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Ordinal logistic regression analysis to assess the association of tumor PDCD1LG2 (PD-L2) expression (predictor) with the overall lymphocytic reaction score (outcome)

Supplementary Figure S2. Distribution of carbohydrates, xenobiotics, cofactors and vitamins, and energy in tumor and normal. (A-D): Heatmap of 8 carbohydrates (A), 7 xenobiotics (B), 7 cofactors and vitamins (C), and 7 energy metabolites (D) sorted by sample types and then tissue types. Heat-map colors represent z-score values of raw detection amount. FFPE, formalin-fixed paraffin-embedded; N, normal; T, tumor.

The presence of Fusobacterium nucleatum (F. nucleatum) in colorectal carcinoma tissue has been associated with microsatellite instability (MSI), lower-level T-cell infiltrates, and poor clinical outcomes. Considering differences in the tumor–immune microenvironment between MSI-high and non–MSI-high carcinomas, we hypothesized that the association of F. nucleatum with immune response might differ by tumor MSI status. Using samples from 1,041 rectal and colon cancer patients within the Nurses’ Health Study and Health Professionals Follow-up Study, we measured F. nucleatum DNA in tumor tissue by a quantitative polymerase chain reaction assay. Multivariable logistic regression models were used to examine the association between F. nucleatum status and histopathologic lymphocytic reactions or density of CD3+ cells, CD8+ cells, CD45RO (PTPRC)+ cells, or FOXP3+ cells in strata of tumor MSI status. We adjusted for potential confounders, including CpG island methylator phenotype; LINE-1 methylation; and KRAS, BRAF, and PIK3CA mutations. The association of F. nucleatum with tumor-infiltrating lymphocytes (TIL) and intratumoral periglandular reaction differed by tumor MSI status (Pinteraction = 0.002). The presence of F. nucleatum was negatively associated with TIL in MSI-high tumors [multivariable odds ratio (OR), 0.45; 95% confidence interval (CI), 0.22–0.92], but positively associated with TIL in non–MSI-high tumors (multivariable OR 1.91; 95% CI, 1.12–3.25). No significant differential association was observed for peritumoral lymphocytic reaction, Crohn-like lymphoid reaction, or T-cell densities. In conclusion, the association of F. nucleatum with immune response to colorectal carcinoma differs by tumor MSI status, suggesting that F. nucleatum and MSI status interact to affect antitumor immune reactions. Cancer Immunol Res; 6(11); 1327–36. ©2018 AACR.See related Spotlight on p. 1290.

Supplementary Figure S1. Flow diagram of the study population in the Nurses'' Health Study and the Health Professionals Follow-Up Study. Supplementary Figure S2. Immunohistochemistry of a colorectal carcinoma case using anti-PDCD1LG2 (PD-L2) antibody (A) and its isotype control (B). Supplementary Figure S3. Kaplan-Meier curves for colorectal cancer-specific mortality (A) and overall mortality (B) according to tumor PDCD1LG2 (PD-L2) expression.

Experimental evidence suggests that the let-7 family of noncoding RNAs suppresses adaptive immune responses, contributing to immune evasion by the tumor. We hypothesized that the amount of let-7a and let-7b expression in colorectal carcinoma might be associated with limited T-lymphocyte infiltrates in the tumor microenvironment and worse clinical outcome. Utilizing the molecular pathological epidemiology resources of 795 rectal and colon cancers in two U.S.-nationwide prospective cohort studies, we measured tumor-associated let-7a and let-7b expression levels by quantitative reverse-transcription PCR, and CD3+, CD8+, CD45RO (PTPRC)+, and FOXP3+ cell densities by tumor tissue microarray immunohistochemistry and computer-assisted image analysis. Logistic regression analysis and Cox proportional hazards regression were used to assess associations of let-7a (and let-7b) expression (quartile predictor variables) with T-cell densities (binary outcome variables) and mortality, respectively, controlling for tumor molecular features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Compared with cases in the lowest quartile of let-7a expression, those in the highest quartile were associated with lower densities of CD3+ [multivariate odds ratio (OR), 0.40; 95% confidence interval (CI), 0.23–0.67; Ptrend = 0.003] and CD45RO+ cells (multivariate OR, 0.31; 95% CI, 0.17–0.58; Ptrend = 0.0004), and higher colorectal cancer-specific mortality (multivariate hazard ratio, 1.82; 95% CI, 1.42–3.13; Ptrend = 0.001). In contrast, let-7b expression was not significantly associated with T-cell density or colorectal cancer prognosis. Our data support the role of let-7a in suppressing antitumor immunity in colorectal cancer and suggest let-7a as a potential target of immunotherapy. Cancer Immunol Res; 4(11); 927–35. ©2016 AACR.

