80.09 The Impact of DNA Repair Genes and Microsatellite Instability on Survival in Colorectal Cancer

Posted on January 18, 2018

S. Narayanan1, T. Kawaguchi1, L. Yan1, X. Peng1, Q. Qi1, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA

Introduction:  Microsatellite instability (MSI) occurs via inactivation of DNA mismatch repair genes resulting in impaired DNA repair function with subsequent accumulation of abnormal genes. Heterogeneity in MSI status is common in colon cancers and has been demonstrated to be an independent predictor of survival, with MSI-high tumors having a better overall prognosis. Previous studies have correlated upregulation of DNA repair genes with increased ability for tumors to metastasize and with chemotherapy and radiation resistance, thereby resulting in poorer survival. We, thus, hypothesized that low expression of DNA repair genes would result in improved survival secondary to increased MSI. 

Methods:  The Cancer Genome Atlas (TCGA) was used to evaluate a cohort of 283 patients with colorectal cancer. RNA sequence gene expression quantification data for colon cancer was retrieved from the Genomics Data Commons (GDC) data portal. Gene expression levels were derived using normalization methods provided in the DESeq2 package and designated as low or high. The expression of DNA repair genes was then compared between MSI-High and Microsatellite stable (MSS) cohorts. Overall survival for patients for each DNA repair gene was determined via Kaplan-Meier analysis. 

Results: We found that low expression of several DNA repair genes correlated with high levels of microsatellite instability. Some of these were the expected mismatch repair genes- MLH1 (p< 0.0001), PMS1 (p= 0.002), PMS2 (p< 0.0001) and MLH3 (p=0.036). However, others were double stranded break DNA repair genes such as ATM (p< 0.0001), PRKDC (p< 0.0001), ATR (p< 0.0001) and BRCA2 (p= 0.0081). Significantly improved overall survival was demonstrated in patients with low expression of ATM (p= 0.04), MLH1 (p= 0.028), PMS2 (p= 0.003) and MLH 3 (p= 0.0029). There was a trend towards improved survival in patients with low expression of BRCA2 and ATR without achieving statistical significance. 

Conclusion: In this analysis of patients with colorectal cancer we found that diminished expression of several DNA repair genes correlated with increased microsatellite instability even those that were not known mismatch repair (MMR) genes. However, improvement in survival primarily correlated with MMR-deficiency and with low expression of other DNA repair genes to a lesser extent. This phenomenon may relate to the increased immunogenicity of tumors with genomic instability and may have implications for further development of immunotherapy targets in the future. 

 

80.06 High Expression of Sphingosine Kinase 1 in Colitis-Associated Cancer

Posted on January 18, 2018

K. Yuza1, M. Nagahashi1, Y. Shimada1, M. Nakano1, Y. Tajima1, H. Kameyama1, M. Nakajima1, H. Ichikawa1, J. Sakata1, T. Kobayashi1, K. Takabe2,3, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 3University At Buffalo Jacobs School Of Medicine And Biomedical Sciences, The State University Of New York,Department Of Surgery,Buffalo, NY, USA

Introduction:
Colorectal cancer (CRC) that developed from inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, is known as colitis-associated cancer (CAC). Significant advances have been made in understanding the link between chronic inflammation and cancer by advancements in molecular biology and animal models that recapitulate human diseases in recent years. It has been implicated that sphingosine-1-phosphate (S1P), a bioactive lipid mediator, is a key mediator of inflammation, tumorigenesis and cancer progression. We have recently shown that S1P produced by upregulation of sphingosine kinase 1 (SphK1) links chronic intestinal inflammation to CAC utilizing animal models (Cancer Cell 2013). However, the role of S1P in human CAC has never been investigated due to the difficulty to measure S1P levels directly in human samples. We have previously shown that high expression of phosphorylated-SphK1 (pSphK1) is associated with higher levels of S1P in human tissue, and detecting pSphK1 by immunohistochemistry could be an alternative method to examine the role of S1P in human patients (JSR 2016). The aim of this study is to determine the expression levels of pSphK1 in sporadic CRC and CAC, and to clarify the importance of S1P in CAC patients.

Methods:
Tissue samples of sporadic CRC (N = 10) and ulcerative colitis-associated cancer (N = 10), respectively, were randomly selected from patients who underwent curative resection between April 2012 and May 2017. The expression of pSphK1 in these samples was examined by immunohistochemistry staining. Semi-quantitative evaluation of the staining of pSphK1 in CRC cells was performed as follows; a staining score was calculated by multiplying two points of staining intensity (0-3) and the ratio (1-4). The staining score of 0 to 5 points was considered as pSphK1-negative, and 6 to 12 was considered as pSphK1-positive.

Results:
Immunohistochemistry analysis of pSphK1 revealed that sporadic CRC had a median staining score of 4 (range 0-12), while ulcerative colitis-associated cancer had a median staining score of 12 (range 4-12) (Mann Whitney test, P<0.05). Based on the staining scoring described in Methods, three out of 10 (30%) patients with sporadic CRC, and nine out of 10 (90%) patients with ulcerative colitis-associated cancer were considered as pSphK1-positive (Fisher’s exact test, P<0.05).

Conclusion:
To our knowledge, this is the first report that compared pSphK1 expression levels in CAC and those in sporadic CRC. The high levels of pSphK1 expression in CAC implicate an important role of S1P in the disease process of CAC.
 

79.16 Elevated Expression of High Mobility Group Protein B1 is Associated with Breast Cancer Survival

Posted on January 18, 2018

K. McDonald1, E. Katsuta1, L. Yan1, Q. Qi1,2, X. Peng1,2, K. Takabe1  1Roswell Park Cancer Institute,Department Of Surgery,Buffalo, NY, USA 2State University Of New York Upstate Medical University,Syracuse, NY, USA

Introduction: HMGB1 is an evolutionarily conserved protein present in the nucleus of eukaryotic cells.  HMGB1 release has been found to be higher in breast cancer cells compared to normal tissue counterparts.  The function of released HMGB1 is diverse and compartment specific.  Inside the nucleus it facilitates DNA repair, while outside the nucleus it functions as a damage associated molecular protein (DAMP), regulating processes such as cell proliferation, autophagy, inflammation and immunity.  HMGB1 released outside the cells is detected by the TLR4 receptor. There is a strong correlation between TLR4 expression and expression of pro-inflammatory mediators.  Depending on the tumor type, the HMGB1/TLR4 interaction can promote or inhibit tumorigenesis. The exact role of HMGB1 in breast cancer has not been fully elucidated. We hypothesize that high HMGB1expression is associated with improved breast cancer survival.

Methods:  All data was obtained from the Cancer Genome Atlas (TCGA). Expression patterns of HMGB1 were retrieved from the Genomic Data Commons (GDC) data portal for analyses. After HMGB1 specific thresholds were derived and used to group estrogen receptor (ER) positive and negative breast cancer patients into a high- or low-expression group, survival data was calculated by using the Cox proportional hazard model. 

