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INTRODUCTION: The link between obesity and elevated breast cancer mortality is well known, however, the underlying mechanisms are poorly understood. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator produced by sphingosine kinases (SphKs) that plays critical roles in inflammation and cancer progression. Previously, we found that obesity increases levels of S1P not only in breast tumors, but also in the lung. "Metastatic niches" are specialized microenvironments in distant organs primed by factors from cancer cells. We hypothesized that S1P secreted from the primary tumor could promote formation of a "metastatic niche" in the lung, which assists circulating cancer cells to form metastatic lesions. Further, HFD-induced obesity increases S1P secretion from the primary tumor, which could promote the formation of "metastatic niches" in the lung and lung metastasis. The aim of this study is to test these hypotheses. METHODS: A mouse model utilizing tail vein injection of E0771 syngeneic breast cancer cells was used. Prior to tail vein injections of naive E0771 cells, mice were treated with conditioned media from E0771 breast cancer cells overexpressing SphK1 (K1-CM) or that from E0771 cells cultured with the vector control (CT-CM). Histological analysis, RT-qPCR, and western blot were used. RESULTS: The lungs after K1-CM treatment demonstrated much more infiltration of macrophages with greater IL-6 secretion than lungs from CT-CM mice in areas without metastasis. Furthermore, SphK1, S1P receptor 1 and IL-6 expression were all significantly higher in the lungs of mice treated with K1-CM than with CT-CM, suggesting that S1P secreted from the primary tumor promotes formation of a metastatic niche in the lung. Next, mice were fed with HFD or ND for 12 weeks before treatment with SphK1-CM, and lungs were examined 7 days after intravenous injection of E0771 cells. Histological analysis demonstrated that there were significantly more lung metastases in mice on HFD than in mice on ND. Importantly, treatment with FTY720, a functional antagonist of S1P receptor 1, significantly reduced the lung metastases in HFD fed animals. Immunofluorescent staining revealed higher expression of IL-6 and greater number of F4/80 positive macrophages in mice fed with HFD compared with mice fed with ND, whereas FTY720 dramatically suppressed both IL-6 and macrophage infiltration in the lung of HFD-fed mice. HFD-induced obesity also increased pERK, pAKT, pStat3, and pp65 in the lung, and FTY720 suppressed these signaling pathways. CONCLUSION: Our results suggest that S1P plays a role in the formation of "metastatic niches" in the lung and lung metastasis of breast cancer, and obesity promotes this process. S1P will be a promising target for treatment of breast cancer metastasis, especially in condition with obesity. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research Grant Number 15H05676 and 15K15471 for M.N and 15H04927 for W.T. M.N. is supported by the Uehara Memorial Foundation, Nakayama Cancer Research Institute, and Tsukada Medical Foundation. K.T. is supported by NIH/NCI grant R01CA160688 and Susan G. Komen Investigator Initiated Research Grant IIR12222224. Citation Format: Nagahashi M, Tsuchida J, Moro K, Tatsuda K, Koyama Y, Takabe K, Wakai T. Sphingosine-1-phosphate signaling promotes metastatic niches and lung metastasis in obesity-related breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-11.

Background: In breast cancer, recent clinical trials have shown that sentinel lymph node biopsy (SLNB) alone without axillary lymph node dissection results in excellent prognosis if there is sentinel lymph node (SLN) metastasis in two or fewer nodes. The aim of the present study was to investigate the association between non-SLN metastasis and clinicopathological factors in case of SLN metastasis in two or fewer nodes in breast cancer. Methods: Patients who underwent SLNB for invasive breast cancer and were found to have positive SLN in two or fewer nodes were evaluated. The associations between non-SLN metastasis and clinicopahological factors were examined. Statistical analyses were performed using the Mann-Whitney and Chi-square tests, with statistical significance set at P < 0.05. Results: A total of 358 patients were enrolled during the study period and all of these patients were female and 54 patients had SLN metastasis (15%). Positive SLN in two or fewer nodes was identified in 44 patients (81.5%). Among these patients, 17 (38.6%) were found to have non-SLN metastasis. Non-SLN metastasis was associated with invasive tumor size (P = 0.015) and lymphatic involvement (P = 0.035). Multivariate analysis showed that tumor size (P = 0.011) and lymphatic involvement (P = 0.019) remained significant independent predictors of non-SLN metastasis, and that an invasive tumor size cut-off point of 28 mm was useful for dividing patients with positive SLN in two or fewer nodes into non-SLN-positive and non-SLN-negative groups. Conclusions: Non-SLN metastasis was found in more than 30% of patients with SLN metastasis present in two or fewer nodes. Large tumor size and the presence of lymphatic involvement were significantly associated with non-SLN metastasis. J Clin Med Res. 2015;7(8):620-626 doi: http://dx.doi.org/10.14740/jocmr2195w

