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Background Lymph node metastasis is more likely in early-stage breast cancer with lower tumor-infiltrating lymphocyte (TIL) density. Therefore, we investigated the correlation between TILs and lymph node metastasis in cT1 breast cancer patients undergoing surgery and the usefulness of TILs in predicting sentinel lymph node metastasis (SLNM) in cT1N0M0 breast cancer. Methods We investigated 332 breast cancer patients who underwent surgery as the first-line treatment after preoperative diagnosis of cT1. A positive diagnosis of SLNM as an indication for axillary clearance was defined as macrometastasis in the sentinel lymph node (SLN) (macrometastasis: tumor diameter > 2 mm). Semi-quantitative evaluation of lymphocytes infiltrating the peritumoral stroma as TILs in primary tumor biopsy specimens prior to treatment was conducted. Results For SLN biopsy (SLNB), a median of 2 (range, 1–8) SLNs were pathologically evaluated. Sixty cases (19.4%) of SLNM (macrometastasis: 46, micrometastasis: 16) were observed. Metastasis was significantly greater in breast cancers with tumor diameter > 10 mm than in those with diameter ≤ 10 mm (p = 0.016). Metastasis was significantly associated with lymphatic invasion (p Conclusions Our results suggest a correlation between lymph node metastasis and tumor immune-microenvironment in cT1 breast cancer. TIL density may be a predictor of SLNM in breast cancer without lymph node metastasis on preoperative imaging.

Background Invasion is often found during postoperative pathological examination of cases diagnosed as ductal carcinoma in situ (DCIS) by histological examinations such as core needle biopsy (CNB) or vacuum-assisted biopsy (VAB). A meta-analysis reported that 25.9% of invasive ductal carcinoma (IDC) cases are preoperatively diagnosed by CNB as DCIS. Risk factors for invasion have been studied by postoperative examination, but no factors have been found that could be obtained preoperatively from blood tests. In this study, we investigated factors predictive of invasion based on preoperative blood tests in patients diagnosed with DCIS by preoperative biopsy. Methods In this study, 118 patients who were diagnosed with DCIS by preoperative biopsy were included. Biopsies were performed with 16-gauge CNB or VAB. Peripheral blood was obtained at the time of diagnosis. This study evaluated absolute platelet count, absolute lymphocyte count, lactate dehydrogenase, carcinoembryonic antigen, and cancer antigen 15–3 (CA15–3). The platelet–lymphocyte ratio (PLR) was calculated by dividing the absolute platelet count by the absolute lymphocyte count, and patients were grouped into high PLR (≥160.0) and low PLR ( Results Invasion was found more frequently after surgery in pathologically high-grade cases than in pathologically not-high-grade cases (p = 0.015). The median PLR was 138.9 and 48 patients (40.7%) were classified into the high PLR group. The high PLR group was significantly more likely to have invasion detected by the postoperative pathology than the low PLR group (p = 0.018). In multivariate analysis of factors predictive of invasion in postoperative pathology, a high PLR (p = 0.006, odds ratio [OR] = 3.526) and biopsy method (VAB vs. CNB, p = 0.001, OR = 0.201) was an independent risk factor. Conclusions The PLR may be a predictor of invasion in the postoperative pathology for patients diagnosed with DCIS by preoperative biopsy.

Background Although many studies have identified several inflammation-based and/or nutritional markers with prognostic value for patients with various types of cancer, the optimal markers and cut-off values for these markers remain obscure. Therefore, this retrospective study aimed to identify optimal markers and their cutoffs. Methods We compared prognostic values among established preoperative inflammation-based and/or nutritional markers in 225 patients who underwent R0 resection for stage III gastric cancer. Inflammation-based and/or nutritional markers comprised C-reactive protein to albumin ratio (CAR), neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), prognostic nutritional index (PNI), Glasgow prognostic score (GPS), and prognostic index (PI). Time-dependent receiver operating characteristic curves were analyzed to assess predictive ability and to determine the optimal cut-off values. Prognostic factors predicting overall survival (OS) and cancer specific survival (CSS) were analyzed using Cox proportional hazards models. Results Multivariate analyses revealed that CAR and PLR cut-off values of 0.47 and 172, respectively, were independent prognostic factors for overall survival (OS) (HR, 2.257; 95% CI, 1.180–4.319; p = 0.014 and HR, 1.478; 95% CI, 1.025–2.133; p = 0.037, respectively) and cancer-specific survival (CSS) (HR, 2.771; 95% CI, 1.398–5.493; p = 0.004 and HR, 1.552; 95% CI, 1.029–2.341; p = 0.036, respectively). These results were different from those we previously reported in patients with stage II. Conclusions Among inflammation-based and/or nutritional markers, CAR and PLR were independent prognostic factors of OS and CSS in patients with stage III gastric cancer. The optimal markers and their cut-off values should be determined in specific populations.

Background/aim Infusion reactions (IRs) often occur with trastuzumab. Although premedication by non-steroidal anti-inflammatory drugs can be effective to a certain extent, IRs are still common and infrequently severe. Therefore, a predictive marker that can select patients requiring further prophylaxis is useful for appropriate prevention, but remains unclear. Patients and methods We conducted a retrospective analysis for 136 consecutive female inpatients aged 18 years and older who received 8 mg/kg of the initial trastuzumab administration for breast cancer with a 25-mg dose of rectal diclofenac before trastuzumab infusion between May 2007 and April 2019, in order to assess IRs. Results Overall, 57 patients were eligible for inclusion in the study. IRs were observed in 17.5% (10/57) of the patients. Univariate analysis showed that patients with a low eosinophil percentage (≤2%) were associated with IRs (p=0.016). Conclusion A low eosinophil percentage can be a useful new predictive marker for trastuzumab-induced IRs in patients with breast cancer.

Background: Since 1965, the Laurén classification has been used most commonly for gastric adenocarcinoma, with two types, intestinal type and diffuse type. Signet ring cell carcinoma (Sig) and non-solid poorly differentiated adenocarcinoma (Por2) are the histological forms of diffuse type and are often found in advanced tumors, and they seem to be associated with a poor prognosis. S-1 based adjuvant chemotherapy for patients with stage II/III gastric cancer has generally been accepted in Japan, but histological type does not alter treatment strategy. The aim of the present study was to investigate the prognostic impact of the histopathologic mixture of Sig and Por2 in patients with stage II/III gastric cancer treated with S-1 adjuvant chemotherapy.Methods: The clinicopathological data of 968 gastric carcinoma patients who underwent gastrectomy between 2007 and 2016 at our department were retrospectively analyzed. In this study, tumors containing Sig or Por2 were classified as Diffuse type, and those not containing them were classified as Intestinal type.Results: There were 307 cases of Diffuse type and 661 of Intestinal type. Diffuse type included 189 cases with Sig. Pathological diagnosis of Sig was an independent risk factor for peritoneal recurrence in patients with stage II/III. Patients with Diffuse type had worse overall survival than those with Intestinal type in Stage III. Of the patients who received S-1 adjuvant chemotherapy, the prognosis of Stage III patients with Sig but not Por2 was Significantly worse compared to patients with Intestinal type.Conclusions: The coexistence of Signet ring cell carcinoma in the primary tumor was associated with a poor prognosis in patients with stage III gastric cancer. These findings suggest that, because mixed Sig gastric cancer had a high risk of peritoneal recurrence even if adjuvant chemotherapy were performed, the pathological diagnosis should be considered when determining the therapeutic strategy for adjuvant chemotherapy in stage III gastric cancer.

