Potential of extravasated platelet aggregation as a surrogate marker for overall survival in patients with advanced gastric cancer treated with preoperative docetaxel, cisplatin and S-1: a retrospective observational study

Seventy-eight patients with advanced gastric cancer who had undergone gastrectomy between 2005 and 2014 were retrospectively evaluated. Thirty-nine of them had received preoperative DCS therapy (p-DCS group), whereas the remaining 39 had not received any preoperative chemotherapy (control group). The 39 patients in the control group had, however, received gastrectomy and postoperative chemotherapy of S-1 alone. Eligibility criteria were as follows: clinical Stage III and resectable Stage IV gastric cancer with fewer than three peripheral hepatic and para-aortic lymph node (PAN) metastases [18] in accordance with the Japanese Classification of Gastric Carcinoma (JCGC), 3rd English edition [19], PAN metastasis being defined as clearly enlarged (≥ 10 mm) on enhanced computed tomography (CT) scans with 2.5 mm slice thickness; absence of peritoneal metastasis on staging laparoscopy; age 20–80 years; Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; no prior chemotherapy or radiotherapy; no prior gastrectomy; no detected bleeding from primary lesion; good oral intake; and adequate hematological, hepatic, and renal function.

Patients were excluded for any of the following reasons: apparent infection; serious comorbidity such as cardiovascular disease, pulmonary fibrosis, pneumonia, bleeding tendency, uncontrolled hypertension, poorly controlled diabetes mellitus, and other serious medical conditions; synchronous or metachronous active malignancy; central nervous system disorder; history of severe drug-induced allergy; and pregnancy or breastfeeding.

In the p-DCS group, patients had received two cycles of preoperative chemotherapy consisting of 35 mg/m² docetaxel as a 1-h intravenous infusion on days 1 and 15; 35 mg/m² cisplatin as a 2-h intravenous infusion on days 1 and 15 with hyperhydration; and 40 mg/m² S-1 twice daily on days 1–14 every 4 weeks. At least 4 weeks after the completion of two cycles of DCS therapy, curative gastrectomy and D2 lymphadenectomy plus PAN dissection (PAND) and hepatectomy had been performed. Lymph node dissection was performed in patients with PAN metastasis diagnosed by enhanced helical CT, which was defined as lymph node station No. 16a2 and b1 (16a2b1PAN) between the upper margin of the celiac artery and lower border of the inferior mesenteric artery [19].

In the control group, administration of S-1 was started within 6 weeks after gastrectomy and continued for 1 year. The treatment regimen consisted of 6-week cycles in which, in principle, 40 mg/m² S-1 twice daily was given for 4 weeks and no chemotherapy was given for the following 2 weeks [2, 20].

After the second course of preoperative DCS therapy, the amount of tumor shrinkage was evaluated based on thin-slice helical CT and the tumor response classified into one of the following four categories in accordance with RECIST [8]: complete response (CR), disappearance of all target lesions; partial response (PR), ≥30% decrease in the combined diameters of target lesions; progressive disease (PD), ≥20% increase in the combined diameters of target lesions; and stable disease (SD), neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD. Patients with CR and PR were regarded as RECIST responders.

In the p-DCS group, the resected specimens were histologically evaluated, and tumor response evaluated according to the histological criteria in JCGC, 3rd English edition [19]. The histological evaluation criteria were classified into one of the following five categories according to the proportion of the tumor affected by degeneration or necrosis: grade 3, no viable tumor cells remaining; grade 2, viable tumor cells remaining in less than one-third of the tumorous area; grade 1b, viable tumor cells remaining in more than one-third but less than two-thirds of the tumorous area; grade 1a, viable tumor cells occupying more than two-thirds of the tumorous area; and grade 0, no evidence of therapeutic effect.

Ten percent or 50% residual tumor per tumor bed has been used as the cutoff percentage in Western countries, in accordance with the criteria proposed by Becker et al. [21]. In contrast, a cutoff of 33% or 67% viable tumor cells per tumor bed is commonly used in Asian countries, in accordance with the definition in JCGC, 3rd English edition [19]. Although the definition of a histological response is controversial, Kurokawa et al. [9, 10] have evaluated the results when histological responses were classified as Grade 2 or 3 and found that the results were similar to Grades 1b, 2 or 3. In this study, a histological response was defined as less than one-third of viable tumor cells (grade 2 or 3). All resected specimens were examined by the same pathologist, who assessed the extent of residual disease, disease stage, and effect of chemotherapy according to the criteria of JCGC, 3rd English edition [19].