Supplementary Figure Legends

Figure S1. Quantitative reverse-transcription PCR assays for let-7a, let-7b, and RNU6-2 using 10-fold dilution series from the same specimen. Figure S2. let-7a and let-7b expression in colorectal cancer. Table S1. Inter-assay coefficients of variation in quantitative reverse-transcription PCR assays for let-7a, let-7b and RNU6-2. Table S2. Clinical, pathological, and molecular characteristics according to tumor let-7b expression in 792 colorectal cancer cases. Table S3. Association of tumor let-7a and let-7b expression with disease stage. Table S4. Spearman's rank-correlation coefficients between the densities of T-cells. Table S5. Tumor let-7a expression and patient mortality in the 518 cases with CD45RO+ T-cell density data.

Expression of the immune checkpoint ligand CD274 (programmed cell death 1 ligand 1, PD-L1, from gene CD274) contributes to suppression of antitumor T cell–mediated immune response in various tumor types. However, the role of PDCD1LG2 (PD-L2, CD273, from gene PDCD1LG2) in the tumor microenvironment remains unclear. We hypothesized that tumor PDCD1LG2 expression might be inversely associated with lymphocytic reactions to colorectal cancer. We examined tumor PDCD1LG2 expression by IHC in 823 colon and rectal carcinoma cases within two U.S.-nationwide cohort studies and categorized tumors into quartiles according to the percentage of PDCD1LG2–expressing carcinoma cells. We conducted multivariable ordinal logistic regression analysis to assess the associations of tumor PDCD1LG2 expression with Crohn-like lymphoid reaction, peritumoral lymphocytic reaction, intratumoral periglandular reaction, or tumor-infiltrating lymphocytes, controlling for potential confounders, including microsatellite instability, CpG island methylator phenotype, long-interspersed nucleotide element-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Tumor PDCD1LG2 expression was inversely associated with Crohn-like lymphoid reaction (Ptrend = 0.0003). For a unit increase in the three-tiered ordinal categories of Crohn-like lymphoid reaction, a multivariable OR in the highest (vs. lowest) quartile of the percentage of PDCD1LG2–expressing tumor cells was 0.38 (95% confidence interval, 0.22–0.67). Tumor PDCD1LG2 expression was not associated with peritumoral lymphocytic reaction, intratumoral periglandular reaction, tumor-infiltrating lymphocytes, or patient survival (Ptrend > 0.13). Thus, tumor PDCD1LG2 expression is inversely associated with Crohn-like lymphoid reaction to colorectal cancer, suggesting a possible role of PDCD1LG2-expressing tumor cells in inhibiting the development of tertiary lymphoid tissues during colorectal carcinogenesis. Cancer Immunol Res; 5(11); 1046–55. ©2017 AACR.

Assessment of aspirin use and statistical analysis to assess an interaction of tumor MIR21 expression level and regular aspirin use after diagnosis in survival analysis

Purpose: Prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2; a target of aspirin) produces inflammatory mediator prostaglandin E2 (PGE2), and contributes to colorectal neoplasia development. PTGS2-driven inflammatory responses can induce tumor expression of microRNA MIR21 (miR-21) that can increase local PGE2 level by downregulating PGE2-metabolizing enzymes. We hypothesized that the prognostic association of tumor MIR21 expression level in colorectal carcinoma might depend on inflammatory tumor microenvironment and be stronger in tumors expressing high-level PTGS2.Experimental Design: Utilizing 765 rectal and colon cancer specimens in the Nurses' Health Study and the Health Professionals Follow-up Study, we measured MIR21 expression by quantitative reverse transcription PCR, and PTGS2 expression by immunohistochemistry. Cox proportional hazards regression model was used to assess statistical interaction between MIR21 and PTGS2 in colorectal cancer–specific survival analysis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutations.Results: Tumor MIR21 expression level was associated with higher colorectal cancer–specific mortality (Ptrend = 0.029), and there was a statistically significant interaction between MIR21 and PTGS2 (Pinteraction = 0.0004). The association between MIR21 expression and colorectal cancer–specific mortality was statistically significant in PTGS2-high cancers (multivariable hazard ratio of the highest vs. lowest quartile of MIR21, 2.28; 95% confidence interval, 1.42–3.67; Ptrend = 0.0004) but not in PTGS2-absent/low cancers (Ptrend = 0.22).Conclusions:MIR21 expression level in colorectal carcinoma is associated with worse clinical outcome, and this association is stronger in carcinomas expressing high-level PTGS2, suggesting complex roles of immunity and inflammation in tumor progression. Clin Cancer Res; 22(15); 3841–8. ©2016 AACR.