BACKGROUND/AIM In addition to its cytocidal effects as a microtubule dynamics inhibitor, eribulin mesylate (eribulin) regulates the tumour microenvironment. We examined the clinical significance of tumour infiltrating lymphocytes (TILs) and transforming growth factor-β (TGF-β), which are local markers of host immunity, and of the neutrophil-lymphocyte ratio (NLR) and absolute lymphocyte count (ALC), which are systemic markers. PATIENTS AND METHODS We administered eribulin chemotherapy to 106 patients with locally advanced or metastatic breast cancer. Of these, 21 had their lesions resected. RESULTS The response to eribulin was significantly associated with ALC (p=0.007). The expression of pSmad2 (an indicator of activation of TGF-β downstream signaling) was significantly decreased before and after eribulin chemotherapy (p

A 50-year-old woman was referred to our hospital due to breast cancer with multiple liver metastasis diagnosed by CT scan. Laboratory findings showed liver dysfunction(T-Bil 7.6mg/dL)with marked elevation of tumor markers(CEA 727.9 ng/mL). Breast tumor biopsy showed an invasive ductal carcinoma(scirrhous type), ER(+), PgR(-), and HER2(3+). Combination therapy with docetaxel, carboplatin and, trastuzumab was administered after the end of 1 course of weekly paclitaxel plus bevacizumab regimen. The patient maintained a good condition without liver dysfunction 8 months after the first visit. Follow-up CT scan showed partial response of breast and hepatic tumors. Our case suggests that careful chemotherapy can improve the prognosis of breast cancer with liver metastasis even if a patient is in an icteric condition.

A 68-year-old woman noticed a tumor in her left breast and visited our hospital. She was diagnosed with left breast cancer cT2N2M0, stage ⅢA, and surgery was performed after the neoadjuvant chemotherapy. Although this case was indicated for radiation therapy(PMRT)after total mastectomy, postoperative radiation was not performed due to an implantable pacemaker placed in the left anterior chest. Therefore, postoperative adjuvant therapy with endocrine therapy was started. After 3 years of treatment, her left axillary lymph node was enlarged, and needle biopsy confirmed metastasis and recurrence. When local excision was performed, postoperative irradiation was deemed necessary because residual microlesions were suspected. Irradiation planning did not deny the possibility that the pacemaker in the anterior chest indwelling could become the irradia- tion range. Therefore, a leadless pacemaker was inserted, and local irradiation(50 Gy/20 Fr)was performed without complications. The next treatment is fulvestrant, and no recurrence has been observed 6months postoperatively.

A 66-year-old-man was hospitalizedfor the treatment of rectal cancer. Preoperative contrast-enhancedcomputedtomography revealedrectal cancer, left-sided inferior vena cava, and distant metastasis. He was diagnosed with cStage Ⅳb rectal cancer. Laparoscopic abdominoperineal resection with standard lymph node dissection was performed. There have been a few reports on laparoscopic colorectal resection for patients with left-sided inferior vena cava. We report a case of laparoscopic abdominoperineal resection for a patient with left-sided inferior vena cava.

Although the S-1 plus CDDP(SP)regimen is the standard treatment for advanced gastric cancer, hydration and admission have been recommended after cisplatin has been administered. In this study, short hydration(SH)method was used and SP was administered in outpatient settings. We evaluated renal toxicity of cisplatin in the SH-SP regimen at our hospital.Eleven of 16 patients(5 underwent only 1 course and so were excluded)received the SH-SP regimen between January 2012 and January 2018 to present and were included. Serum creatinine(Cr)and estimated glomerular filtration rate(eGFR)were used to assess renalfunction.Median course was 5. Rate of 5-course accomplishment was 72.7%. Grade 1 Cr elevation was observed in only 3 patients and there was no severe renal disorder.The SHSP regimen could be administered in outpatient settings and was considered safe as it did not cause renal toxicity.

An 86-year-old woman visited her nearby hospital for a mass on the left side of her sternum. She had undergone total mastectomy for left breast cancer 32 years prior and multiple small nodules had been found in both lungs 2 years ago. She was sent to our hospital for suspected breast cancer recurrence. Chest computed tomography showed multiple small nodules in both lungs and a mass with sternal sclerosis on the parasternal bone of her left 2-3 intercostal space. A core needle biopsy was performed on the chest wall tumor by ultrasonography. Not only pathological but also immunohistological examination findings were similar to those of the surgical specimens 32 years ago(estrogen receptor: positive, progesterone receptor: positive, HER2: negative, Ki-67 score: low). Therefore, she was diagnosed with a late recurrence of her breast cancer. She started letrozole but the disease had progressed 6 months later. We experienced a case of late breast cancer recurrence 32 years after surgery.

A 72-year-old woman was diagnosed with cecal cancer. Preoperative enhanced abdominal computed tomography(CT) and three-dimensional(3D)-CT angiographyrevealed a celiacomesenteric trunk(CMT). We performed a laparoscopic ileocecal resection without anyproblems owing to the obtained preoperative 3D-CT images. The postoperative course was uneventful. The celiac arteryhas manyanomalies, and 6 categories have been defined. Patients can be divided into 28 groups bycombining these anomalies with mutations in the hepatic artery, as explained byAdachi. Our case was a Type Ⅳ anomaly according to the Adachi classification, which accounts for 2.4% of all cases. We herein report this extremelyrare case.

Orbital metastasis from breast cancer is a rare condition. Here, we describe 2 cases of orbital metastasis from breast cancer. The first patient was a 26-year-old woman diagnosed with triple-negative invasive ductal carcinoma. She underwent surgery after neoadjuvant chemotherapy. One year after surgery, she had multiple bone metastases and then multiple liver metastases developed. During chemotherapy for metastatic disease, she complained ofheadaches and visual disturbances. Findings ofa MRI scan suggested a metastatic tumor in the left orbit. A total of 30 Gy of radiation therapy was administered, but she died a month after the orbital metastasis was discovered. The second patient was a 42-year-old woman, who had advanced breast cancer with bone metastasis. Diplopia developed 8 months after initiation of chemotherapy. Meningeal dissemination was suspected because ophthalmological examination revealed swelling ofbilateral optic discs. She lost her sight within a month. She died 2 months after the diagnosis oforbital metastasis. There was no evidence ofcentral nervous system metastasis in either case. Loss ofvision severely impairs patients' quality oflif e. It is important to know that there is rarely such a rapid progression ofdisease, especially in young patients with triple-negative disease.

In the treatment of advanced breast cancer, onlya few drugs confer overall survival(OS)benefit. Eribulin is a drug that was shown to extend OS in an international phase Ⅲtrial; however, the underlying mechanism is thought to involve cancer microenvironment regulation. The concept of "breast cancer subtype discordance" implies the biological changes that accompany treatment. Herein, we encountered a case of advanced breast cancer in a 54-year-old woman that showed biological changes after eribulin chemotherapy. The patient noticed a lump in her left breast and visited a nearby doctor, who referred her to our hospital for close examination and treatment. Ultrasonographyrevealed a large mass at the center of the left mammarygland and axillaryly mph node swelling. Core-needle biopsyconfirmed an invasive ductal carcinoma(ER stronglypositive, PgRnegative, HER-2 negative, Ki-67 low expression). CT findings showed multiple lung metastases. Letrozole was administered for cT4N2M1, stage Ⅳ, Luminal A, which showed progression to the left side with advances in breast cancer. Six months later, the primarytumor and axillaryly mph nodes showed progression. Subsequent treatment with eribulin was started, and partial response was obtained; however, new lymph node metastasis developed in the axilla after 11 cycles. The primary tumor and axillaryly mph nodes showed stronglypositive ER expression, were PgR-negative and HER2-positive, and showed Ki-67 low expression and HER2-positive conversion.

