58 0.47 Etiology (viral/nonviral) Fibrosis grade (0-2/3-4) -0.68 0.50 0.17-1.41 0.19 -0.82 0.92 0.75-1.12 0.42 Tumor size, cm -0.38 0.96 0.30-3.02 0.94 Tumor stage (I-II/III-IV) 0.48 1.61 0.51-5.09 0.41 Tumor Multiplicity -0.50 0.60 0.13-2.69 0.51 Microvascular invasion Histological dieff rentiation 0.48 1.63 0.36-7.22 0.52 (well-to-moderate/poor) 1.36 3.91 1.24-12.32 0.02 1.16 3.18 0.99-10.22 0.05 Prior TACE/RFA Type of hepatectomy -0.38 0.682 0.21-2.14 0.51 (major/minor) 1.77 5.90 0.77-44.94 0.09 INR≥ 1.0 0.60 1.83 0.66-5.06 0.24 AFP> 20 ng/mL PIVKA II> 40, mAU/L -0.25 0.77 0.28-2.14 0.62 0.20 1.01 0.99-1.04 0.09 AST, U/L -0.005 0.99 0.97-1.01 0.61 ALT, U/L Albumin, g/L 0.20 1.23 0.46-3.24 0.67 0.14 1.15 0.34-3.92 0.81 TB, mg/dL -0.08 0.92 0.83-1.01 0.10 Preop. platelet count× 10 /𝜇 L -0.15 0.85 0.74-0.99 0.04 Postop. platelet count× 10 /𝜇 L Preop. sPDGF-BB , 1.33 3.78 1.20-11.94 0.02 0.84 2.32 0.71-7.54 0.16 pg/mL 1.87 6.52 1.82-23.24 0.004 1.73 5.64 1.56-20.30 0.008 Postop. sPDGF-BB ,pg/mL -0.77 0.46 0.06-35.0 0.45 Clavien-Dindo severe morbidity ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; TB, total bilirubin; AFP, alpha-fetoprotein; PIVKA II, protein induced by vitamin K absence/antagonist-II; TACE, transarterial chemoembolization; RFA, radiofrequency ablation; INR, international normalized ratio;Preop., preoperative; Postop., postoperative; sPDGF-BB, serum platelet derived growth factor-BB; , categorical variable. be a desirable therapeutic goal where new vessel may inhibit injury[24, 25]. The dimeric form of PDGF including at least formation and tumor growth thereby arrested[21]. Similarly, one B chain (PDGF-AB and PDGF-BB) was found to be more PDGF-BB-induced pericyte detachment was observed in two potent for HSC with predominant expression of PDGF-R different cancer cell lines[22]. In another study by McCarty 𝛽 subunits[26]. The “good” prohealing action of PDGF-BB et al., overexpression of PDGF-BB decreased colorectal and observed earlier has now been translated for clinical appli- pancreatic cancer growth by increasing the tumor pericyte cations, establishing an inverse association between sPDGF- content [23]. The authors observed that the tumor with BB concentrations and advanced br fi osis[14]. Takayema et al. highest PDGF-BB expression in vitro had the slowest tumor observed an association between decreased concentration of growth rate in vivo. Another compelling evidence on oppos- sPDGF-BB and poor outcomes in patients with fulminant ing effects of PDGF-BB levels in tumor microenvironment hepatic failure (FHF) suggesting a protective role of PDGF- warrants a caution while selecting the patients for anti-PDGF BB in liver repair[27]. This observation, to some extent, drugs[22]. These mechanisms somehow justify the n fi dings rationalizes the peculiar results we obtained in our cohort; of our study; a similar phenomenon such as the increased however, we did not observe a signicfi ant association between proportion of cancer cells leaking through the less stabilized fibrosis stage and sPDGF-BB concentrations. Nevertheless, tumor vasculature in patients with low PDGF-BB could have the ubiquitous mechanism of fibroproliferative response led to early postresection HCC recurrence. interlaced with PDGF-BB and the resultant phenotypic Investigations on liver b fi rogenesis, particularly with the change that may occur during the process of cancer recur- identification of the role played by PDGF on hepatic stellate rence cannot be completely ignored. cells (HSC), have clearly suggested PDGF as the most potent Our results provide rfi st in-human evidence for an mitogen in the series of tissue repair process aer ft liver inverse association between perioperative sPDGF-BB and Journal of Oncology 7 PRE OP Ethical Approval 1.0 sPDGF-BB > 2062.34 pg/ml All procedures performed in studies involving human par- 0.8 ticipants were in accordance with the ethical standards of P < 0.01 the institutional and/or national research committee and 0.6 with the 1964 Helsinki Declaration and its later amendments sPDGF-BB < 2062.34 pg/ml or comparable ethical standards. The institutional ethics 0.4 committee (Kagoshima University # 24-155/26-77, Kirishima Medical Center # 2505, and Kagoshima Medical Center # 25- 0.2 30) approved analyses of blood samples and patient data. 0.0 0.0 5.0 10.0 15.0 20.0 25.0 Consent Time ( months ) 0 All patients gave signed, informed consent. 0-censored Disclosure 1-censored (a) Teruto Hashiguchi and Yutaka Imoto are joint senior authors. POST OP 1.0 sPDGF-BB > 2133.29 pg/ml Conflicts of Interest + + 0.8 All the authors have no conflicts of interest to disclose. P < 0.001 0.6 sPDGF-BB < 2133.29 pg/ml Acknowledgments 0.4 + Dr. Bibek Aryal (https://orcid.org/0000-0003-0257-7439) is 0.2 a fellow of Japan Society for the Promotion of Science (JSPS). The authors thank Nobue Uto, Ryoko Narimatsu 0.0 (Department of Laboratory and Vascular Medicine), and Miyuki Sugo (Kirishima Medical Center) for their techni- 0.0 5.0 10.0 15.0 20.0 25.0 cal support. This study was funded by Grants-in-Aid for Time ( months ) Scientific Research, JSPS KAKENHI (Grant nos. 16F16420, 18F16420, and 16H05229). 0-censored 1-censored Supplementary Materials (b) Supporting Figure 1: intraplatelet (IP) PDGF-BB concentra- Figure 3: Kaplan-Meier disease-free survival curves based on tions in recurrent and nonrecurrence cases (A) before (PRE preoperative (PRE OP) (a) and postoperative (POST OP) (b) serum OP) and (B) 4 weeks after liver resection (POST OP). IP PDGF-BB (sPDGF-BB) concentration. concentrations are expressed per 10 platelets. Supporting Figure 2: intraplatelet (IP) P-selectin concentrations in recur- rent and nonrecurrence cases (A) before (PRE OP) and (B) 4 postresection HCC recurrence. Given the relatively small weeks after liver resection (POST OP). IP concentrations are sample size of this pilot study and with no direct mechanistic expressed per 10 platelets. 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