Results: When evaluating TLR4 expression in breast cancers on survival, there was no survival benefit in ER-positive (n-High = 284, n-low = 526; p=.201) or ER-negative (n-High = 207, n-low = 31; p=.221) cancers in our cohort.  However, high HMGB1 expression was associated with a statistically significant improvement in survival for ER-positive cancers (n-High = 312, n-low = 498; p=.006).  In the sub-analysis, both luminal A and B breast cancers with high HMGB1 expression had a statistically significant improvement in survival (n-High =389, n-low =30 ; p=.023; n-High =145 , n-low =46 ; p=.034 respectively).  On the other hand, a survival advantage in ER-negative cancers that overexpressed HMGB1 was not statistically significant (n-High =112, n-low =126; p=.092).  

Conclusion: By utilizing a big dataset (TCGA) with sufficient statistical power, we found that high expression of HMGB1 in breast tumor samples was associated with better overall survival in ER-positive, luminal A and B breast cancers. Our study supports the novel role of HMGB1 as a tumor suppressor in breast cancer. 

 

79.15 Methods to improve establishment of breast cancer patient-derived xenografts

Posted on January 18, 2018

M. Okano1, T. Kawaguchi1, M. Oshi1, I. Okano1, E. Katsuta1, K. Takabe1  1Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA

Introduction:  Despite massive expenditures in drug development to treat breast cancer, the number of compounds that was proved to be effective in animal models but failed in humans remains high. This is partly because currently used murine models do not mimic human patient cancer biology. Patient-Derived Xenograft (PDX) model, where tumor fragments from the patients are implanted into immunocompromised mice, has emerged as pre-clinical model to address these issues, however, even the optimal site of implantation remain controversial. We hypothesized that patient-derived breast tumor survive and grow better when implanted orthotopically in mammary fat pad (MFP) compared to dorsal subcutaneous space (SQ).

Methods:  We xenografted 9 patient breast cancer tumors into NSG mice. 3 tumors (including 1 brain metastatic tumor) were ER(+)HER2(-) and 6 tumors were triple negative (TN). 2 of the TN samples were previously established PDX tumor provided from University of Utah. Using 2-3 mice per each patient sample, 1mm(3) tumor fragments were implanted surgically into both four sites in SQ and four sites in MFP, the bilateral second and forth fat pads. Tumors were resected at the day when the biggest tumor grew up to 1.5cm and they were passaged to another 2-4 mice as the next generation. The size and weight of the tumor were measured. Tumor “take” was defined as tumorigenesis of palpable tumor after implantation regardless of time it took.

Results: PDX tumors were established in 5 out of 9 patient tumors. Except brain metastasis tumor, the average time for tumor take was significantly longer in 1st generation compared to 2nd or 3rd generation (117.3 days vs 60.5 days, p<0.0001). The overall tumor take rate was 51.8% (156/301 implantation site). Take rate from TN tumors was 57.4% (156/272 site), on the other hand, that from ER positive tumors was 0% (0/39 site). Interestingly, tumor take rate was 89.6% (86/96 site) in brain metastatic tumor although it was ER positive tumor. Tumor take rate was significantly better in MFP implantation compared from SQ (61.5%, 99/161 vs 40.1%, 57/140, p<0.001). Tumor weight were significantly heavier in MFP compared to SQ (0.58g vs 0.097g, p<0.0001). With increase of passage, the weight difference between MFP tumor and SQ tumor significantly increased (weight MFP/SQ of 1st generation vs 2nd and 3rd generation were 3.2 vs 5.3, p<0.0001). Even in brain metastasis tumor, take rate was significantly better in MFP implantation than SQ (100%, 48/48 vs 79.2%, 38/48, p=0.0012), and tumor weight were significantly heavier in MFP compared to SQ (0.21g vs 0.50g, p<0.0001). Time to tumor taken was significantly shorter in metastatic tumor compared from the other tumors (51days vs 74days, p=0.018). 

Conclusion: Triple negative breast cancers were more successful than ER positive tumors in establishing PDX. Brain metastasis tumor had the best take rate. MPF is better implantation site than SQ to develop breast PDX model.

 

79.13 High Amplification of MYC are Associated with Poor Survival in non-Triple Negative Breast Cancer

Posted on January 18, 2018

K. Takabe1, E. Katsuta1, L. Yan2, M. Nagahashi3  1Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NY, USA 3Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, , Japan

Introduction:  Mutation of MYC that causes elevated expression of this oncogenic transcription factor is found in many cancers. This leads to the unregulated expression of many genes that result in cell proliferation and tumorigenesis. It has been reported that MYC amplification is associated with Triple Negative Breast Cancer (TNBC). However, clinical relevance of MYC amplification in breast cancer remains mostly unexplored. We hypothesized that high amplification of MYC is a prognostic biomarker that represent worse biology and poor survival.  

Methods:  Genomic and clinical data were obtained from the Cancer Genome Atlas (TCGA) through cBioportal. MYC amplification was defined based upon Genomic Identification of Significant Targets in Cancer (GISTIC) copy-number; GISTIC 2 was defined as tumor with amplification, remaining GISTIC -1, 0 and 1 were defined as tumor without amplification. 

Results: Among 1080 patients with DNA copy-number data, 229 tumors (21.2%) showed MYC amplification which was the most common copy-number alteration in whole TCGA cohort of breast cancer. 156 patients (15.5%) were classified as TNBC based on ER, PgR, immunohistochemistry status and HER2 immunohistochemistry and FISH method. In agreement with previous report, there was greater proportion of tumors with high amplification of MYC in TNBC compared to non-TNBC (37% vs 18%, p<0.001). MYC mRNA expression was significantly higher in tumor with amplification compare to tumor without amplification in whole cohort as well as TNBC and non-TNBC (p<0.001, p=0.002 and p<0.001, respectively). There was no significant difference in overall survival (OS) between tumor with vs without amplification in whole breast cohort (p=0.112). To our surprise, tumors with high amplification showed significantly worse prognosis in non-TNBC patients (5-year OS rates: 80.2% vs 86.6%, p<0.037), whereas there was no significant difference in TNBC (p=0.68). These findings imply that altered MYC plays a role to promote cancer progression in non-TNBC. In non-TNBC cohort, there was greater proportion of higher AJCC Stage patients in tumors with MYC amplification group (Stage III/IV: 35.9% vs 24.8%, p<0.007).

Conclusion: Breast tumors with MYC amplification has worse prognosis in non-TNBC, but not in TNBC. MYC amplification may play different role between TNBC and non-TNBC.

 

79.12 Sphingosine-1-phosphate Affects Tumor-associated Immune Cells in Human Breast Cancer Patients

Posted on January 18, 2018

J. Tsuchida1, M. Nagahashi1, K. Moro1, A. Otani1, M. Endo1, M. Ikarashi1, M. Nakajima1, Y. Koyama1, J. Sakata1, T. Kobayashi1, H. Kameyama1, Q. Qi3, L. Yan3, K. Takabe2,4, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery,Buffalo, NEW YORK, USA 3Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NEW YORK, USA 4University At Buffalo Jacobs School Of Medicine And Biomedical Sciences, The State University Of New York,Department Of Surgery,Buffalo, NEW YORK, USA

Introduction: It is known that immune cells such as certain T cells and macrophages assist cancer progression. Those tumor-associated immune cells (TAICs) are considered to be one of the key players in the mechanisms how cancer invades and metastasizes. Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, has emerged as a new player in cancer progression. S1P is produced by sphingosine kinases (SPHKs) inside cells and exported out of the cells. Extracellular S1P exerts its functions by binding to S1P specific receptors on the cell surface, and regulates various cellular functions such as cell proliferation, migration, and angiogenesis. Although previous in vivo studies indicated that S1P regulates immune function in many pathological conditions, the role of S1P in tumor-associated immune function has not been fully investigated in patients. Here, we test our hypothesis that S1P signaling affects TAICs in human breast cancer.