This study evaluated the prognostic significance of preoperative neutrophil-to-lymphocyte ratio (NLR) and CD8A total of 154 patients with BTCs who underwent surgery were enrolled in this study. We obtained neutrophil and lymphocyte counts, and calculated NLR from preoperative peripheral blood samples. CD8The overall survival (OS) and recurrence-free survival (RFS) of patients with high NLR were shorter than those with low NLR. The OS and RFS of patients with high CD8NLR and CD8

Background/aim The host's systemic inflammatory response is thought to affect the progression of cancer and the antitumor effects of chemotherapy. Meta-analyses have reported that the peripheral blood platelet-to-lymphocyte ratio (PLR) is a prognostic indicator of this effect. Therefore, we hypothesized that PLR may differ, depending on sentinel lymph node metastasis (SLNM) in patients diagnosed with cT1N0M0 breast cancer by preoperative imaging. This study investigated the ability of preoperative PLR to predict SLNM in patients diagnosed with cT1N0M0 breast cancer. Patients and methods This study included 475 patients with cT1N0M0 breast cancer diagnosed by preoperative imaging. Peripheral blood was obtained at diagnosis, i.e., before surgery. PLR was calculated from preoperative blood tests, by dividing the absolute platelet count by the absolute lymphocyte count. Results The probability of SLNM was significantly higher (p=0.002) in cases where the tumor diameter was larger than 10 mm. The incidence of SLNM was significantly high in the high (preoperative) PLR group (p=0.031). Multivariate analysis revealed that high PLR [compared to low PLR, p=0.021, odds ratio (OR)=1.815, 95% confidence interval (CI)=1.093-3.090] and large tumor size (compared to small tumor size, p=0.001, OR=2.688, 95%CI=1.524-4.997) were independent factors influencing SLNM. Conclusion PLR may act as a predictor of SLNM in cT1N0M0 breast cancer.

Background The selection of treatment for a patient with breast cancer largely relies on the cancer subtype. However, this process is complicated by changes in tumor biology at relapse. Smoking has been identified as a risk factor for breast cancer. The direct effect of a tobacco component delivered via blood circulation on the mammary gland tissue and subsequent DNA damage have been proposed to explain the association between cigarette smoking and breast cancer carcinogenesis. This postulation is supported by both tissue culture and animal studies demonstrating that the associated DNA damage further alters breast cancer cells, as indicated by an increased proliferative capacity and malignant transformation. In this study, we aimed to explore the relationship between changes in Estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) each receptor at recurrence, and smoking and the prognosis after recurrence. Methods This retrospective study included 989 patients with primary breast cancer who developed relapse after surgery and 50 patients who underwent regenerative biopsy or surgery from December 2007 to March 2018. ER, PgR, and HER2 expression in the primary and recurrent lesions was evaluated using immunohistochemistry, and the correlations of these expression patterns with smoking history (pack-years) were examined. Results When ER was evaluated in recurrent tumors, negative and positive conversions were recognized in 3 (6.0%) and 1 patient (2.0%), respectively. When PgR was evaluated, negative conversion was recognized in 15 patients (30.0%). When HER2 was evaluated, positive conversion was recognized in 6 patients (12.0%). Consequently, we observed a change in the intrinsic subtype in in 5 patients with recurrent tumors (10.0%). Although most clinical factors were not correlated with smoking, a positive conversion of HER2 in recurrence was significantly more frequent among smokers than among non-smokers (p = 0.024). Conclusions Biological changes during breast cancer recurrence should be given careful clinical consideration because they affect treatment after recurrence. Our results suggest that smoking may induce increased HER2 expression in recurrent breast tumors.

Background Neoadjuvant treatment with imatinib mesylate (IM) has not been established for gastrointestinal stromal tumor (GIST). A case of a giant gastric GIST that was radically resected following neoadjuvant therapy with IM is reported. Case report A 71-year-old woman with abdominal distension was referred to our hospital for treatment. Contrast-enhanced computed tomography showed a large (>30 cm), heterogeneous mass, with dilated vessels around the tumor. The diagnosis was a gastric GIST by endoscopic ultrasound-fine needle aspiration, and neoadjuvant therapy with 400 mg IM daily was started. Radical surgery was performed after 13 months. Since the tumor was not invasive, complete resection by partial gastrectomy was achieved without the need for combined resection of other organs. The patient remains alive without recurrence 12 months after surgery. Conclusion A giant GIST, larger than 30 cm in size, is quite rare. Neoadjuvant IM treatment should be considered in the management of large GISTs.

Background/aim Neoadjuvant chemoradiotherapy (CRT) is a standard treatment for patients with clinical Stage II/III rectal cancer. However, the benefit of postoperative adjuvant chemotherapy for patients after neoadjuvant CRT is uncertain. Recently, neoadjuvant rectal (NAR) score was suggested as an independent prognostic factor for patients with rectal cancer after neoadjuvant CRT. The aim of this study was to examine the prognostic significance of NAR score in rectal cancer patients who underwent neoadjuvant CRT followed by surgery, and to investigate which patients may benefit from postoperative adjuvant therapy. Patients and methods A total of 72 patients who underwent neoadjuvant CRT followed by R0 resection for clinical stage II /III rectal cancer were evaluated. The correlation between NAR score, various clinicopathological factors and disease recurrence were evaluated. Results Disease recurrence was significantly more often observed in patients with incomplete neoadjuvant CRT, tumor regression grade (TRG) 3-4, and high NAR score. Multivariate analysis revealed that NAR score was an independent predictor of disease recurrence. Conclusion NAR score may be one of the predictive markers for disease recurrence in patients who underwent neoadjuvant CRT followed by surgery for rectal cancer. Patients with a low NAR score may benefit form postoperative adjuvant chemotherapy.

Additional file 1: Supplementary Table 1. Correlation between TILs and clinicopathological features in cT1N0M0 breast cancer patients undergoing SLNB by intrinsic subtype.

Additional file 1: Table S1. The recurrence cases of organs that was not biopsied at the simultaneously.

Endoscopic submucosal dissection (ESD) of lesions using expanded indications for early gastric cancer (EGC) has been accepted as an alternative treatment for cases without lymph node metastasis. We herein report a rare case of metastatic lymph node tissue in mixed adenoneuroendocrine carcinoma (MANEC) after curative ESD using the expanded pathological criteria. A 70-year-old man underwent ESD for two EGC lesions. A pathological examination revealed lesions that required curative resection based on the expanded pathological criteria of the Japanese classification of gastric carcinoma. However, lymph node metastasis was detected at 26 months after ESD. Additional surgical resection was performed and MANEC was pathologically diagnosed in the metastatic lymph node. The patient subsequently underwent additional chemotherapy and remains alive at 2 years after surgery. Even though MANEC is a rare tumor, this case suggests that periodic follow-up is important when patients undergo curative resection by ESD based on the expanded indications because of the high malignant potential and the poor prognosis.

Tumor‑associated neutrophils (TANs) have an immunosuppressive function and play an important role in tumor progression. However, the detailed mechanism is largely unknown. The present study investigated the immunosuppressive ability of TANs in gastric cancer. Tumor tissue culture supernatant (TTCS) and non‑tumor tissue culture supernatant (NTCS) were purified and added to neutrophils. Expression of programmed cell death ligand‑1 (PDL‑1), 7‑amino‑actinomycin D and human leukocyte antigen‑DR (HLA‑DR), and the levels of hydrogen peroxide (H2O2) were determined. Levels of programmed cell death‑1 (PD‑1) and CD25 were assessed in T cells co‑cultured with neutrophils. Furthermore, CD4+ T cells were co‑cultured with dendritic cells and neutrophils to examine their proliferation. CD15 and PD‑1 immunohistochemical staining was also performed to explore the positional relationship. The results revealed that the neutrophils incubated with TTCS showed upregulation of PDL‑1 expression, as well as a decreases in the ratio of apoptotic cells, expression of HLA‑DR, and levels of H2O2. CD4+ T cells co‑cultured with neutrophils conditioned with TTCS showed a decrease in proliferation, upregulation of PD‑1 expression, and downregulation of CD25 expression. IHC showed that PD‑1+ T cells formed clusters and TANs infiltrated around the clusters. In conclusion, neutrophils in gastric cancer tissue inhibit the proliferation of CD4+ T cells and may form a local immunosuppressive environment through the PD‑1/PDL‑1 pathway.