Regular aspirin use after diagnosis and colorectal cancer mortality according to tumor MIR21 expression level among 579 patients with stage I to III colorectal cancer

Up to 70% of patients with late-stage breast cancer have bone metastasis. Current treatment regimens for breast cancer bone metastasis are palliative with no therapeutic cure. Disseminated tumor cells (DTCs) colonize inside the osteogenic niches in the early stage of bone metastasis. Drug delivery into osteogenic niches to inhibit DTC colonization can prevent bone metastasis from entering its late stage and therefore cure bone metastasis. Here, we constructed a 50% DSS6 peptide conjugated nanoparticle to target the osteogenic niche. The osteogenic niche was always located at the endosteum with immature hydroxyapatite. Arsenic-manganese nanocrystals (around 14 nm) were loaded in osteogenic niche-targeted PEG-PLGA nanoparticles with an acidic environment-triggered arsenic release. Arsenic formulations greatly reduced 4T1 cell adhesion to mesenchymal stem cells (MSCs)/preosteoblasts (pre-OBs) and osteogenic differentiation of osteoblastic cells. Arsenic formulations also prevented tumor cell colonization and dormancy via altering the direct interaction between 4T1 cells and MSCs/pre-OBs. The chemotactic migration of 4T1 cells toward osteogenic cells was blocked by arsenic in mimic 3D osteogenic niche. Systemic administration of osteogenic niche-targeted arsenic nanoparticles significantly extended the survival of mice with 4T1 syngeneic bone metastasis. Our findings provide an effective approach for osteogenic niche-specific drug delivery and suggest that bone metastasis can be effectively inhibited by blockage of tumor cell colonization in the bone microenvironment., Graphical abstract Osteogenic niche-targeted nanoparticles selectively delivered arsenic trioxide into the pre-metastatic niche. Locally released arsenic effectively inhibited breast cancer cell colonization and diminished the number of pre-metastatic niches in the bone.Image 1

Background: We hypothesized that the risk of colorectal cancer in night-shift workers might be different according to insulin receptor substrate status. Methods: Among 77,470 eligible women having night work assessed in the Nurses' Health Study, we documented a total of 1,397 colorectal cancer cases, of which 304 or 308 had available data on IRS1 and IRS2, respectively. We used duplication-method Cox proportional hazards regression analysis for competing risks to calculate HRs and 95% confidence intervals (CI) for each colorectal cancer subtype. We measured tumor IRS1 or IRS2 expression by immunohistochemistry (IHC). Results: Compared with women who never worked night shifts, those working ≥15 years night shifts had a marginal trend of increased overall risk of colorectal cancer (Ptrend = 0.06; multivariable HR = 1.20; 95% CI, 0.99–1.45). Longer duration of night-shift work was associated with a higher risk of IRS2-positive tumors (multivariable HR = 2.69; 95% CI, 1.48–4.89; Ptrend = 0.001, ≥15 years night shifts vs. never) but not with IRS2-negative tumors (multivariable HR = 0.90; 95% CI, 0.54–1.51; Ptrend = 0.72; Pheterogeneity for IRS2 = 0.008). Similarly, the corresponding multivariable HRs were 1.81 for IRS1-positive tumors (95% CI, 0.94–3.48; Ptrend = 0.06) and 1.13 for IRS1-negative tumors (95% CI, 0.71–1.80; Ptrend = 0.56; Pheterogeneity for IRS1 = 0.02). Conclusions: Our molecular pathologic epidemiology data suggest a potential role of IRS in mediating carcinogenesis induced by night-shift work. Impact: Although these findings need validation, rotating night shift might increase colorectal cancer risk in women with abnormal insulin receptor pathways.