INTRODUCTION: While obesity is an established independent prognostic factor for breast cancer patients, the underlying mechanisms how obesity promotes breast cancer progression are not well understood. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator produced by sphingosine kinases (SphKs) that plays critical roles in inflammation and cancer progression. Our hypothesis is that obesity increases levels of S1P in both tumor and its microenvironment, which promotes obesity-induced inflammation and breast cancer progression. The aim of this study is to test the hypothesis. METHODS: A high fat diet (HFD) was fed C57Bl/6 mice, which is the most commonly used murine diet-induced obesity model. E0771 cells derived from C57Bl/6 mice were implanted into mammary fat pads of mice that were fed with HFD or normal diet (ND) for 12 weeks prior to the implantation. Western blot, immunofluorescent staining, RT-qPCR and LC-ESI-MS/MS assays were used. RESULTS: Mice fed with HFD for 20 weeks developed severe obesity with an almost 2 fold increase of body weight compared with ND fed mice. E0771 mouse breast cancer cells were implanted into mammary fat pad of C57Bl/6 mice, and the mice fed with HFD developed significantly larger tumors within 30 days than those fed with ND. Expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the tumors of HFD fed mice, compared to those fed with ND. Furthermore, immunofluorescent analysis with anti-F4/80 antibody showed that tumors from HFD fed animals recruited significantly more tumor associated macrophages than those from ND fed animals. Expression of SphK1 and S1PR1, but not SphK2, was increased in the tumors from mice fed HFD. Mass spectrometry analysis revealed that while S1P levels in the normal breast mammary fat pad were increased with HFD feeding, S1P levels were even higher in breast tumors. Consistent with increased SphK1 and S1P in tumors, S1P was also significantly increased in the tumor interstitial fluid, which is a component of the tumor microenvironment and bathes cancer cells in the tumor. S1P levels were also increased in the serum of the tumor-implanted animals fed with HFD compared with those fed with ND, while minimal changes in S1P were evident in the serum of non-tumor bearing mice fed with HFD. Furthermore, S1P levels in the lungs of tumor-implanted animals fed with HFD were significantly higher than those fed with ND. CONCLUSION: S1P is increased not only in tumor, but also in tumor microenvironment such as tumor interstitial fluid by obesity. Our results suggest that S1P has a role in obesity-induced inflammation and the cancer progression. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research Grant Number 15H05676 and 15K15471 for M.N and 15H04927 for W.T. M.N. is supported by the Uehara Memorial Foundation, Nakayama Cancer Research Institute, and Tsukada Medical Foundation. K.T. is supported by NIH/NCI grant R01CA160688 and Susan G. Komen Investigator Initiated Research Grant IIR12222224. Citation Format: Tsuchida J, Nagahashi M, Moro K, Tatsuda K, Koyama Y, Takabe K, Wakai T. Obesity increases the lipid mediator, sphingosine-1-phosphate in the tumor and tumor microenvironment, and promotes tumor progression. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-03.

Background: The purpose of this study was to elucidate the association between sentinel lymph node (SLN) metastasis and Ki67 labeling index and to elucidate whether Ki-67 was useful or not for prediction of SLN metastasis in breast cancer. Methods: We identified 343 invasive breast cancer patients with sentinel lymph node biopsy (SLNB) from 2003 to 2012. The association between SLN status and clinicopathological features, molecular subtypes and Ki-67 labeling index were evaluated. Results: SLN metastasis was detected in 79 patients (23.0%). SLN metastasis was significantly associated with clinical T-stage (p = 0.0003), lymphovascular involvement (LVI) (p 0.0001). Ki-67 labeling index of primary tumor was significantly lower in SLN positive patients (p = 0.0331), and Ki-67 cut-off point of 7.5% was useful for dividing SLN positive from negative (p = 0.0197). Conclusion: Low value of Ki-67 labeling index, in addition to progression of clinical T-stage and presence of LVI, is significantly associated with SLN metastasis, and it seems to be useful to consider Ki-67 labeling index for SLN metastasis prediction.