Tertiary lymphoid structures (TLSs), which consist of B cells, T cells, follicular dendritic cells and high endothelial venules, have recently been found to be associated with effective antitumor immune responses in patients with cancer. Tumor-infiltrating T cells and B cells have each been demonstrated to be associated with survival in patients with cancer. We hypothesized that TLSs, an assembly of immune cells, may be important for the initiation and/or maintenance of T cell and B cell responses against tumors. The aim of the present study was to examine the cellular mechanism of B cells in TLSs within gastric cancer and to understand the antitumor immune response of TLSs. Each B cell subset in a tumor was examined using flow cytometry to evaluate B cell differentiation and the functional status of B cells. In addition, B cell clonality was investigated by analyzing the B cell antigen receptor gene using PCR, and the function and formation/maintenance of TLSs were evaluated using reverse transcription-quantitative PCR. Tumor-infiltrating B cells were more differentiated compared with that in distant non-tumor tissues and tumor-draining lymph nodes. The PCR results revealed specific BCR gene expression in tumor-infiltrating B cells. The expression of co-stimulatory factors, CD80 and CD86, was observed, in addition to the constantly expressed major histocompatibility complex molecules (HLA-ABC and HLA-DR). CD70 was expressed in addition to CD27 in both CD20+ B cells and CD8+ T cells, indicating that these factors are activated together through their interaction. The mRNA expression levels of CCL21, CXCL13, PD-L1, perforin and granzyme B in TLSs was significantly higher compared with that in non-TLSs. The majority of tumor-infiltrating B cells in gastric cancer exist in the form of TLSs around the tumor and have been antigen-sensitized and differentiated, and proliferated in TLSs but not in the lymph nodes. In addition, B cells in TLSs might primarily function as antigen-presenting cells and be associated with the induction of cytotoxic T cells.

Background/aim A single-arm phase II clinical trial was conducted to evaluate the safety and efficacy of adding bevacizumab to standard capecitabine-based neoadjuvant chemoradiotherapy (CRT) for the treatment of locally advanced rectal cancer (LARC). Patients and methods Twenty-five patients were enrolled. Patients received capecitabine-based CRT for 5 weeks and 3 days. Bevacizumab was administered every 2 weeks during CRT. Within 6-10 weeks after completion of CRT, surgery was performed. Results With regard to CRT-related acute toxicities, most of the adverse events were limited to grade 1. A pathological complete response was obtained in four (16%) patients. In total, six patients (24%) developed postoperative complications. Six out of five (83%) patients healed without the need for surgical intervention. Conclusion Although acute toxicity during CRT with bevacizumab was minimal and postoperative complications do not seem to increase, the addition of bevacizumab apparently offers no clinically-significant benefit for patients with LARC.

As a result of recent major advances in chemotherapy for metastatic colorectal cancer, the prognosis for patients with metastatic colorectal cancer has improved. However, elderly patients often cannot receive intensive therapy. There are still many problems to solve regarding treatment for elderly patients with metastatic colorectal cancer. We herein report a case of complete response of pulmonary metastases from rectal cancer to tegafur-uracil (UFT)/leucovorin (LV) + bevacizumab (Bmab) in an elderly patient. An 80-year-old woman who had undergone curative surgery for rectal cancer 5 years ago was diagnosed with pulmonary metastases. Taking into account her advanced age and low renal function (creatinine clearance: 41.2 mL/min), UFT/LV + Bmab therapy was selected. The patient received UFT (300 mg/m2/day) and LV (75 mg/day) on days 1–5, 8–12, and 15–19 and Bmab (7.5 mg/kg) on day 1. The treatment cycle was repeated every 21 days. Following 17 courses of treatment without adverse events, a complete response was observed. Furthermore, there was no recurrence within 6 months after the final course of therapy. This case indicates that UFT/LV + Bmab is suitable for the treatment of elderly patients with metastatic colorectal cancer.

// Takahiro Toyokawa 1 , Kazuya Muguruma 1 , Tatsuro Tamura 1 , Katsunobu Sakurai 2 , Ryosuke Amano 1 , Naoshi Kubo 2 , Hiroaki Tanaka 1 , Masakazu Yashiro 1 , Kosei Hirakawa 1 and Masaichi Ohira 1 1 Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Abeno-ku, Osaka 545-8585, Japan 2 Department of Gastroenterological Surgery, Osaka City General Hospital, Miyakojima-ku, Osaka 534-0021, Japan Correspondence to: Takahiro Toyokawa, email: t-toyokawa@med.osaka-cu.ac.jp Keywords: gastric cancer; gastrectomy; prognostic factor; nutrition; inflammation Received: October 05, 2017 Accepted: May 07, 2018 Published: June 29, 2018 ABSTRACT Background: The aim of this study was to evaluate and compare the prognostic value of preoperative established inflammation-based and/or nutritional markers, C-reactive protein-to-albumin ratio (CAR), neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, Prognostic Nutritional Index (PNI), Glasgow Prognostic Score, and prognostic index in patients with stage II gastric cancer. We then developed a new prognostic index based on the results of our investigation. Methods: This study retrospectively reviewed 240 consecutive patients who underwent R0 resection for stage II gastric cancer. Time-dependent receiver operating characteristic curve analyses were performed to assess discrimination ability and to determine optimal cut-off values. Prognostic factors predicting overall survival (OS) were analyzed using Cox proportional hazards models. Results: Among inflammation-based and/or nutritional markers, multivariate analyses demonstrated CAR and PNI as independent prognostic factors for OS (hazard ratio (HR) 1.707, 95% confidence interval (CI) 1.016-2.867, p =0.044 and HR 0.415, 95%CI 0.234-0.736, p =0.003, respectively). CAR-PNI score, constructed as the combination of CAR and PNI, was significantly associated with OS, relapse-free survival and cancer-specific survival (p

Background Immune responses in a tumour microenvironment can be evaluated by analysing tumour-infiltrating lymphocyte (TIL) density; this has been verified in the clinical setting. Although there are many reports on TIL density in primary tumours, little is known about its density in recurrent tumours. Methods Of 300 patients treated with neoadjuvant chemotherapy during the study period, 29 were considered for evaluation of TIL density in primary and recurrent tumours. We performed a retrospective analysis of the association between TIL density and prognosis. Results TIL density was significantly lower in recurrent tumours than in primary tumours (P = 0.007). There was no correlation between post-recurrence survival and TIL density in core-needle biopsy specimens obtained from primary tumours (P = 0.837). However, patients with high TIL density in recurrent tumours had significantly better post-recurrence survival than did the corresponding group with low TIL density (P = 0.041). Multivariate analysis revealed that high TIL density contributed significantly towards improving post-recurrence survival in all patients (P = 0.035; hazard ratio, 0.167). Conclusions In recurrent breast cancer, a decrease in TILs density was observed as compared to the primary tumour, and this affects the poor prognosis after relapse.