Cinnamomum cassia (L.) Presl (cinnamon), an important folk medicine is widely used to prevent osteoporosis for long time in China. Our study aimed to investigate the anti-osteoporosis activity and mechanisms of cinnamon extracts obtained by supercritical CO2 extraction (SFE) and identify activity associated chemical components by gas chromatography-mass spectrometry. The cinnamon SFE exhibited superior anti-osteoporosis efficacy in an ovariectomised mice model to common alcohol extracts. It could induce calcified nodules and ALP activity, upregulate the mRNA expression of ALP, BMP-2, and RUNX2 in MC3T3-E1 cells. The major chemical classes of cinnamon extracts were alcohol esters (28.2%), and terpenes (16.1%). The spectrum-activity analysis indicated that the potential chemical-markers of extracts could be (E)-Cinnamaldehyde, γ-Sitosterol, and (Z, Z)-9,12-Octadecadienoic acid, which could induce the proliferation and ALP activity in MC3T3-E1 cells. Our study revealed the promising applications of the cinnamon SFE in prevention of osteoporosis, and identified its anti-osteoporosis associated compounds.

Multi-drug resistance (MDR) in breast cancer poses a great threat to chemotherapy. The expression and function of the ATP binding cassette (ABC) transporter are the major cause of MDR. Herein, a linear polyethylene glycol (PEI) conjugated with dicyandiamide, which called polymeric metformin (PolyMet), was successfully synthesized as a simple and biocompatible polymer of metformin. PolyMet showed the potential to reverse MDR by inhibiting the efflux of the substrate of ATP-binding cassette (ABC) transporter from DOX resistant MCF-7 cells (MCF-7/DOX). To test its MDR reversing effect, PolyMet was combined with DOX to treat mice carrying MCF-7/DOX xenografts. In order to decrease the toxicities of DOX and delivery PolyMet and DOX to tumor at the same time, PolyMet was complexed with poly-γ-glutamic acid-doxorubicin (PGA-DOX) electrostatically at the optimal ratio of 2:3, which were further coated with lipid membrane to form lipid/PolyMet-(PGA-DOX) nanoparticles (LPPD). The particle size of LPPD was 165.8 nm, and the zeta potential was +36.5 mV. LPPD exhibited favorable cytotoxicity and cellular uptake in MCF-7/DOX. Meanwhile, the bioluminescence imaging and immunohistochemical analysis indicated that LPPD effectively conquered DOX-associated MDR by blocking ABC transporters (ABCB1 and ABCC1) via PolyMet. Remarkably, LPPD significantly inhibited the tumor growth and lowered the systemic toxicity in a murine MCF-7/DOX tumor model. This is the first time to reveal that PolyMet can enhance the anti-tumor efficacy of DOX by dampening ABC transporters and activating the AMPK/mTOR pathway, which is a promising strategy for drug-resistant breast cancer therapy.

The purpose of the present study was to compare metabolites from formalin‑fixed and paraffin‑embedded (FFPE) pancreatic tissue blocks with those identified in optimal cutting temperature (OCT)‑embedded pancreatic tissue blocks. Thus, ultra‑performance liquid chromatograph‑mass spectrometry/mass spectrometry‑based metabolic profiling was performed in paired frozen (n=13) and FFPE (n=13) human pancreatic adenocarcinoma tissue samples, in addition to their benign counterparts. A total of 206 metabolites were identified in both OCT‑embedded and FFPE tissue samples. The method feasibility was confirmed through reproducibility and a consistency assessment. Partial least‑squares discriminant analysis and heatmap analysis reliably distinguished tumor and normal tissue phenotypes. The expression of 10 compounds, including N‑acetylaspartate and creatinine, was significantly different in both OCT‑embedded and FFPE tumor samples. These ten compounds may be viable candidate biomarkers of malignant pancreatic tissues. The super‑categories to which they belonged exhibited no significant differences between FFPE and OCT‑embedded samples. Furthermore, purine, arginine and proline, and pyrimidine metabolism used a shared pathway found in both OCT‑embedded and FFPE tissue samples. These results supported the notion that metabolomic data acquired from FFPE pancreatic cancer specimens are reliable for use in retrospective and clinical studies.