Methods: The expression level of each enzyme-encoding gene involved in S1P production was evaluated by retrieving RNA sequencing and gene expression quantification data using the Genomics Data Commons (GDC) data portal of the The Cancer Genome Atlas cohort. Gene expression levels were derived using normalization methods provided in the DESeq2 package. We compared the difference in expression levels of S1P related genes between HER2-negative and sphingosine kinase 1 (SPHK1)-high breast tissue (n = 263), and HER2-negative and SPHK1-low breast tissue (n = 277). We also compared the expression level of TAIC-related genes in between the same groups of the patients.

Results: The expression of some of the S1P-related genes; S1PR1, S1PR2, S1PR4, S1PR5, and SPNS2, were significantly increased in patients with high SPHK1 compared to those with low SPHK1. In contrast, expression of some of the S1P-related genes; S1PR3, ABCG2, SGPL1 and ORMDL3 were significantly decreased in patients with high SPHK1 compared to those with low SPHK1. Importantly, we found a significant increase in the expression of the TAIC-related genes, including CD4, CD8A, CD8B, PTPRC as T cell markers in SPHK1-high HER2-negative breast cancer tissues than in SPHK1-low. Macrophage markers including CCL2, CD163, CD68, CXCL8, IL-10, MSR-1, TGFB1, VEGFA, but not PLIN1, are increased in SPHK1-high HER2-negative breast cancer tissues. Moreover, adepocyte-related genes, such as HOXC9, LEP, SERPINE1, TNF, and TNFRSF9, but not UCP1, AGT, and ADIPOQ, were also increased in SPHK1-high HER2-negative breast cancer tissue. Taken together, these data revealed that SPHK1 is associated with increased expression of most of the TAIC-related genes in HER2-negative breast cancer.

Conclusion: Our results suggest that S1P affects TAICs in HER2-negative breast cancer patients, indicating the important roles of S1P in the complicated immune system related to tumor progression. Further investigations are needed to understand the underlying mechanisms.

79.09 Intrinsic heterogeneity of triple-negative breast cancer cells triggers vascular mimicry in 3D culture

Posted on January 18, 2018

A. MAITI1, A. MAITI1  1Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA

Introduction: Within the same tumor microenvironment phenotypic and functional heterogeneity arise due to plasticity of cancer cells as a consequence of environmental differences, genetic changes and reversible epigenetic changes. Individual tumor cells growing in culture also display heterogeneity in their intrinsic ability to progress and metastasize, however, molecular mechanism is still unknown. Tumor growth and metastasis are thought to be angiogenesis related processes. Recent reports suggested that cancer cells feed themselves by an angiogenesis independent pathway, known as Vacsulogenic mimicry (VM). We have examined the ability of matrigel to stimulate complex cell behavior by its heterogeneous composition.

Methods: Cells are mixed with matrigel and plated in low attachment plates. In order to understand differential gene expression pattern we stained MDA MB 231cells in formaldehyde fixed 3D matrigel matrix environment. In order to under the differential gene expression pattern, we isolated two phenotypically different groups of cells (Vessel and Tumorsphere forming cells) from the 3D matrigel culture by using microscopic suction procedure for gene expression analysis by qPCR. Epigenetic mechanisms mediated suppression of tumor suppressors or anti-angiogenesis marker genes are hall mark of VM formation and cancer progression,  we sought to examine whether re-expression of those genes with Entinostat (MS-275), a selective inhibitor of class I histone deacetylase (HDAC) could abolish VM structures in 3D matrigel cell culture.

Results:When MDA-MB-231 cells are mixed with matrigel and cultured in low attachment plates,  around 80% cells formed vessel like phenotype known as vascular mimicry (VM) and  20% cells form spheroids. Since CD44, a stem cell marker of enhances tumor cell plasticity, we examine CD44 expression in MDA-MB-231 cells grown in 3D matrigel matrix environment. We have observed that VM forming cells are stained CD44 positive while spheroid forming cells are negative in 3D matrigel culture. Both group of cells stained positive for VEGFc and HIF1α . Gene expression data suggested that VM forming cells have more expression of CD44 and HIF1α compared to spheroid forming cells. When we treated cells with MS-275, VM structure is totally abolished. QPCR data suggested that MS-275 treatment epigenetically re-express anti-angiogenic genes; SERPINF1, THBS1 and THBS2 and tumor suppressor genes; APC, PTEN and p21. While MS-275 treatment also downregulated Vimentin, VEGF-A and CD44 .

Conclusion:Our results suggest that the VM phenotype arises in a subpopulation of cells from a conserved transcriptional response in 3D matrigel environment. Epigenetically re-expression of anti-angiogenic genes  could be a mechanism to control VM formation in triple-negative breast cancer cells.

79.10 Higher CD73 Expression is Associated with Poor Prognosis in Breast Cancer

Posted on January 18, 2018

E. Katsuta1, L. Yan2, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NY, USA

Introduction:  CD73 is a surface enzyme that converts AMP into adenosine. Accumulated extracellular adenosine in tumor microenvironment generates immunosuppression and pro-angiogenic environment that promotes the onset and progression of cancer. Further, tumors that express high levels of CD73 have worse prognosis in some types of malignancies. However the impact of CD73 expression levels on breast cancer patient survival remains controversial. It was also reported that the impact of CD73 expression on patients survival was different among the subtype of breast cancer.

Methods:  Gene expression was compared between cancer and non-cancer tissue using GENT (Gene Expression across Normal and Tumor tissue). Overall survival (OS) and disease-free survival (DFS) were compared between CD73 high and low expression groups based upon RNA-seq data of The Cancer Genome Atlas (TCGA) as the treatment- naïve cohort. Gene set enrichment analysis (GSEA) was conducted between CD73 high and low patients in TCGA. Relapse-free survival (RFS) was compared between CD73 high and low expression patients who received neoadjuvant chemotherapy using GSE25066 cohort. Gene expression was compared between before and after chemotherapy using GSE28844 cohort.

Results: CD73 expression level was significantly lower in cancer than normal breast tissue (p<0.001). Patients were classified as Luminal A (n=419), Luminal B (n=194), Her2 (n=67), Basal (n=140) and normal (n=24) by PAM50 classification. The expression of CD73 was significantly higher in Normal and Basal subtype compare to Her2, Luminal A, and Luminal B. Patients with high expression of CD73 showed worse survival compared with low expression group in both OS (5-year OS rate: 60.5% vs 83.4%, p<0.001) and DFS (5-year DFS rate: 71.5% vs 82.1%, p=0.049) in whole cohort, as well as in Luminal A+B patients OS (5-year OS rate: 54.5% vs 85.9%, p<0.001).  However, there was no significant difference between these two groups neither in Her2 (p=0.180), Basal (p=0.962) or Normal (p=0.172) subtypes. High expression of CD73 group also showed worse relapse-free survival in neoadjuvant chemotherapy patients cohort (3-year RFS rate: 72.2% vs 83.6%, p=0.003). CD73 expression was significantly elevated in tumors after chemotherapy compared from before the treatment (p<0.001). It may imply that CD73 high expressed cells were resistant to chemotherapy. GSEA results revealed that epithelial-mesenchymal transition (EMT) and angiogenesis related gene sets were significantly enriched in CD73 high patient tumors.