Although single-port laparoscopic resection of gastrointestinal stromal tumor (GIST) offers advantages over multiport procedures in terms of reduced pain, faster recovery, and superior cosmetic results, its efficacy and indications are not fully established. In this study, we compared the safety and efficacy of single-port and multiport laparoscopic resection for GIST. We evaluated 85 consecutive patients (age > 18 years) with GISTs who underwent gastric wedge resection at an area hospital between January 2000 and December 2014. The surgical outcomes of patients undergoing single-port (S group) and multiport (M group) laparoscopic resections were compared. There were 61 patients in the M group and 24 in the S group. No statistically significant differences in patient characteristics (sex ratio, age, performance status, physical status), surgical factors (operative time, blood loss), postoperative course (time to resumption of oral intake, length of hospital stay), or treatment outcome (receipt of adjuvant therapy, tumor recurrence) were found between the M and S groups (all p > 0.05). Patients in the M group had larger tumors than those in the S group (mean 36.6 ± 14.8 mm vs. 26.4 ± 7.9 mm; p

Scirrhous gastric cancer (SGC) exhibits aggressiveness of the rapid infiltrating tumor cells with abundant fibroblasts. Experimental studies using SGC cell lines have obtained useful information about this cancer. Our literature search divulged a total of 18 SGC cell lines; two cell lines were established from primary SGC and the other lines were established from a metastatic lesion of SGC. Fibroblast growth factor receptor 2 (FGFR2) and transforming growth factor-beta receptor (TβR) are linked to the rapid development of SGC. Cross-talk between the cancer cells and cancer-associated fibroblasts (CAFs) has been shown to contribute to the progression of SGC. Chemokine (C-X-C motif) receptor 1 (CXCR1) from SGC cells might be associated with the abundant CAFs in cancer microenvironments. The in vivo models established using SGC cell lines are expected to serve as a useful tool for the development of drugs such as FGFR2 inhibitors, TβR inhibitors, and CXCR1 inhibitors, which might be promising as SGC treatments. However, the number of available SGC cell lines is insufficient for the clarification of the entire biologic behavior of SGC. Since the mechanisms responsible for the characteristic aggressiveness of SGC are not fully elucidated, the establishment of new SGC cell lines could help clarify the biological behavior of SGC and contribute to its treatment.

Purpose Quality-of-life (QOL) has been reported to affect the prognosis of many types of cancer, and several studies used various QOL assessment tools to determine the relationship between QOL and cancer prognosis. In this study, QOL-Questionnaire for Cancer Patients Treated with Anti-Cancer Drugs-the Breast (QOL-ACD-B) was modified to be suitable for preoperative chemotherapy (POC) and was named the QOL-ACD-BP. Methods A total of 300 patients were treated with POC after being diagnosed with breast cancer between February 2007 and December 2016 at our institute. We evaluated novel evaluation scale for QOL (QOL-ACD-BP) before and after POC in a retrospective manner. Results In the multivariate analysis with overall survival, the high QOL before [p = 0.048, hazard ratio (HR) 0.441] or after POC (p = 0.030, HR 0.273) was an independent factor. Conclusion Our study shows that QOL after POC may also affect prognosis and supported the importance of maintaining QOL in cancer treatment. In patients with breast cancer treated with POC, QOL-ACD-BP, which is a new QOL evaluation index, was found to be a useful tool for predicting the patients’ prognosis. Electronic supplementary material The online version of this article (10.1007/s00432-018-2670-0) contains supplementary material, which is available to authorized users.

Background Tumor-Associated Neutrophils (TANs) may be able to induce lymphangiogenesis and angiogenesis, although the detailed roles of TANs remain unclear. The Neutrophil-Lymphocyte Ratio (NLR) is an inflammation-based prognostic factor for gastric cancer. This study aimed to investigate the distribution of CD15+neutrophils in the primary tumor and Tumor-Draining Lymph Nodes (TDLNs), and to examine the association of TANs with the clinicopathological features (including NLR) of patients with gastric cancer. Results Immunohistochemical staining showed that the median number of CD15+TANs was 18 and 24 per high-power field (HPF) in primary tumors and TDLNs, respectively. Patients were divided into high and low infiltration groups based on the median number. A high number of infiltrating CD15+TANs in the primary tumors and in the TDLNs were associated with depth of invasion and lymph node metastasis. Kaplan-Meier analysis revealed that a poor overall survival was associated with high numbers of CD15+TANs, and the multivariate analyses revealed that a high number of CD15+TANs in the TDLNs was an independent prognostic factor. The numbers of CD15+TANs in the primary tumors and TDLNs showed weak positive correlation. The number of CD15+TANs in the primary tumors was positively correlated with the preoperative NLR, (P = 0.001, R = 0.327) and immunohistochemical staining revealed that C-X-C motif chemokine receptor 2 (CXCR2) +neutrophils might be the origin of the CD15+TANs. Flow cytometry analysis indicated that infiltrating neutrophils increased in the tumor and TDLN compared to non-cancerous tissue. Neutrophils treated with cancer supernatant upregulated TWIST and IL-6 genes in vitro. Conclusion Our findings suggested that local infiltration of CD15+TANs may be correlated with inflammation in TDLNs and systemic response to cause metastasis in gastric carcinoma.

Introduction: The anterior approach to the inferior vena cava (IVC) by the liver hanging maneuver is effective in resecting large retrohepatic tumors without mobilizing the right lobe. Case presentation: A 50-year-old man was referred to our hospital with a diagnosis of pheochromocytoma. He had severe congestive heart failure and cardiac ejection fraction was 15%. Abdominal magnetic resonance imaging (MRI) and ultrasonography (US) showed an adrenal mass about 80 mm in diameter. The tumor-infiltrated posterior segment of the right hepatic lobe and tumor were widely attached to the IVC. After treatment of congestive heart failure with conservative therapy, surgery was planned. Right adrenectomy and right hepatectomy were performed, the latter using the liver hanging maneuver to avoid mobilizing the right lobe, and we were able to minimize blood pressure fluctuations and perform the operation safely. The histopathologic diagnosis was malignant pheochromocytoma. Conclusions: We performed right hepatectomy without mobilizing the right lobe by the liver hanging maneuver and minimized stimulation of the tumor. We could perform the operation safely using the liver hanging maneuver, which seems effective in such cases.

Neoadjuvant therapy for locally advanced rectal cancer is becoming increasingly common. However, biomarkers predicting the response to neoadjuvant therapy have not been established. Tumor‐infiltrating lymphocytes (TILs) have a crucial effect on tumor progression and survival outcome as the primary host immune response, and an antitumor immune effect has been reported to contribute to the response to radiotherapy and chemotherapy. We investigated the significance of TILs before and after neoadjuvant treatment and the change in the density of those TILs. Sixty‐four patients who underwent radical resection after neoadjuvant treatment for locally advanced rectal cancer were enrolled. The number of TIL subsets was examined using immunohistochemical staining of pretreatment biopsy samples and post‐treatment resected specimens. In both the neoadjuvant chemotherapy cohort and the neoadjuvant chemoradiotherapy cohort, a low density of CD8+ TILs in pretreatment biopsy samples was associated with a poor response, and a low density of CD8+ TILs in post‐treatment resected specimens was similarly associated with a poor response. In the neoadjuvant chemoradiotherapy cohort, the density of CD8+ TILs in post‐treatment resected specimens was significantly increased compared with that in pretreatment biopsy samples. We concluded that T lymphocyte‐mediated immune reactions play an important role in tumor response to neoadjuvant treatment for rectal cancer, and the evaluation of TILs in pretreatment biopsy samples might be a predictor of the clinical effectiveness of neoadjuvant treatment. Furthermore, neoadjuvant therapy, especially chemoradiotherapy, could induce the activation of the local immune status.