Background High-level plasma 25-hydroxyvitamin D [25(OH)D] has been associated with lower colorectal cancer incidence and mortality. Considering evidence indicating immunomodulatory effects of vitamin D, we hypothesised that survival benefits from high systemic vitamin D level might be stronger for colorectal carcinoma with lower immune response to tumour. Methods Using 869 colon and rectal cancer cases within the Nurses' Health Study and Health Professionals Follow-up Study, we assessed the prognostic association of postdiagnosis 25(OH)D score [derived from diet and lifestyle variables to predict plasma 25(OH)D level] in strata of levels of histopathologic lymphocytic reaction. The Cox proportional hazards regression model was adjusted for potential confounders, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, PTGS2 (cyclooxygenase-2) expression and KRAS, BRAF and PIK3CA mutations. Results The association of postdiagnosis 25(OH)D score with colorectal cancer-specific mortality differed by levels of peritumoural lymphocytic reaction (pinteraction = 0.001). Multivariable-adjusted mortality hazard ratios for a quintile-unit increase of 25(OH)D score were 0.69 [95% confidence interval (CI), 0.54–0.89] in cases with negative/low peritumoural lymphocytic reaction, 1.08 (95% CI, 0.93–1.26) in cases with intermediate peritumoural reaction and 1.25 (95% CI, 0.75–2.09) in cases with high peritumoural reaction. The survival association of the 25(OH)D score did not significantly differ by Crohn's-like lymphoid reaction, intratumoural periglandular reaction or tumour-infiltrating lymphocytes. Conclusions The association between the 25(OH)D score and colorectal cancer survival is stronger for carcinomas with lower peritumoural lymphocytic reaction. Our results suggesting interactive effects of vitamin D and immune response may contribute to personalised dietary and lifestyle intervention strategies.

Background Valtrate is a novel epoxy iridoid ester isolated from Chinese herbal medicine Valeriana jatamansi Jones with anti-proliferative activity against various human cancer cell lines. However, its efficacy and molecular mechanisms against pancreatic cancer (PC) cells are largely unclear. Purpose To investigate the anti-cancer effects of valtrate on PC cell lines and its underlying mechanisms. Methods MTT assay was first performed to detect the effect of valtrate on cell viability in human PC cell lines and normal pancreatic epithelial cells HPDE. Cell apoptosis and cycle phase assay were detected by flow cytometry. The relative mRNA expressions of Bax, Bcl-2, c-Myc, and CyclinB1 were tested by quantitative PCR (qPCR) assay. The expression of relative proteins was detected by Western blotting (WB). A PANC-1luc cells xenograft mouse model in nu/nu female mice was used to elucidate the effect of valtrate on tumor growth in vivo. Results Valtrate significantly inhibited the growth of PC cells without affecting the growth of normal pancreatic epithelial cells HPDE, induced significant apoptosis and cell cycle arrest in G2/M phase. Moreover, valtrate inhibited the tumor growth of PC cell PANC-1 in xenograft mice by 61%. Further mechanism study demonstrated that valtrate could increase the expression level of Bax, suppress Bcl-2 as well as c-Myc and Cyclin B1, inhibit the transcriptional activity of Stat3, while valtrate decreased the expression level of Stat3 and phosphated-Stat3 (Tyr705) and induced the high molecular aggregation of Stat3. Molecular docking analysis predicted that valtrate might interact with Cys712 of Stat3 protein. Valtrate could also induce a transient depleted intracellular glutathione (GSH) level and increased reactive oxygen species (ROS). NAC (N-acetylcysteine), a reducer reversed valtrate-induced the depletion of Stat3, p-Stat3, c-Myc, and Cyclin B1. Conclusion Valtrate exerts anti-cancer activity against PC cells by directly targeting Stat3 through a covalent linkage to inhibit Stat3 activity, which causes apoptosis and cell cycle arrest.