Conclusion: Tumors with high expression of CD73 have worse prognosis in treatment-naïve patients as well as patients who underwent neoadjuvant chemotherapy. The worse prognosis of the patients with high expression of CD73 may be able to be explained by metastatic potential with up-regulated EMT as well as promoted angiogenesis.

79.08 Ceramides Are Elevated with Activation of Ceramide Biosynthesis Pathways in Human Breast Cancer

Posted on January 18, 2018

K. Moro1,4, T. Kawaguchi2, J. Tsuchida1, E. Gabriel2, Q. Yan3, L. Yan3, N. Sato4, T. Wakai1, K. Takabe2,5, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NEW YORK, USA 3Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NEW YORK, USA 4Niigata Cancer Center Hospital,Surgery,Niigata, NIIGATA, Japan 5University At Buffalo Jacobs School Of Medicine And Biomedical Sciences,Surgery,Buffalo, NEW YORK, USA

Introduction: Sphingolipids have emerged as key regulatory molecules that control various aspects of cell biology. Among them, sphingosine-1-phosphate (S1P) and ceramide are known to form a “rheostat” where former promote cell growth and survival, and the latter apoptosis in cancer. There are three biosynthesis pathways that generate ceramide; De novo pathway; Sphingomyelin pathway; and Salvage pathway, and it is metabolized to S1P through Catabolic pathway. Despite their critical roles, the levels of sphingolipids have never been measured in patients due to lack of methods to precisely quantify them until recently. We have recently published high levels of S1P not only in breast tumor, but also in tumor microenvironment, such as tumor interstitial fluid, and reported that S1P plays pivotal roles in breast cancer progression. On the other hand, the levels of ceramide, a bioactive metabolite of S1P, in breast cancer patients have not yet well investigated to date. The aim of this study is to clarify the ceramide levels and its biosynthesis pathways in breast cancer patients.

Methods: Breast cancer, peri-tumor normal breast defined as tissue within 1 cm from the gross edge of cancer and normal breast tissue samples were collected from surgical specimens from a series of 44 patients with breast cancer. Sphingolipids, including ceramides (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:0) and their metabolites of monohexosylceramides, dihydroceramide and sphingomyelin in the tissue samples were determined by mass spectrometry. Results were analyzed for statistical significance with the Kruskal-Wallis test. The Cancer Genome Atlas (TCGA) was used to analyze gene expressions related to the sphingolipid metabolism.

Results: Ceramide levels were higher in breast cancer compared from both normal and peri-tumor breast tissue. Substrates and enzymes that generate ceramide were significantly increased in all three ceramide biosynthesis pathways in cancer; Monohexocylceramide and glucosylceramides beta in Salvage pathway; Sphingomyelin and sphingomyelin phosphodiesterase 2 and 4 in Sphingomyelin pathway; Dihydroceramide and dihydroceramide desaturase 1 in De novo pathway. Sphingosine and ceramide synthases 2, 4, 5 and 6 in Catabolic pathway were also significantly elevated in cancer. On the contrary, gene expression of enzymes that catalyze ceramide, sphingomyelin synthase 2 and ceramide kinase were significantly suppressed, all contribute to ceramide increase in cancer.

Conclusion: This is the first study to reveal the clinical relevance of ceramide metabolism in breast cancer patients. We demonstrated that ceramide levels in breast cancer tissue are significantly higher than those in normal tissue, with activation of the three ceramide biosynthesis pathways. Our finding is in agreement with the classic notion that apoptotic cell destruction signal is activated in cancer, however, it is not enough to overcome its proliferative drive.

 

79.07 Triple-negative Breast Cancer that expresses high level of Annexin A1 have worse prognosis

Posted on January 18, 2018

M. Okano1, E. Katsuta1, M. Oshi1, K. Takabe1  1Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA

Introduction:  Annexin A1 (ANXA1) is a calcium-dependent phospholipid-linked protein, involved in anti-inflammatory effects, regulation of cellular differentiation, proliferation and apoptosis. Recently, we reported that ANXA1 is associated with triple-negative breast cancer (TNBC) and its poor prognosis. It was also reported that ANXA1 relates to epithelial mesenchymal transition (EMT). We hypothesized that ANXA1 expression associate with EMT that leads to poor prognosis of TNBC. 

Methods:  Clinical and RNA-seq data were all obtained from the Cancer Genome Atlas (TCGA). Patients were classified as either high or low expression of ANXA1 determined by automated scanning and selecting the threshold yielding the lowest p-value. Overall survival (OS) and Gene set enrichment analysis (GSEA) were conducted comparing high and low expression group. To validate the relationship between ANXA1 expression and survival, ANXA1 protein expression was assessed by Immunohistochemistry (IHC) in 48 TNBC patients. Patients were classified into either positive or negative based upon IHC score. Clinicopathological factors and survival were compared between them. 

Results: TNBC patients had significantly higher levels of ANXA1 expression compare to non-TNBC patients in TCGA cohort (p<0.001). ANXA1 high and low expression group were 140 and 20 patients in TNBC, and 540 and 245 in non-TNBC in TCGA cohort, respectively. High expression of ANXA1 group showed significantly worse OS (5-year OS rate: 68.6% vs 100%, p=0.035) in TNBC patients. On the contrary, high expression of ANXA1 demonstrated better OS in non-TNBC patients (5-year OS rate: 88.4% vs 78.7%, p=0.004). This finding was validated in protein expression level by IHC. Among 48 cases of TNBC patients 17 cases (35.4%) were classified as ANXA1 positively group. OS was significantly shorter in patients with ANXA1 positive tumors compared with ANXA1 negative tumor (p=0.008). To explore the mechanism of worse survival of TNBC patients with ANXA1 high expression, GSEA was conducted between ANXA1 high and low expression group. GSEA demonstrated that high expression of ANXA1 group enriched not only EMT related genes (NES=1.916, p=0.004), but also IL2/STAT5 (NES=2.04, p<0.001) and TNF alpha signaling (NES=2.02, p<0.001) related genes as well.

Conclusion: High expression of ANXA1 in TNBC patients associated with worse OS. It may be able to be explained by its metastatic potential with up-regulated EMT signaling and aggressive characteristics with up-regulated TNF alpha and IL2/STAT5 signaling.