Background: Crosstalk between cancer cells and fibroblasts is crucial for tumour progression. It has been reported that exosomes derived from cancer cells play an important role in the intracellular communications involved in the development of carcinoma. However, the role of exosomes from fibroblasts remains unclear. This study aimed to clarify the effect of exosomes from fibroblasts on the motility of gastric cancer cells. Methods: 5 gastric cancer cell lines were used: OCUM-12, NUGC-3, MKN45, FU97 and MKN74. 2 cancer-associated fibroblasts (CAFs) were used. CD9 expression of exosomes from fibroblasts was examined by western blot. The effect of exosomes on the motility of cancer cells was analysed by migration assays. MMP2 was examined by RT-PCR or gelatin zymography. Then, CD9 and MMP2 expressions of 619 gastric cancers were analysed by immunohistochemistry. Results: Exosomes from CAFs were taken into scirrhous-type gastric cancer cells, namely OCUM-12 cells and NUGC-3 cells, but not into other types of gastric cancer cells. Exosomes from CAFs were positive for CD9. Exosomes from CAFs significantly stimulated the migration and invasion of OCUM-12 and NUGC-3 cells, which was inhibited by anti-CD9 antibody or CD9-siRNA. MMP2 expression of OCUM-12 and NUGC-3 cells was significantly decreased by CD9-siRNA. 116 CD9-positive cases were significantly correlated with scirrhous-type gastric cancer, lymph node metastasis and venous invasion. The 5-year survival rate of patients with CD9-positive tumours was significantly lower (P

Background/Aims: Lysyl oxidase (LOX) family members play a key role in modifying the primary tumor microenvironment by crosslinking collagens and elastin in the extracellular matrix. The aim of this study was to analyze the LOX-like (LOXL)1, LOXL3, and LOXL4 expressions in gastric cancer tissue by immunohistochemical staining. Methods: The correlations between the clinicopathological features of 597 primary gastric carcinomas and LOX family members – LOXL1, LOXL3, and LOXL4 – were investigated by immunohistochemical studies. The effect of the transforming growth ­factor β1 (TGFβ1) on the expressions of LOXL1, LOXL3, and LOXL4 in gastric cancer was examined using diffuse-type gastric cancer cell lines in vitro. Results: The expressions of LOXL1, LOXL3, and LOXL4 were correlated with T invasion, lymph node metastasis, and lymphatic and venous invasion. LOXL1 expression was associated with histological intestinal-type and expanding growth patterns. The overall survival of patients with LOXL1-, LOXL3-, or LOXL4-positive cancer was poorer than those with negative cancer. LOXL3 and LOXL4 mRNA expressions were significantly high in diffuse-type gastric cancer cells with high invasion ability. TGFβ decreased the LOXL1 expression and increased LOXL3 and LOXL4 expression. Conclusion: LOXL1, LOXL3, and LOXL4 expressions are associated with distant metastasis of gastric cancer.

Background We sought to clarify the clinical value of the examination of cancer cells exposed to gastric serosa by our novel method of serosal stamp cytology and a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Method A total of 70 patients who underwent gastrectomy were enrolled. Stamp cytology specimens were obtained by stamping the gastric serosa at the primary gastric tumor lesion, followed by Papanicolaou's staining. Samples obtained by brushing the serosa at the primary gastric tumor were analyzed by our RT-PCR of carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20). Results Among the 70 patients, 11 patients were diagnosed as stamp cytology-positive. Eight and five patients were found to be CEA-positive and CK20-positive, respectively. Since 21 of the 70 patients were either stamp cytology-positive or RT-PCR analysis-positive, these 21 patients were considered to be positive for cancer cells exposed to serosa of primary gastric tumor. The 3-year recurrence-free survival rate of the patients with a single positive result by our method (41.7%) was significantly (log rank p = 0.0002) worse than that of the patients with both negative results (81.0%). Our method showed 58.8% sensitivity and 79.2% specificity. A multivariate analysis revealed that a stamp cytology and/or RT-PCR result was an independent prognostic factor for recurrence. Conclusion The examination of cancer cells exposed to gastric serosa by our serosal stamp cytology and RT-PCR system will be useful for the identification of patients at high risk for peritoneal recurrence after curative surgery for gastric cancer.

The aim of this study was to analyze the significance of glucose metabolism-related enzymes in the proliferation of gastric cancer under hypoxia. Four hypoxia-resistant gastric cancer cell lines and four parent cell lines were used. Reverse transcription-PCR was used to evaluate the mRNA expression levels of the following metabolism-related enzymes: pyruvate kinase isozyme M2 (PKM2), glutaminase (GLS), enolase 1 (ENO1), glucose-6-phosphate dehydrogenase (G6PDH), and PKM1. The effects of these enzymes on the proliferation of gastric cancer cells were examined using siRNAs, shikonin as a PKM2 inhibitor, or BPTES as a GLS inhibitor, in vitro and in vivo. Levels of both PKM2 and GLS mRNA were significantly high in all hypoxia-resistant cell lines, compared with those of their parent cells. Knockdown of PKM2 and GLS significantly decreased the proliferation of all hypoxia-resistant cells. The combination of siPKM2 and siGLS significantly decreased proliferation compared with treatment by siPKM2 or siGLS alone. The knockdown of ENO1, G6PDH, or PKM1 did not decrease the proliferation of all hypoxia-resistant cells. Combination treatment using shikonin and BPTES inhibited the proliferation of all hypoxia-resistant cancer cells more than that by either agent alone. The in vivo study indicated that the tumor size treated by the combination of shikonin and BPTES was significantly smaller than that of vehicle-treated group. These findings suggested that PKM2 and GLS might play important roles in the proliferation of hypoxic gastric cancer cells. A combination of PKM2 and GLS inhibitors could be therapeutically promising for the treatment of gastric cancer.

Background Tumor-infiltrating lymphocytes (TILs) have been reported to reflect the anti-tumor immune status. However, recent investigations have demonstrated that intratumoral fibrosis is important as a factor affecting the infiltration of TILs. This study investigated the organ specificities of TIL infiltration and intratumoral fibrosis in primary colorectal cancer and distant metastases, as well as the relationship between the distribution of TILs and intratumoral fibrosis. Methods Patients who underwent resection of primary tumors or distant metastases for colorectal cancer with distant metastases were enrolled. We evaluated the TIL infiltration by immunohistochemical staining with CD3&CD8 and intratumoral fibrosis by immunohistochemical staining with α-SMA positive cancer-associated fibroblasts and Masson’s trichrome staining against collagen fibers. The "ImageJ" was used to evaluate fibrosis, and the density of TILs in the dense and sparse areas of fibrosis was calculated. The Immunoscore (IS) was obtained based on the density of CD3+/CD8+TILs in the tumor center and invasive margin of the primary tumor. Results The degree of CD3+/CD8+TIL infiltration in peritoneal metastases was significantly lower than that in liver and lung metastases. The area ratio of α-SMA positive cancer-associated fibroblasts and collagen fibers in peritoneal metastases was significantly higher than that of liver and lung metastases. Furthermore, the density of TILs in the high-fibrosis area was significantly lower than that in the low-fibrosis area. In the high-IS group of primary tumors, the degree of TIL infiltration in distant metastases was significantly higher than that in the low-IS group. Conclusion The infiltration of T lymphocytes into tumors is prevented in peritoneal metastases of colorectal cancer due to the high intratumoral fibrosis, which may lead to treatment resistance and a poor prognosis.