Background Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, has been implicated as a key regulatory molecule in cancer through its ability to promote cell proliferation, migration, angiogenesis, and lymphangiogenesis. Previous studies suggested that S1P produced by sphingosine kinase 1 (SphK1) in breast cancer plays important roles in progression of disease and metastasis. However, the associations between S1P and clinical parameters in human breast cancer have not been well investigated to date. Materials and methods We determined levels of S1P and other sphingolipids in breast cancer tissue by electrospray ionization-tandem mass spectrometry. Associations between S1P levels and clinicopathologic features of the tumors were analyzed. Expression of phospho-SphK1 (pSphK1) in breast cancer tissues was determined by immunohistochemical scoring. Results Levels of S1P in breast cancer tissues were significantly higher in patients with high white blood cell count in the blood than those patients without. S1P levels were lower in patients with human epidermal growth factor receptor 2 overexpression and/or amplification than those patients without. Furthermore, cancer tissues with high pSphK1 expression showed significantly higher levels of S1P than cancer tissues without. Finally, patients with lymph node metastasis showed significantly higher levels of S1P in tumor tissues than the patients with negative nodes. Conclusions To our knowledge, this is the first study to demonstrate that high expression of pSphK1 is associated with higher levels of S1P, which in turn is associated with lymphatic metastasis in breast cancer.

Background Sphingolipids, including sphingosine-1-phosphate (S1P) and ceramide, have emerged as key regulatory molecules that control various aspects of cell growth and proliferation in cancer. Although important roles of sphingolipids in breast cancer progression have been reported in experimental models, their roles in human patients have yet to be determined. The aims of this study were to determine the levels of sphingolipids including S1P, ceramides, and other sphingolipids, in breast cancer and normal breast tissue and to compare the difference in levels of each sphingolipid between the two tissues. Materials and methods Tumor and noncancerous breast tissue were obtained from 12 patients with breast cancer. Sphingolipids including S1P, ceramides, and their metabolites of sphingosine, sphingomyelin, and monohexosylceramide were measured by liquid chromatography–electrospray ionization–tandem mass spectrometry. Results The levels of S1P, ceramides, and other sphingolipids in the tumor were significantly higher than those in normal breast tissue. There was a relatively strong correlation in the levels of S1P between the tumor and those of normal breast tissue from the same person. On the other hand, there was no correlation in the levels of most of the ceramide species between the tumor and those of normal breast tissue from the same person. Conclusions To our knowledge, this is the first study to reveal that levels of sphingolipids in cancer tissue are generally higher than those of normal breast tissue in patients with breast cancer. The correlation of S1P levels in these tissues implicates the role of S1P in interaction between cancer and the tumor microenvironment.

A 71-year-old woman diagnosed with Fournier's gangrene caused by penetration of a rectal carcinoma was referred to our hospital. Emergency drainage and sigmoid colostomy were performed. Pathological examination of a biopsy sample showed moderately differentiated tubular adenocarcinoma. Abdominoperineal resection with bilateral salpingo-oophorectomy and hysterectomy was performed with curative intent after 4 courses of chemotherapy with S-1 and oxaliplatin. The postoperative pathological diagnosis was StageⅡ (pT4bN0M0). The patient received tegafur/uracil and Leucovorin as adjuvant chemotherapy. Seven months after the curative operation, partial pneumonectomy was performed for the recurrence of the rectal carcinoma in the right lung. Eight months after pneumonectomy, recurrent tumors were observed in the right lung and subcutaneous fat layer of the right buttock along the drainage site. The tumor in the right buttock was excised along with part of the gluteus maximus, and partial pneumonectomy was then performed. Three years and 6 months after the emergency drainage, the patient is alive with no evidence of recurrence. After drainage for rectal carcinoma complicated by Fournier's gangrene, the possibility of recurrence around the drainage site should be considered.

A 58-year-old woman presenting with abdominal distension was diagnosed with a tumor in the right ovary. A chest-abdominal-pelvic computed tomography scan revealed multiple lung metastases, multiple liver metastases, and peritoneal dissemination. Invasion of the rectum by peritoneal dissemination of the Douglas' pouch was suspected. She was diagnosed with Stage Ⅳ right ovarian cancer and was treated with preoperative chemotherapy. After chemotherapy, debulking surgery of the abdominal cavity (total hysterectomy, bilateral salpingo-oophorectomy, partial omentectomy, and Hartmann's procedure) was performed. Because there was swelling observed in multiple mesorectal lymph nodes, lymph node dissection was performed based on methods used for rectal cancer surgery. Postoperative histopathological examination revealed multiple mesorectal lymph node metastases arising from ovarian cancer. We suggest that mesorectal lymph node dissection be considered a part of debulking surgery for ovarian cancers that have invaded the rectum.