 

79.05 Breast Tumors that Express CCL5 and CXCL10 Attract CTLs and are Associated with Improved Survival

Posted on January 18, 2018

E. Katsuta1, L. Yan2, P. Kalinski3, K. Takabe1  1Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NY, USA 3Roswell Park Cancer Institute,Department Of Medicine,Buffalo, NY, USA

Introduction:  Cytotoxic T-lymphocytes (CTLs) infiltration into tumor of has been shown to predict better prognosis in breast cancer. Triple negative breast cancer (TNBC) has been shown to have higher immunogenicity and to show enhanced CTL attraction. However, the mechanism of CTL attraction to breast cancer remains elusive. CD8A is a surface marker of CTLs and Granzyme B (GZMB) is a serine protease, secreted by CTLs to mediate apoptosis of the target cells. Chemokines such as CCL5 and CXCL10 are known to attract CTLs into other types of cancer. However, the expression of CCL5 and CXLC10 and their role in the CTL attraction to breast cancer remain unknown. Therefore, we hypothesized that breast cancers that express CCL5 and CXLC10 attract CTLs and have better survival.

Methods:  RNA-seq and clinical data were obtained from the Cancer Genome Atlas (TCGA). CD8A and GZMB expression was analyzed as CTL markers. Overall survival (OS) was analyzed based upon gene expression of RNA-seq data. The cutoff value was determined by automated scanning and selecting the threshold yielding the lowest p-value.

Results: We observed that high expression of CTL markers showed significantly better OS; 5-year OS rates of CD8A high and low expression: 83.5% vs 74.7%, p<0.001, 5-year OS rates of GZMB high and low expression: 82.2% vs 66.0%, p<0.001. High expression of chemokines was also associated with better OS; 5-year OS rates in CCL5 high vs low cohorts was 82.2% vs 72.1%, p<0.001, 5-year OS rates in CXCL10 high vs low cohorts: 95.3% vs 80.8%, p=0.020. We found that CCL5 expression highly correlated with CD8A (R=0.793, p<0.001) as well as GZMB expression (R=0.756, p<0.001), while CXCL10 expression showed a weaker, although highly-significant correlation with CD8A (R=0.340, p<0.001) and GZMB (R=0.495, p<0.001) expression, indicating the roles of these chemokines in CTL attraction. These results support our notion, thus we further hypothesized that tumor that express high CCL5 and CXCL10 as well as CD8A and GZMB should have best survival. Tumors with high expression of CD8A, GZMB, CCL5 and CXCL10 were classified as high CTL infiltrating tumor (high-CTL), low expression of all of them as low-CTL, and others as middle-CTL. High-CTL group showed significantly better prognosis than other two groups, and middle-CTL group showed better prognosis than low-CTL group, with 89.3%, 81.2% and 61.8% of 5-year OS rates, respectively (high vs middle: p=0.016, middle vs low: p<0.001). There was greater proportion of high-CTL tumor in TNBC compare to non-TNBC patients (24.3% vs 4.4%, p<0.001). Mutation count was significantly higher in high-CTL tumors compare to other two groups, as well as mid-CTL vs low-CTL (Mutation count: 74, 63, 30, respectively, p<0.001).

Conclusion: Breast tumors with high mutation count and TNBC tumors highly express CCL5 and CXCL10, and attract CTLs, that result in better prognosis and may predict their responsiveness to immunotherapies. 

79.04 Breast Cancer Cell Metabolism is Regulated by Sphingosine Kinases

Posted on January 18, 2018

M. Nagahashi1, M. Nakajima1, M. Abe2, T. Saito3, M. Komatsu3, T. Soga4, J. Tsuchida1, K. Yuza1, K. Takabe5,6, K. Sakimura2, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Brain Research Institute, Niigata University,Department Of Cellular Neurobiology,Niigata City, NIIGATA, Japan 3Niigata University Graduate School Of Medical And Dental Sciences,Department Of Biochemistry,Niigata City, NIIGATA, Japan 4Keio University,Institute For Advanced Biosciences,Tsuruoka City, YAMAGATA, Japan 5Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 6University At Buffalo Jacobs School Of Medicine And Biomedical Sciences, The State University Of New York,Department Of Surgery,Buffalo, NY, USA

Introduction:
Cancer cells reprogram their metabolism to promote proliferation, survival, and long-term maintenance. The common feature of this altered metabolism is the increased glucose uptake and fermentation of glucose to lactate, which has been known as “Warburg effect”. However, the mechanism how cancer cells regulate their metabolism has not yet revealed. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that regulates many physiological and pathological processes. S1P exerts its function either intracellularly or extracellularly after produced by sphingosine kinases (SphK1 and SphK2) inside the cells. Previously our group and others have demonstrated that S1P and SphKs play important roles in cancer cell survival. We have recently published that expression of SphK1 associates with worse prognosis of breast cancer patients. Thus, we hypothesized that SphKs regulates cancer cell-specific metabolism, including “Warburg effect”.

Methods:
SphK1 or SphK2 knock-out (KO) E0771 murine breast cancer cell lines were generated with a CRISPR/Cas9 mediated targeted deletion of the SphK1 or SphK2 gene. Proliferation was assessed by WST-8. Metabolic changes in SphK1KO and SphK2KO E0771 cells were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) between SphK1/SphK2KO and their corresponding control E0771 cells.

Results:
Proliferation assays revealed significantly less cell proliferation of SphK1KO E0771 cells compared to the control cells. On the other hand, SphK2KO E0771 cells showed significantly more proliferation than the control cells. CE-TOFMS analysis revealed the metabolomics profiles of both SphK1KO and SphK2KO E0771 cells, which were dramatically changed in the glycolysis pathway and tricarboxylic acid (TCA) cycle compared to the control cells. Interestingly, SphK1KO E0771 cells contained lower amount of glutathione (GSH) than control cells, while SphK2KO E0771 cells contained significantly higher amount of GSH than control cells. Considering that GSH plays roles in oxidative stress and drug resistance, our findings indicate an important role of SphK1 and SphK2 in not only cell survival, but also oxidative stress and drug resistance.

Conclusion:
Our results indicate that SphKs play pivotal roles in cancer specific metabolism, which strengthen resistance to oxidative stress and cancer cell survival. SphKs will be a promising target for patients with breast cancer.
 

72.03 Clinical Significance of BRAF Non-V600E Mutations in Colorectal Cancer

Posted on January 18, 2018

Y. Shimada1, Y. Tajima1, M. Nagahashi1, H. Ichikawa1, M. Nakano1, H. Kameyama1, J. Sakata1, T. Kobayashi1, Y. Takii2, S. Okuda3, K. Takabe4,5, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, , Japan 2Niigata Cancer Center Hospital,Department Of Surgery,Niigata, , Japan 3Niigata University Graduate School Of Medical And Dental Sciences,Division Of Bioinformatics,Niigata, , Japan 4Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA 5University At Buffalo Jacobs School Of Medicine And Biomedical Sciences,Department Of Surgery,Buffalo, NY, USA

Introduction: Recent advances of comprehensive genomic sequencing (CGS) enables to detect not only BRAF V600E mutation but also BRAF non-V600E mutations in a single assay. While it has been proved that BRAF V600E mutation in colorectal cancer shows poor prognosis and poor response to anti-EGFR therapy, clinical significance of BRAF non-V600E mutation has not been fully investigated. The present work aimed to describe clinicopathological characteristics and clinical outcome of BRAF non-V600E mutant type compared with BRAF wild-type and BRAF V600E mutant-type.