Background: Cancer progression has been recognized as not only the proliferation of tumor cells but also an interaction between cancer cells and the surrounding stroma in the tumor microenvironment. Among the tumor stromal cells, cancer-associated fibroblasts (CAF) have been reported to be closely associated with tumor development in various solid carcinomas. We previously reported that chemokine (C-X-C motif) receptor 2 (CXCR2) signaling might play an important role for the pathogenic construction of CAF in the gastric cancer (GC) microenvironment, suggesting that GC cells might alter their adjacent stroma to form a permissive environment for tumor progression, and also we reported that chemokine (C-X-C motif) ligand 1 (CXCL1) from cells stimulated the recruitment of bone marrow cells into GC microenvironment via CXCR2 signaling, resulting in the malignant progression of GC. Following these findings, we reported that among the CXCR2 ligands, CXCL1 might play an important role in the malignant progression of GC via CXCR2 signaling. And then, some studies suggest that CXCR2-CXCL1 plays an important role in not only GC but also other solid cancer. However, there is no report about the clinicopathological significance of CXCR2-CXCL1 signal in cholangiocarcinoma. Then, we examined the clinicopathological significance of CXCR2-CXCL1 signal in cholangiocarcinoma in this study. Patients and Method: We retrospectively analyzed the total cases of 168 patients with cholangiocarcinoma. The expressions of CXCR2 and CXCL1 were investigated by immunohistochemistry, and we then analyzed the correlation between the clinicopathological features of 168 patients and the CXCR2 and CXCL1 expression level. Results: The CXCR2 and CXCL1 positive expression wad as follows: CXCR2 was 18.5% (31/168), CXCL1 was 72.8% (126/173), both CXCR2 and CXCL1 was 17.9% (30/168). Metastasis negative was correlated with CXCL1 expression. Low depth invasion was correlated with CXCR2 expression. The prognoses of the both CXCR2 and CXCL1 positive patients were significantly better than those of CXCR2 and/or CXCL1 negative patients (p Conclusion:CXCLR2 and CXCL1 expression might be a better prognostic factor of patients with cholangiocarcinoma, and CXCR2-CXCL1 signaling might play an important role for tumor-suppressing effects of cholangiocarcinoma. Citation Format: Yurie Yamamoto, Gen Tsujio, Tomohiro Sera, Atsushi Sugimoto, Syuhei Kushiyama, Sadaaki Nishimura, Kenji Kuroda, Ryota Tanaka, Kenjiro Kimura, Ryousuke Amano, Masaichi Ohira, Masakazu Yashiro. Clinicopathologic significance of CXCR2-CXCL1 signaling in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2677.

Background/Aim: Intratumoral heterogeneity has been reported in various cancer types and may cause cancer metastasis and chemotherapy treatment resistance, including resistance to molecular targeted therapy. In gastric cancer, the intratumoral heterogeneity of cancer-associated molecules, such as HER2, has been reported and relates to the acquisition of chemotherapy resistance. To reveal whether intratumoral heterogeneity is related to cancer etiology, such as the mechanism of metastasis, and the development of new treatment strategies, such as combination therapy, we investigated intratumoral heterogeneity of gastric cancer with operative specimen and Cancer Genome Atlas (TGCA) data. Methods: The study included 503 patients who underwent gastrectomy and immunohistochemical staining for gastric cancer between 2001 and 2006 and TGCA data. We conducted clustering analysis based on protein and gene expression data for FGFR2IIIb (FGFR2), HER2(ERBB2), CXCR2(CXCR2), CXCR4(CXCR4), cMet(MET), TGFβ1(TGFB1), IGF1R(IGF1R), and p-smad2(SMAD2), and we analyzed the relation of FGFR2IIIb (FGFR2) and HER2(ERBB2) expression with the expression of other proteins and genes. Additionally, we investigated clinicopathological feature correlations. Result/Discussion: We divided gastric cancer specimens into nine clusters by immunohistochemical staining and 11 clusters using TCGA data. According to immunohistochemical staining, the high HER2 and FGFR2IIIb expression groups formed a cluster with high expression of various proteins. Analysis of stage 3-4 samples revealed more high protein expression clusters than the analysis of stage1-2, and this suggests that cancer acquires heterogeneity with cancer progression. In the TGCA data of stage 3-4, high FGFR2 and MET and high ERBB2 and SMAD2 or IGF1R expression formed clusters, which is consistent with the clustering analysis that was performed with immunohistochemical staining results. Conclusion: Our findings suggest that the existence of a combination of intratumoral heterogeneity and clusters may change with cancer progression. Citation Format: Gen Tsujio, Masakazu Yashiro, Yurie Yamamoto, Tomohiro Sera, Atsushi Sugimoto, Shuhei Kushiyama, Sadaaki Nishimura, Mami Yoshi, Tasuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Kazuya Muguruma, Masaichi Ohira. Analysis of intratumoral protein and gene expression heterogeneity in gastric cancer with a clustering analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3125.

Introduction: Scirrhous gastric cancer shows an extremely poor prognosis with rapid growth and infiltration activity. One of the reasons for poor prognosis is a lack of the identification of driver gene for scirrhous gastric cancer. FGFR2 (fibroblast growth factor receptor 2), E-cadherin, TGFβR (transforming growth factor β receptor), CD44/IGFIR (insulin-like growth factor I receptor), have been reported as candidates of driver for scirrhous-type of gastric cancer, however useful molecular-targeted therapeutic agents against these driver molecules have not yet been clinically approved. In this study, we identified the characteristic genes of scirrhous-type gastric cancer by RNA-seq of gastric cancer cell lines. Methods: RNA-seq was performed using 6 scirrhous gastric cancer cell lines (OCUM1, OCUM2MLN, OCUM8, OCUM12, OCUM13, OCUM14) using a next-generation sequencer (ION S5 system, Thermo Fisher Scientific). Furthermore, RNA-seq public data of 4 strains of scirrhous gastric cancer cell lines (KATO3, NUGC4, SNU601, SNU668) and 10 strains of non-scirrhous gastric cancer cell lines (MKN7, MKN45, MKN74, HS746T, SNU1, SNU16, SNU638, AZ521, HGC27, TMK1) on the Sequence Read Archive were added, and the genes expressed in scirrhous gastric cancer were compared and examined. The analysis tools used were kallisto and sleuth. We set p-value < 0.05 and log2 (fold change) > 2 as the cut-offs to screen out differentially expressed genes (DEGs). Pathway enrichment and protein-protein interaction (PPI) network analysis were performed based on metascape databases. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was also performed for the genes. Results: A total of 234 DEGs were identified from this analysis. A gene co-expression network was constructed containing 8 DEGs. The genes were mainly associated with response to chemokine, and cytokine-mediated signaling pathway. Several critical genes were disclosed, such as C-C motif chemokine ligand 20 (CCL20) and C-X-C subfamiliy. Genes highly expressed in scirrhous gastric cancer cell lines were CCL20 (10.6-fold change, p=9.89×10-4) and C-X-C Motif Chemokine Ligand 3 (CXCL3, 19.5-fold change, p=1.67×10-5). The mean of abundances across samples (=log(counts+0.5)), which represent the average expression levels of all samples, were CCL20 and CXCL3, 4.04 and 4.53, respectively. KEGG pathway enrichment analysis showed that viral protein interaction with cytokine and cytokine receptor, Cytokine-cytokine receptor interaction and IL-17 signaling pathway were significantly over-represented. Conclusion: CCL20 and CXCL3 might be characteristic genes for scirrhous-type of gastric cancer. Citation Format: Tomohiro Sera, Masakazu Yashiro, Gen Tsujio, Yurie Yamamoto, Atsushi Sugimoto, Shuhei Kushiyama, Sadaaki Nishimura, Masaichi Ohira. Identification of characteristic genes of scirrhous-type gastric cancer cells by RNAseq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 258.