A 62-year-old man presented with pruritus caused by obstructive jaundice. He was diagnosed with carcinoma of the ampulla of Vater and underwent pylorus-preserving pancreaticoduodenectomy. Histologic examination revealed signet-ring cell carcinoma without nodal metastasis. The patient underwent a partial resection of the small bowel for small bowel obstruction 38 months after the initial resection. Histologic examination disclosed signet-ring cell carcinoma at the surface of the resected small bowel, and the diagnosis of peritoneal recurrence was confirmed. He received oral S-1(100 mg/day) every other day for 14 months until bowel obstruction caused by peritoneal carcinomatosis became apparent. The patient died of the disease 58 months after the initial resection.

The patient was a 73-year-old woman. She underwent right hemicolectomy and D3 lymph node dissection for cecal cancer in June 2003. Although a peritoneal dissemination was intraoperatively noted around the primary tumor lesion, it was resected concurrently and thus R0 surgery was accomplished. Postoperative adjuvant chemotherapy was not performed. During the follow-up on an outpatient basis, a solitary left lung metastasis was found and partial left upper lobectomy of the lung was performed in December 2004. A solitary liver metastasis was identified in the liver (S3), and lateral segmentectomy of the liver was performed in June 2007. The patient was alive with no evidence of recurrence 11 years and 9 months after resection of the primary lesion and 7 years and 9 months after the hepatectomy. Long-term survival can be achieved by performing resection without residual cancer even in some cases with metachronous metastatic recurrences in multiple organs. Metastasectomy should be considered proactively when the patient is in a good general condition and R0 resection is possible.

A 51-year-old man underwent right nephrectomy for renal cell carcinoma. Six months later, a solitary metastatic tumor was detected in the pancreatic tail and he underwent distal pancreatectomy and splenectomy. One year and 6 months after the initial resection, a solitary metastatic tumor was detected in the pancreas head, and he underwent partial pancreatectomy. A solitary metastatic tumor was detected again in the remnant pancreatic body 3 years and 10 months after the initial resection. Partial resection of the distal part of the remnant pancreas was performed. The patient remains alive and well with no evidence of remnant disease 4 years after the initial resection.

It is unknown how long the risk of developing secondary resistance to imatinib persists in patients with gastrointestinal stromal tumors (GISTs). Here we report a case of a patient with a metastatic gastric GIST who developed secondary resistance to imatinib 70 months after initiation of imatinib therapy. A 62-year-old woman with a gastric GIST underwent total gastrectomy with pancreaticosplenectomy. Immunohistochemistry revealed a KIT-positive GIST. The mitotic index of the tumor was 13/50 high-power fields, indicating a high-risk malignancy. After surgery, the patient developed a solitary liver metastasis and underwent right hepatic lobectomy. Four months later, a metastatic tumor was found at the left adrenal gland, and imatinib therapy was initiated in December 2004. Imatinib therapy led to marked tumor shrinkage and complete clinical remission in the patient. However, in October 2010, computed tomography scans revealed a peritoneal metastasis in the ileocecal area. The tumor progression was clinically determined to be due to the development of secondary resistance to imatinib, and the patient's treatment was switched to sunitinib. This case illustrates secondary resistance to imatinib can develop even after a sustained and marked treatment response. Long-term therapy and close monitoring are recommended for the management of patients with metastatic GISTs.

low or below reference ranges. Vitamin A was associated with sex, metastases, CRP and albumin (all p < 0.01). Vitamin B1 was associated with venous invasion and WCC (p < 0.05 and

SummaryBackground and aimsTransthoracic esophagectomy using 3-field lymphadenectomy (TTE-3FL) for esophageal cancer is one of the most aggressive gastrointestinal surgeries. Early enteral nutrition (EN) for TTE-3FL patients is useful and valid for early recovery; however, EN using a fat-containing formula risks inducing chyle leak. In the present study, we retrospectively examined esophageal cancer patients treated byTTE-3FL and administered postoperative EN to elucidate the validity of lowering the fat levels in elemental formulas to prevent postoperative chyle leak and improve postoperative recovery.MethodsA total of 74 patients who received TTE-3FL for esophageal cancer were retrospectively examined. Patients were classified into two groups according to the type of postoperative EN: Group LF patients received a low-fat elemental formula, and Group F patients received a standard fat-containing polymeric formula. The following clinical factors were compared between the groups: EN start day, maximum EN calories administered, duration of respirator use, length of ICU stay, incidence of postoperative infectious complications, use of parenteral nutrition (PN), and incidence of postoperative chyle leak.ResultsPatients in Group LF were started on EN significantly earlier after surgery and they consumed significantly higher maximum EN calories compared to Group F patients (P