Methods:  One-hundred-eleven Stage IV CRC patients were analyzed. We investigated genetic alterations using 415-gene panel, which includes BRAF V600E and non-V600E mutations. The differences of clinicopathological characteristics and genetic alterations were analyzed among BRAF wild-type, BRAF V600E mutant-type, and BRAF non-V600E mutant-type using Fisher’s exact test. Overall survival (OS) and Progression-free survival (PFS) in response to targeted therapies were analyzed among the 3 groups using log-rank test. 

Results: CGS revealed that 98 patients (88%), 7 patients (6%), and 6 patients (6%) were BRAF wild-type, BRAF V600E mutant-type, and BRAF non-V600E mutant-type, respectively. The variants of BRAF non-V600E in each 6 patients were as follows: G469A, G469A and V502I, D594G, I326V, N581Y, D594G. BRAF V600E mutant-type were more frequently right-sided, histopathological grade 3, mucinous type, and with multiple peritoneal metastases distant from primary lesion. BRAF non-V600E mutant-type were more frequently left-sided, non-mucinous type, and with bilateral multiple lung metastases. While BRAF V600E mutant-type showed significantly worse OS than BRAF wild-type and non-V600E mutant-type (P < 0.001 and P = 0.038, respectively), BRAF non-V600E mutant-type showed no significant difference compared with BRAF wild-type. Two of 6 patients with BRAF non-V600E mutation underwent R0 resection and showed no evidence of disease at final follow-up. In 47 patients with anti-EGFR therapy, while BRAF V600E mutant-type showed significantly worse PFS than BRAF wild-type (P = 0.013), BRAF non-V600E mutant-type showed no significant difference compared with BRAF wild-type. In 73 patients with anti-VEGF therapy, there was no significant difference on PFS among the 3 groups.

Conclusion: BRAF non-V600E mutant-type demonstrates different clinicopathological characteristics and clinical outcome from BRAF V600E mutant-type. Further preclinical and clinical investigations are needed to clarify the role of BRAF non-V600E mutation in colorectal cancer.

 

72.01 PTEN Mutation Is Associated With Worse Prognosis In Stage III Colorectal Cancer

Posted on January 18, 2018

Y. Tajima1, Y. Shimada1, M. Nagahashi1, H. Ichikawa1, H. Kameyama1, M. Nakano1, J. Sakata1, T. Kobayashi1, H. Nogami2, S. Maruyama2, Y. Takii2, S. Okuda3, K. Takabe4,5, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Niigata Cancer Center Hospital,Department Of Surgery,Niigata, NIIGATA, Japan 3Niigata University Graduate School Of Medical And Dental Sciences,Division Of Bioinformatics,Niigata, NIIGATA, Japan 4Roswell Park Cancer Institute,Breast Surgery,Buffalo, NEW YORK, USA 5The State University Of New York,Department Of Surgery, University At Buffalo Jacobs School Of Medicine And Biomedical Sciences,Buffalo, NEW YORK, USA

Introduction:
he PI3K/AKT/mTOR pathway is related with cell proliferation and frequently activated in many human cancers. On the other hand, PTEN is a tumor suppressor gene inhibiting PI3K-initiated signaling. Loss of PTEN can be occurred in various types of tumor and associated with progression and worse prognosis. However, the association between PTEN mutation and prognosis in colorectal cancer (CRC) remains unclear. Our aim was to analyze the clinical impact of PTEN mutation in patients with colorectal cancer.

Methods:
Two-hundred-one Stage I–IV CRC patients who underwent colorectal resection were analyzed. We investigated genetic alterations associated with CRC using 415-gene panel. The association between PTEN mutation status and clinicopathological characteristics was analyzed using Fisher’s exact test. The association between PTEN mutation status and relapse-free survival (RFS) was analyzed using log-rank test and Cox proportional hazards model.

Results:
Fifty-five (27%) of 201 patients had PTEN mutation. Tumor diameter < 50 mm, lymphatic invasion, venous invasion, distant metastasis, poorly differentiated cluster grade 2/3 and Ki67 < 60 % were significantly associated with PTEN mutation (P < 0.001, P = 0.036, P = 0.003, P = 0.002, P = 0.009 and P = 0.003, respectively). Univariate analysis showed that PTEN mutation was significantly associated with the worse RFS in patients with Stage III CRC (P = 0.002) (Fig.1). On the other hand, PTEN mutation was not significantly associated with the RFS in patients with Stage I/II CRC and the overall survival in patients with Stage IV CRC (Fig.1). Of 415 genes, 18 genes had mutations in over 10% of patients with Stage III CRC. Those 18 genes were ACVR2A (10.9%), APC (71.7%), BRAF (10.9%), BRCA2 (10.9%), CDH1 (10.9%), CIC (10.9%), ERBB2 (13.0%), FAT1 (10.9%), FBXW7 (23.9%), KRAS (41.3%), PIK3CA (15.2%), PTEN (15.2%), RNF43 (19.6%), SMAD2 (10.9%), SMAD4 (21.7%), SPEN (13.0%), STK11 (15.2%), TP53 (78.3%). Among the 18 genes, PTEN mutation was significantly associated with CIC mutation (P = 0.020). Univariate analysis in patients with Stage III CRC showed that the RFS was significantly worse in mucinous type, CIC mutation, ERBB2 mutation and PTEN mutation (P = 0.005, P = 0.009, P = 0.037, P = 0.002, respectively). Multivariate analysis in patients with Stage III showed that only PTEN mutation was significantly affected the RFS (hazard ratio 6.18, 95% confidence interval 1.63–23.5, P = 0.007).

Conclusion:
PTEN mutation was associated with worse prognosis in Stage III CRC. We speculate that PTEN is one of potential driver genes in CRC.
 

6.02 Comparison of Outcomes of Minimally Invasive and Open Pneumonectomy

Posted on January 18, 2018

A. Kumar1, H. Devishetty1, T. Demmy1, S. Yendamuri1  1Roswell Park Cancer Institute,Department Of Thoracic Surgery,Buffalo, NY, USA

Introduction:
Over the last decade minimally invasive surgery has been increasingly liberally utilized for the treatment of non-small cell lung cancer (NSCLC). This approach is increasingly used for the conduct of pneumonectomies. We sought to examine short and long term outcomes of the use of minimally invasive surgery for pneumonectomy in the United States.

Methods:
The National Cancer Database was queried for patients undergoing pneumonectomy between 2004 and 2014 and trends examined. As coding for surgical approach is only available from 2010 onwards, this subset was examined for the impact of surgical approach on short and long term outcomes of minimally invasive pneumonectomy. Univariate and multivariate analyses were performed to examine the impact of surgical approach using SPSS.

Results:
18,926 patients had a pneumonectomy between 2004 and 2014. The proportion of patients having a pneumonectomy steadily declined from 9.1% to 4.9% of all anatomic resections over this time period. From 2010 to 2014, 7407 pneumonectomies were performed. 2.0% and 13.5% of these cases were attempted to be performed robot-assisted and by VATS respectively and 1.4% and 8.5% were completed as intended. Intent to treat analysis did not show a difference between minimally invasive (MIS) and open approaches with respect to 30 day and 90 day mortality (6.7% vs 7.0%; P=0.90 and 11.7 vs 12.4%; P=0.51 respectively). There were no differences in age, gender, race, stage, laterality, length of stay and readmission rates between both approaches. Of note, there was no difference in the number of lymph nodes examined between both approaches (21.4 vs 21.9; P=0.56). However, overall survival was higher in patients undergoing MIS pneumonectomy vs. open pneumonectomy in univariable (57 months vs. 40 months; P=0.013) and multivariable analyses (Figure 1).