Accumulating evidence suggests that metabolic reprogramming has a critical role in carcinogenesis and tumor progression. The usefulness of formalin-fixed paraffin-embedded (FFPE) tissue material for metabolomics analysis as compared with fresh frozen tissue material remains unclear. LC/MS-MS–based metabolomics analysis was performed on 11 pairs of matched tumor and normal tissues in both FFPE and fresh frozen tissue materials from patients with colorectal carcinoma. Permutation t test was applied to identify metabolites with differential abundance between tumor and normal tissues. A total of 200 metabolites were detected in the FFPE samples and 536 in the fresh frozen samples. The preservation of metabolites in FFPE samples was diverse according to classes and chemical characteristics, ranging from 78% (energy) to 0% (peptides). Compared with the normal tissues, 34 (17%) and 174 (32%) metabolites were either accumulated or depleted in the tumor tissues derived from FFPE and fresh frozen samples, respectively. Among them, 15 metabolites were common in both FFPE and fresh frozen samples. Notably, branched chain amino acids were highly accumulated in tumor tissues. Using KEGG pathway analyses, glyoxylate and dicarboxylate metabolism, arginine and proline, glycerophospholipid, and glycine, serine, and threonine metabolism pathways distinguishing tumor from normal tissues were found in both FFPE and fresh frozen samples. This study demonstrates that informative data of metabolic profiles can be retrieved from FFPE tissue materials. Implications: Our findings suggest potential value of metabolic profiling using FFPE tumor tissues and may help to shape future translational studies through developing treatment strategies targeting metabolites.

Background Evidence indicates not only carcinogenic effect of cigarette smoking but also its immunosuppressive effect. We hypothesized that the association of smoking with colorectal cancer risk might be stronger for tumors with lower anti-tumor adaptive immune response. Methods During follow-up of 134 981 participants (3 490 851 person-years) in the Nurses' Health Study and Health Professionals Follow-up Study, we documented 729 rectal and colon cancer cases with available data on T-cell densities in tumor microenvironment. Using the duplication-method Cox regression model, we examined a differential association of smoking status with risk of colorectal carcinoma subclassified by densities of CD3+ cells, CD8+ cells, CD45RO (PTPRC)+ cells, or FOXP3+ cells. All statistical tests were two-sided. Results The association of smoking status with colorectal cancer risk differed by CD3+ cell density (Pheterogeneity = .007). Compared with never smokers, multivariable-adjusted hazard ratios for CD3+ cell-low colorectal cancer were 1.38 (95% confidence interval = 1.09 to 1.75) in former smokers and 1.59 (95% confidence interval = 1.14 to 2.23) in current smokers (Ptrend = .002, across smoking status categories). In contrast, smoking status was not associated with CD3+ cell-high cancer risk (Ptrend = .52). This differential association appeared consistent in strata of microsatellite instability, CpG island methylator phenotype, or BRAF mutation status. There was no statistically significant differential association according to densities of CD8+ cells, CD45RO+ cells, or FOXP3+ cells (Pheterogeneity > .04, with adjusted α of 0.01). Conclusions Colorectal cancer risk increased by smoking was stronger for tumors with lower T-lymphocyte response, suggesting an interplay of smoking and immunity in colorectal carcinogenesis.

OBJECTIVES Dysregulation of the cell cycle has been observed and implicated as an etiologic factor in a range of human malignancies, but remains relatively unstudied in neuroendocrine tumors (NETs). We evaluated expression of key proteins involved in cell cycle regulation in a large cohort of NETs. METHODS We evaluated immunohistochemical expression of CDKN1B, CDKN1A, CDKN2A, CDK2, CDK4, CDK6, cyclin D1, cyclin E1, and phosphorylated retinoblastoma protein (phospho-RB1) in a cohort of 267 patients with NETs. We then explored associations between cell cycle protein expression, mutational status, histologic features, and overall survival. RESULTS We found that high expression of CDK4, CDK6, CCND1, and phospho-RB1 was associated with higher proliferative index, as defined by MKI67. We additionally observed a trend toward shorter overall survival associated with low expression of CDKN1B. This association seemed strongest in SINETs (multivariate hazards ratio, 2.04; 95% confidence interval, 1.06-3.93; P = 0.03). We found no clear association between CDKN1B mutation and protein expression. CONCLUSIONS Our results suggest that dysregulation and activation of the CDK4/CDK6-CCND1-phospho-RB1 axis is associated with higher proliferative index in NETs. Investigation of the therapeutic potential of CDK4/CDK6 inhibitors in higher grade NETs is warranted.