In the p-DCS group, primary cancer lesions were biopsied by esophagogastroduodenoscopy (EGD) before commencement of preoperative chemotherapy. In the control group, biopsies were performed by EGD on diagnosis. Biopsies were taken from the edge of ulcerations associated with gastric cancer, not from the bases of such ulceration. More than five biopsy specimens were collected from each patient and evaluated immunohistochemically. Immunohistochemistry using 3-μm-thick, 10% formalin-fixed, paraffin-embedded tissue sections was performed using Dako Envision System dextran polymers conjugated to horseradish peroxidase (Dako, Carpinteria, CA, USA) to prevent any endogenous biotin contamination. The specimens were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase was blocked by immersing sections in 3% H₂O₂ in 100% methanol for 20 min at room temperature. Antigen retrieval was activated by microwaving sections at 95 °C for 10 min in 0.001 M citrate buffer (pH 7.6). After blocking the endogenous peroxidase, sections were incubated with Protein Block Serum-Free (Dako) at room temperature for 10 min to block nonspecific staining. Subsequently, sections were incubated for 2 h at room temperature with a 1:100 diluted anti-platelet antibody (anti-CD42b rabbit monoclonal, EPR6995; Abcam, Tokyo, Japan); a 1:50 diluted anti-podoplanin antibody (anti-D2–40 mouse monoclonal, Code IR072/IS072; Dako, Tokyo, Japan); a 1:50 diluted anti-forkhead box (FOX)P3 antibody (anti-FOXP3 mouse monoclonal, 236A/E7; Abcam), and a 1:50 diluted anti-SNAIL antibody (anti-SNAIL rabbit polyclonal antibody, ab180714; Abcam). Peroxidase activity was detected using 3-amino-9-ethylcarbazole enzyme substrate. Sections were incubated in Tris-buffered saline without primary antibodies as negative controls. Samples were faintly counterstained with Meyer hematoxylin.

To evaluate the expression of CD42b in the biopsy specimens, the immunostained cells in five non-overlapping intratumoral fields were counted at 400× magnification. The average expression of CD42b was evaluated: ≥10% was defined as positive and <10% as negative [22]. In the biopsy specimens stained by D2–40, the immunostained cells were counted at 200× magnification. The percentage of podoplanin-positive (PP) cells and staining intensity (SI) were evaluated and an immunoreactivity score (IRS) calculated for each tumor as IRS = PP × SI (0 negative, 1–3 weak, 4–7 moderate, and 8–15 high). Scores were allocated as follows: 0 PP 0%, 1 PP 1%–20%, 2 PP 21%–40%, 3 PP 41%–60%, 4 PP 61%–80%, and 5 PP 81%–100%; and 0 SI negative, 1 weak, 2 moderate, and 3 strong. For IRS, ≥4 was defined as positive and <3 as negative [23].

For analysis of SNAIL, IRS was calculated by multiplication of intensity (0–3) by the percentage of stained cells (0–4). Tissue samples with scores of 0 were classified as SNAIL negative and those with scores of 1–12 as SNAIL positive [24].

To evaluate infiltration of FOXP3, five non-overlapping intratumoral fields were counted at 400× magnification and the mean number per field defined as the number of FOXP3 infiltrates for the tumor. The average number of FOXP3-positive T cells was evaluated; ≥5.5 being defined as positive and <5.5 as negative [25].

Fisher’s exact test was used to measure the statistical significance of correlations between CD42b expression and chemotherapeutic response. Patient survival was calculated by the Kaplan–Meier method and the log-rank test was used to compare the survival rates between subgroups. Variables found to have possible associations with chemoresistance and prognosis by univariate analysis (P < 0.10) were subjected to multivariate analysis using multi logistic regression analysis and the Cox proportional hazards regression model, respectively. Statistical significance was set at P < 0.05. Data management and statistical analysis were performed using SPSS version 23 (SPSS, Chicago, IL, USA).