Conclusion:
Minimally invasive pneumonectomy appears safe with peri-operative outcomes comparable to open surgery.  MIS may improve long term pneumonectomy outcomes but confirmation by controlled studies is needed to reduce biases like high conversion and declining utilization rates observed in our cohort.
 

5.07 Recurrence in Patients Who Achieved Pathological Complete Response by Neoadjuvant Chemotherapy

Posted on January 18, 2018

M. Asaoka1, K. Narui3, A. Yamada3, N. Suganuma4, T. Chishima5, K. Takabe2, T. Ishikawa1  1Tokyo Medical University Hospital,Department Of Breast Cancer,Tokyo, TOKYO, Japan 2Roswell Park Cancer Institute,Buffalo, NY, USA 3Yokohama City University Hospital,Department Of Breast Cancer,Yokohama, KANAGAWA, Japan 4Kanagawa Cancer Center,Department Of Mammary Gland Endocrine Surgery,Yokohama, KANAGAWA, Japan 5Yokohama Rosai Hospital,Department Of Oncology,Yokohama, KANAGAWA, Japan

Introduction:

Recent studies have indicated that patients who achieved a pathological complete response (pCR) by neoadjuvant chemotherapy (NAC) have better long-term outcomes than those who did not. Recently, the pCR rate is approaching to 50% particularly in patients with hormone-receptor negative disease. If the disease is not recurred locally in cases with pCR and pCR could be accurately diagnosed preoperatively, it may be possible to treat some population of patients without surgery after chemotherapy. We analyzed the outcomes of patients who had achieved pCR by NAC with a special attention to local recurrence and risk factors of recurrence.

Methods:

We investigated disease free survival in 395 patients who were identified as having a pCR from 1599 patients with primary operable breast cancer treated by NAC in 4 institutions (pCR rate of 24.7%; 395/1599). As for subtypes in 395 cases, pCR cases were 50 in Luminal type (pCR rate of 7.2%), 98 in Luminal-HER2 type (32.1%), 116 patients in HER2 type (52.5%), and 131 in triple negative (TN) type (34.2%). 

Results:

The median follow-up was 41 months. Recurrent diseases including local recurrence or distant metastasis was found in 5.80% (23/395). According to subtypes, these were 2.00% (1/50) in Luminal type, 4.08% (4/98) in Luminal-HER2 type, 10.3% (12/116) in HER2 type, and 4.58% (6/131) in TN type. Local recurrence was found in 1.2% of all cases (5/395). It was prominent that brain metastasis was frequently observed in HER2 type (12/116). Clinical stage before NAC and nodal status after NAC were found as a risk factor of recurrence in the univariate analysis, and only clinical stage remained statistically significant in the multivariate analysis.

Conclusion:

Except HER2 type, recurrence was not frequent in cases obtained pCR, particularly in cases with an early clinical stage. Local recurrence was rarely observed in any subtype.  Based on this result, we think that it is possible to omit surgery in patients highly expected pCR. We have already conducted a multicenter feasibility study to treat without surgery. For cases diagnosed as clinical complete response after NAC by contrast-enhanced magnetic resonance imaging, ultrasound-guided core needle biopsy (CNB) is performed before starting the surgery. The concordance of pathological results between CNB and surgical specimen is examined. The enrollment was completed lately.

 

5.05 Expression of miR-9 Predicts Breast Cancer Survival

Posted on January 18, 2018

J. C. Sporn1, E. Katsuta1, L. Yan2, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Biostatistics & Bioinformatics,Buffalo, NY, USA

Introduction:

miRNAs are a diverse family of RNA molecules. They are typically about 18 to 24 nucleotides in length and regulate translation and stability of partially complementary target mRNAs. It has been shown that miRNA expression is severely dysregulated in cancer cells and that altered expression of certain miRNAs can promote tumorigenesis. Some miRNAs were found to be overexpressed in cancer cells and shown to promote proliferation in vitro and tumorigenesis in vivo, other miRNAs may be downregulated in cancer cells subsequently failing to inhibit the expression of oncogenic protein and thus promoting tumorigenesis. miR-9 was shown to increase breast cancer cell motility and invasiveness in vitro and to promote metastasis formation in mice by inhibition of E-cadherin expression. Interestingly, miR-9 was also linked to downregulation of two sirtuins, SIRT1 and SIRT3. Sirtuins (of which there are seven in humans) are NAD+-dependent enzymes involved in a variety of cellular pathways including stress-response and chromatin-silencing. SIRT1 is downregulated in BRCA1-associated breast cancer compared with normal controls and inhibition of BRCA1-mutant tumor growth by the anticancer agent resveratrol upregulated SIRT1 activity.

Methods:

We utilized the TCGA dataset to analyze the expression of miR-9-5p in human breast cancer patients. To link the expression levels with the provided TCGA survival data, we used the OncoLnc platform. 988 patients were divided at the 50th percentile into two equal groups of 494 according to their expression levels (low expression versus high expression). Cox regression was performed and a Kaplan plot was calculated. We further compared the expression levels of SIRT1 to SIRT7 between the groups with low and high expression of miR-9-5p.

Results:

The miR-9-5p expression ranged from 22.98 to 562.5 in the ‘low’ and from 567.51 to 164644.95 in the ‘high’ expression group. Survival analysis showed that high expression of miR-9-5p was associated with worse prognosis (p-value= 0.00801). This is in line with previous in vitro findings linking increased miR-9 expression with invasiveness and metastatic potential in breast cancer. While there was a trend towards lower expression of SIRT1 in the group with high expression of miR-9-5p, it did not reach statistical significance. However, there was a statistically significant decrease in SIRT3 levels in the group with high expression of miR-9-5p (p<0.001).

Conclusion:

Our analysis supports an important link between miR-9 and sirtuins and identifies miR9-5p as a novel biomarker to predict survival in breast cancer patient.

5.03 Bone Morphogenetic Protein Expression Significantly Affects Breast Cancer Prognosis

Posted on January 18, 2018

A. A. MAAWY1, E. Katsuta1, L. Yan2, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Biostatistics And Bioinformatics,Buffalo, NY, USA

Introduction:
Bone morphogenetic proteins are members of the TGFβ family of signaling pathways and are known to be essential in fetal development, tissue differentiation and a multitude of cellular functions. Differential expression noted in some forms of breast cancer and is known to regulate the epithelial to mesenchymal transition, tissue infiltration and metastasis. Under-expression of BMP7 is known to be associated with a more metastatic phenotype and development of bone metastases. Studies in these patients have revealed aberrations of both BMP expression and signaling, which correlate clinically with disease progression. Identifying these tumor subsets can act as prognostic markers and may prove to be a therapeutic target with implications on disease progression and overall prognosis. This study investigates the association of BMP gene expression with breast cancer survival using a ‘big data’ approach employing RNA sequencing from the Cancer Genome Atlas (TCGA).