Background: Peritoneal dissemination is one of common causes of poor prognosis of patients with gastric cancer (GC). The mechanism of peritoneal metastasis has not been fully elucidated. Recent studies have reported that extracellular vesicles (EVs) play an important role for the intercellular communication in GC microenvironment. The peritoneal dissemination possesses some intercellular communication steps between cancer cells and hostile cells including peritoneal mesothelial cells (PMCs), however, the effect of EVs derived from PMCs on GC cells has not been validated. In the present study, to clarify the intercellular communication we evaluated the effect of EVs from PMCs on the proliferation, migration and invasion of GC cells. Materials and Methods: Four GC cell lines (OCUM12, NUGC3, MKN45, MKN74) and 4 PMCs cell lines were used. PMCs cell lines were primary cultured from ascites of gastric cancer patients. The EVs derived from PMCs were isolated from conditioned medium of PMCs by ultracentrifugation. The EVs derived from PMCs were confirmed by the transmission electron microscope (TEM) using uranyl acetate and the western blotting using EV markers such as CD9, CD63, and CD81. The EVs derived from PMCs were fluorescently labelled using PKH26 and the uptakes of EVs were viewed under a fluorescence microscope. The effect of EVs from PMCs on the proliferation, migration and invasion of GC cells was evaluated by MTT assay, wound healing assay and Matrigel invasion assay respectively. Results: The EVs derived from PMCs showed typical morphology of EVs by TEM, and were positive for EV markers such as CD9, CD63, and CD81 in western blotting. The EVs derived from PMCs significantly suppressed the proliferation of GC cells. On the other hand, the EVs derived from PMCs significantly promoted the migration of OCUM12 and NUGC3 cells, but not MKN45 and MKN74 cells. Furthermore, The EVs derived from PMCs significantly promoted the invasion of OCUM12 and NUGC3 cells. PKH26 labelled EVs derived from PMCs were found in cytoplasm of OCUM12 and NUGC3 cells. Conclusion: The EVs derived from PMCs significantly promoted the migration and invasion of GC cells. The EVs derived from PMCs might be one of factors which associate with the intercellular communication steps in peritoneal dissemination possesses in GC. Citation Format: Atsushi Sugimoto, Masakazu Yashiro, Tomohisa Okuno, Yuichiro Miki, Gen Tsujio, Yurie Yamamoto, Tomohiro Sera, Shuhei Kushiyama, Sadaaki Nishimura, Kenji Kuroda, Shingo Togano, Masaichi Ohira. Extracellular vesicles derived from mesothelial cells promotes the migration and invasion of gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2895.

[Background] Tumor-infiltrating lymphocytes (TILs) have been reported to reflect the anti-tumor immune status and correlate with the prognosis and therapeutic outcomes. However, recent investigations have demonstrated that the level of TIL infiltration varies depending on the metastatic organ, and intratumoral fibrosis is important as a factor affecting the infiltration of TILs. The present study investigated the organ specificities of TIL infiltration and intratumoral fibrosis in primary colorectal cancer and distant metastases, including peritoneal metastases, as well as the relationship between the distribution of TILs and intratumoral fibrosis. [Methods] Patients who underwent resection of primary tumors or distant metastases for colorectal cancer with distant metastases from 1994 to 2019, including 67 cases of peritoneal metastases, 39 cases of primary tumors with simultaneous resection of peritoneal metastases, 138 cases of liver metastases, and 25 cases of lung metastases, were enrolled. We evaluated the TIL infiltration by immunohistochemical staining with anti-CD3 and CD8 antibodies and intratumoral fibrosis by immunohistochemical staining with anti-α-SMA antibody against cancer-associated fibroblasts (CAFs) and Masson's trichrome staining against collagen fibers. The software program "ImageJ" was used to evaluate fibrosis, by which the intensity and range of fibrosis were determined, and the density of TILs in the dense and sparse areas of fibrosis was calculated. [Results] The level of CD3+TIL infiltration in peritoneal metastases was significantly lower than that in primary tumors, liver metastases, and lung metastases, and the level of CD8+TIL infiltration in peritoneal metastases was significantly lower than that in liver metastases and lung metastases (median CD3+TILs: peritoneal metastases 9.0 vs. primary tumors 15.2 vs. liver metastases 13.1 vs. lung metastases 20.0; median CD8+TILs: peritoneal metastases 1.8 vs. primary tumors 3.4 vs. liver metastases 5.4 vs. Lung metastases 8.4;/x400HPF). The area ratio of α-SMA+CAF and collagen fibers in peritoneal dissemination was significantly higher than that of primary lesions, liver metastases, and lung metastases (median α-SMA+CAF: peritoneal metastasis 23.53% vs. primary lesion 18.55%. vs. liver metastases 11.25% vs. lung metastases 12.78%; median collagen fibers: peritoneal metastases 15.64% vs. primary lesions 11.49% vs. liver metastases 8.13% vs. lung metastases 9.18%). Furthermore, the density of TILs in the high-fibrosis area was significantly lower than that in the low-fibrosis area. [Conclusion] Lymphocytes have difficulty infiltrating areas of high fibrosis, and the infiltration of T lymphocytes into tumors is prevented in peritoneal metastases of colorectal cancer due to the high intratumoral fibrosis, which may lead to treatment resistance and a poor prognosis. Citation Format: En Wang, Masatsune Shibutani, Hisashi Nagahara, Tatsunari Fukuoka, Yasuhito Iseki, Yuki Okazaki, Shinichiro Kashiwagi, Hiroaki Tanaka, Kiyoshi Maeda, Kosei Hirakawa, Masaichi Ohira. A study on characteristics of stromal fibrosis and tumor-infiltrating lymphocytes in peritoneal metastasis of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3155.

Background: In recent years, it has been revealed that the immunological state of the host affects with the effect of cancer treatments. The neutrophil-to-lymphocyte ratio (NLR) as a systemic inflammatory marker and the density of tumor-infiltrating lymphocytes (TILs) at cancer microenvironment have been reported to be useful prognostic indices in various cancers, including colorectal cancer (CRC). The aim of this study is to evaluate the prognostic significance of immunological indices in patients who underwent curative resection of pulmonary metastases of CRC. Patients and Methods: The medical records of 17 patients who underwent potentially curative resection for pulmonary metastases of CRC were retrospectively reviewed. The correlation between the relapse-free survival (RFS) or the overall survival (OS) after curative resection of pulmonary metastases of CRC and the clinicopathological factors, NLR or TILs were evaluated. The NLR was calculated from the blood sample which was obtained within 2 weeks before pulmonary resection by dividing the absolute neutrophil count by the absolute lymphocyte count. According to the receiver operating characteristic (ROC) curve analysis, patients with an NLR of ≥ 2.5 were classified as high-NLR group. The density of TILs was examined by immunohistochemistry of pulmonary metastatic tumor sample, by staining anti-CD3 and anti-CD8 antibody. The number of CD3+TILs and CD8+TILs at the border of tumor was counted in a randomly selected field at a magnification of ×400. The mean of the values obtained in five different areas was used. According to the ROC curve analysis, the patients with the number of CD3+TILs ≥ 31 were classified as high- CD3+TILs group and patients with the number of CD8+TILs ≥ 21 were classified as high- CD8+TILs group. Results: The RFS and OS after resection of pulmonary metastases were significantly worse in the high-NLR group than in the low-NLR group (p=0.001, p=0.003, respectively). The RFS after resection of pulmonary metastases was significantly better in the high-CD3+TILs group than in the low-CD3+TILs group (p=0.041). Multivariate analysis revealed that the number of pulmonary metastases and the NLR before resection of pulmonary metastases were independent prognostic factors of the RFS (p=0.007, p=0.002, respectively) and the NLR before resection of pulmonary metastasis was the independent prognostic factor of the OS (p=0.009). Conclusion: The immunological indices such as the NLR and the density of TILs, may have prognostic significance in patients who underwent curative resection of pulmonary metastases of CRC. Citation Format: Yuki Okazaki, Masatsune Shibutani, En Wang, Hisashi Nagahara, Tatsunari Fukuoka, Yasuhito Iseki, Shinichiro Kashiwagi, Hiroaki Tanaka, Kiyoshi Maeda, Kosei Hirakawa, Masaichi Ohira. The prognostically significant immunological indices for patients who underwent curative resection of pulmonary metastases of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2828.