From 2005 to 2014, 78 patients with advanced gastric cancer were found to be eligible, 39 of whom had received preoperative DCS therapy followed by curative gastrectomy with D2 lymphadenectomy plus PAND and/or hepatectomy (p-DCS group). The remaining 39 patients had not received preoperative DCS therapy prior to undergoing curative gastrectomy with D2 lymphadenectomy plus hepatectomy and had received postoperative chemotherapy of S-1 alone (control group). Patient characteristics are summarized in Table 1. In the p-DCS group, baseline CT showed that 16 (41%) had PAN metastases and nine (23%) hepatic metastases. The tumor stages were as follows: 13 (33%) clinical Stage III and 26 (67%) clinical Stage IV. In the control group, baseline CT showed that none had PAN metastases and one (3%) had hepatic metastases. The tumor stages were as follows: 38 (97%) clinical Stage III and one (3%) clinical Stage IV.

The responses to preoperative DCS therapy were assessed by RECIST and histological evaluation criteria (Table 1). The response rates were 74% with RECIST and 56% with histological criteria.

In the p-DCS group, biopsy specimens were obtained from the primary gastric cancers before commencing preoperative chemotherapy. Expression of CD42b, a platelet marker, was observed around cancer-associated fibroblasts (CAFs) in the biopsy specimens (Fig. 1a) and podoplanin expression was found on the membranes of CAFs (Fig. 1b).

There were no significant associations between CD42b expression and Borrmann macroscopic type, tumor differentiation, clinical T stage, clinical N stage, PAN metastases, or hepatic metastases in either group (Tables 2 and 3).

Univariate analysis of expression of three factors (CD42b, SNAIL, and FOXP3) that are reportedly associated with chemoresistance showed significant associations between CD42b expression (P = 0.025) and SNAIL expression (P = 0.029) and chemoresistance (Table 4). These two variables were therefore considered to be potential predictors of chemoresistance and were subjected to multivariate analysis, which identified a correlation between CD42b expression and chemoresistance (odds ratio: 5.102, 95% confidence interval: 1.039–25.00, P = 0.045) (Table 4).

In the p-DCS group, the EMT marker SNAIL was mainly expressed in the nuclei of cancer cells. Positive SNAIL expression was found in 30/39 cases (77%) (Fig. 1c); however, SNAIL expression was not correlated with CD42b expression (P = 0.230). There was a significant relationship between SNAIL expression and chemoresistance (P = 0.026) but no significant relationship between SNAIL expression and OS (P = 0.248).

In the p-DCS group, the regulatory T (Treg) cell marker FOXP3 was found in 7/39 cases (18%) (Fig. 1d). FOXP3 expression was not significantly correlated with CD42b expression (P = 0.686), chemoresistance (P = 0.205), or OS (P = 0.698).

Overall survival (OS) curves for the patients in the both groups are shown in Fig. 2. In the p-DCS group, comparison of survival rates in RECIST responders and non-responders by log-rank test revealed no significant difference in prognosis (P = 0.212) (Fig. 2a). In contrast, OS was significantly longer in histological responders than non-responders (P = 0.016) (Fig. 2b) and in CD42b-negative than CD42b-positive patients (P = 0.012) (Fig. 2c). In the control group, the OS was significantly longer for CD42b-negative than CD42b-positive patients (P = 0.033) (Fig. 2d).

Relapse-free survival curves for the patients in the both groups are shown in Fig. 3. In the p-DCS group, there was no significant difference in prognosis between the RECIST responders and non-responders (P = 0.112) (Fig. 3a). Histological evaluation and CD42b expression showed that relapse-free survival was significantly longer in responders than non-responders (P = 0.004, P = 0.013, respectively) (Fig. 3b, c). In the control group, the relapse-free survival was significantly longer in CD42b-negative than in CD42b-positive patients (P = 0.015) (Fig. 3d).

In the p-DCS group, univariate analysis showed that histological findings (P = 0.023) and CD42b expression (P = 0.021) were significantly associated with OS. The four variables (sex, hepatic metastasis, histological evaluation, and CD42b expression) that were found to be significant by univariate analysis and therefore had prognostic potential (P < 0.10) were subjected to multivariate analysis. Multivariate analysis identified that male sex (hazard ratio: 0.281, 95% confidence interval: 0.093–0.846, P = 0.024) was correlated with good prognosis and CD42b expression (hazard ratio: 4.406, 95% confidence interval: 1.325–14.65, P = 0.016) with poor prognosis (Table 5).