Methods:
A total of 1096 patient with breast cancer had treatment naïve samples of the tumors undergo genetic sequencing and the results of their sequencing stored in The Cancer Genome Atlas (TCGA) dataset. Overall survival (OS) was compared between high and low expression of indicated BMP related genes; BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15 and BMP receptors 1A and 1B, based upon RNA-sequencing data of TCGA.

Results:

The TCGA cohort was representative of national breast cancer patients with respect to stage, pathology and survival. Using Kaplan Meier analysis, BMP1 (p<0.001), BMP3 (p=0.002), BMP5 (p=0.02), BMP6 (p<0.001), BMP7 (p<0.001), BMP8a (p=0.054), BMP8b (p=0.001), BMPR1A (<0.001)and BMPR1B (p=0.005) all significantly impacted OS. BMP2, BMP4, BMP10 and BMP15 did not significantly impact OS. High expression of BMP1, BMP2, BMP3, BMP4, BMP5, BMP7 and BMPR1A was protective and was associated with improved OS. Conversely, high expression of BMP6, BMP8a, BMP8b and BMPR1B was associated with poorer outcomes and worse OS.

Conclusion:

BMP expression profiles may be of value in prognostication. Evidence suggests that BMPs play a role in breast tumorigenesis, function and progression by modulating the cell cycle, interactions with and remodeling of the extracellular matrix, interactions with the bone metastatic microenvironment and the epithelial to mesenchymal transition. Intervention in this pathway may serve to improve outcomes, manage metastatic disease and assist in clinical decision making on optimal therapy based on risk of recurrence or metastasis.

40.18 Dysregulation of Sphingolipids in Human Hepatocellular Carcinoma

Posted on January 18, 2018

K. Miura1, M. Nagahashi1, Y. Hirose1, T. Kobayashi1, J. Sakata1, H. Kameyama1, Y. Shimada1, H. Ichikawa1, K. Takabe2,3, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 3University At Buffalo Jacobs School Of Medicine And Biomedical Sciences, The State University Of New York,Department Of Surgery,Buffalo, NY, USA

Introduction:  Hepatocellular carcinoma (HCC) is now the third leading cause of cancer deaths worldwide, with over 500,000 people affected. Sphingolipids including sphingosine-1-phosphate (S1P) and ceramide have emerged as key regulatory molecules, controlling various aspects of cancer biology and implicated in the mechanism of action of cancer chemotherapeutics. S1P is known to play important roles in cancer cell survival and progression. We previously demonstrated that S1P is a crucial mediator of cancer-induced angiogenesis and lymphangiogenesis and promote metastasis (Cancer Research 2012). On the other hand, ceramide has been considered to mediate anti-proliferative responses, such as cell growth inhibition, apoptosis induction, senescence modulation and autophagy. Ceramide can be de-acylated to give sphingosine, and sphingosine in turn can be phosphorylated to produce S1P. The dynamic balance between S1P and sphingosine/ceramide is referred to as the “sphingolipid rheostat” and influences cancer cell fate. Although important roles of sphingolipids in cancer progression has been revealed in experimental models, its roles in human HCC patients are yet to be determined. In this study, we measure the levels of sphingolipids including S1P and ceramides in tumor and normal liver tissue by state-of-the-art mass spectrometry.

Methods:  Tumor and normal liver tissue were obtained from 20 patients with HCC. Sphingolipids were measured by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Wilcoxon matched-pairs signed rank test was performed to compare the levels of each sphingolipid between tumor and normal tissue, and P<0.05 was considered as statistically significant.

Results: The levels of sphingolipids including sphingosine, dihydro-sphingosine, S1P, dihydro-S1P, and ceramides were detected successfully in the tumor and normal liver tissue from 20 HCC patients. The levels of S1P and dihydro-S1P in tumor tissue were significantly higher than those in normal liver tissue with almost four-fold increase (P<0.0001 and P<0.0001). Further, sphingosine and dihydro-sphingosine in tumor tissue were also significantly higher than those in normal tissue (P<0.001 and P<0.01). Finally, the levels of each ceramide species (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:0) in tumor tissue were significantly higher than those in normal tissue (P<0.0001 for each species).

Conclusion: Our results indicate important role of sphingolipids in HCC. Further study will be required to investigate the distinct roles of each sphingolipid in human patients. 

 

40.17 Human Biliary Tract Cancer Contains High Levels of S1P

Posted on January 18, 2018

Y. Hirose1, M. Nagahashi1, K. Yuza1, K. Miura1, J. Sakata1, T. Kobayashi1, H. Ichikawa1, Y. Shimada1, H. Kameyama1, K. Takabe2,3, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 3University At Buffalo Jacobs School Of Medicine And Biomedical Sciences, The State University Of New York,Department Of Surgery,Buffalo, NY, USA

Introduction: Biliary tract cancer, including gallbladder cancer, intra- and extrahepatic bile duct cancer, is one of the most lethal diseases among gastrointestinal cancer. Previous studies have suggested that chronic inflammation is involved in the disease process of biliary tract cancer. Sphingosine-1-phosphate (S1P) has emerged as a pleiotropic sphingolipid mediator that regulates many cellular functions associated with inflammation and cancer. Indeed, it has been previously suggested that S1P plays important roles in bile duct cancer progression in experimental model. However, the roles of S1P in human biliary tract cancer has yet to be clarified. The aim of this study is to determine the levels of sphingolipids including S1P and their metabolites in biliary tract cancer tissues and normal biliary tract tissues, and to clarify the difference in the levels of each sphingolipid between the two tissues.

Methods: We examined biliary tract cancer tissues (gallbladder cancer, N=5; intrahepatic bile duct cancer, N=2; and extrahepatic bile duct cancer, N=8) and normal biliary tract tissues (normal gallbladder mucosa, N=5; normal extrahepatic bile duct mucosa, N=12) in patients with biliary tract cancer. Sphingolipids including S1P and their metabolites of dihydro-S1P (DHS1P), sphingosine (Sph), and dihydro-Sph (DHSph) were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The levels of each sphingolipid were compared between cancer tissues and normal tissues using the Mann-Whitney U test. All tests were two-sided and P<0.05 were considered statistically significant.

Results:In the comparison between all biliary tract cancer tissues (N=15) and all normal biliary tract tissues (N=17), the levels of S1P, DHS1P, Sph, and DHSph were significantly higher in cancer tissues than normal tissues (S1P, P=0.004; DHS1P, P=0.030; Sph, P=0.011; DHSph, P<0.001). In the comparison between intra- and extrahepatic bile duct cancer tissues (N=10) and normal bile duct mucosa (N=12), the levels of S1P, DHS1P, Sph, and DHSph were significantly higher in cancer tissues than normal tissues (S1P, P=0.004; DHS1P, P=0.030; Sph, P=0.011; DHSph, P<0.001). In the comparison between gallbladder cancer tissues (N=5) and normal gallbladder mucosa (N=5), the levels of DHSph were significantly higher in cancer tissues than normal tissues (P=0.016), but there were no significant difference in the levels of S1P, DHS1P and Sph.

Conclusion:To our knowledge, this is the first study to reveal the levels of sphingolipids including S1P in human biliary tract cancer patients by mass spectrometry. The high levels of sphingolipids in the cancer tissue may indicate the important roles of S1P in disease process of biliary tract cancer in human patients.

 

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