// Mancang Gu 1, 2 , Reiko Nishihara 1, 3, 4, 5, 6 , Yang Chen 1, 7 , Wanwan Li 1 , Yan Shi 1, 7 , Yohei Masugi 1 , Tsuyoshi Hamada 1 , Keisuke Kosumi 1 , Li Liu 1, 3 , Annacarolina da Silva 1 , Jonathan A. Nowak 6 , Tyler Twombly 1 , Chunxia Du 1 , Hideo Koh 1 , Wenbin Li 1 , Jeffrey A. Meyerhardt 8 , Brian M. Wolpin 8 , Marios Giannakis 8 , Andrew J. Aguirre 8 , Adam J. Bass 8, 9, 10 , David A. Drew 11, 12 , Andrew T. Chan 11, 12 , Charles S. Fuchs 13, 14, 15, * , Zhi Rong Qian 1, 6, * and Shuji Ogino 1, 4, 6, * 1 Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA 2 College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, P.R. China 3 Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA 4 Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA 5 Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA 6 Program in MPE Molecular Pathological Epidemiology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA 7 Medical Oncology Department 2, Chinese People’s Liberation Army General Hospital, Beijing, P.R. China 8 Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA 9 Broad Institute of MIT and Harvard, Cambridge, MA, USA 10 Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA 11 Clinical and Translational Epidemiology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA 12 Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, USA 13 Yale Cancer Center, New Haven, CT, USA 14 Department of Medicine, Yale School of Medicine, New Haven, CT, USA 15 Smilow Cancer Hospital, New Haven, CT, USA * These authors contributed equally to this work Correspondence to: Zhi Rong Qian, email: zhirong_qian@dfci.harvard.edu Shuji Ogino, email: shuji_ogino@dfci.harvard.edu Keywords: anti-tumor effect, colorectal cancer, isogenic cell model, NSAID, PI3K Received: May 31, 2017 Accepted: August 31, 2017 Published: September 18, 2017 ABSTRACT Evidence suggests that nonsteroidal anti-inflammatory drug aspirin (acetylsalicylic acid) may improve patient survival in PIK3CA -mutant colorectal carcinoma, but not in PIK3CA -wild-type carcinoma. However, whether aspirin directly influences the viability of PIK3CA -mutant colon cancer cells is poorly understood. We conducted in vitro experiments to test our hypothesis that the anti-proliferative activity of aspirin might be stronger for PIK3CA -mutant colon cancer cells than for PIK3CA -wild-type colon cancer cells. We measured the anti-proliferative effect of aspirin at physiologic concentrations in seven PIK3CA -mutant and six PIK3CA -wild-type human colon cancer cell lines. After exposure to aspirin, the apoptotic index and cell cycle phase of colon cancer cells were assessed. In addition, the effect of aspirin was examined in parental SW48 cells and SW48 cell clones with individual knock-in PIK3CA mutations of either c.3140A>G (p.H1047R) or c.1633G>A (p.E545K). Aspirin induced greater dose-dependent loss of cell viability in PIK3CA -mutant cells than in PIK3CA -wild-type cells after treatment for 48 and 72 hours. Aspirin treatment also led to higher proportions of apoptotic cells and G0/G1 phase arrest in PIK3CA -mutant cells than in PIK3CA -wild-type cells. Aspirin treatment of isogenic SW48 cells carrying a PIK3CA mutation, either c.3140A>G (p.H1047R) or c.1633G>A (p. E545K), resulted in a more significant loss of cell viability compared to wild-type controls. Our findings indicate that aspirin causes cell cycle arrest, induces apoptosis, and leads to loss of cell viability more profoundly in PIK3CA -mutated colon cancer cells than in PIK3CA -wild-type colon cancer cells. These findings support the use of aspirin to treat patients with PIK3CA -mutant colon cancer.

A deep insight into the function and kinetics of ATP-binding cassette (ABC) transporters may aid in the development of pharmaceutics that can minimize the particular facet of chemo-resistance. We utilized bioluminescence imaging to monitor the ABC transporter mediated intracellular drug efflux function. We also investigated the potential association between the intracellular bioluminescent pharmacokinetic profiles and the anti-tumor efficacy of the coix seed extract and gemcitabine against pancreatic cancer cells in vitro and in vivo. The bioluminescent pharmacokinetic parameters and pharmacodynamic index (IC50 and TGI) were determined. The expression levels ABCB1 and ABCG2 were assessed. Results showed that coix seed extract could synergistically enhance the anti-cancer efficacy of gemcitabine (p &lt, 0.05). Meanwhile coix seed extract alone or in combination with gemcitabine could significantly increase the AUCluc while decreasing the Kluc (p &lt, 0.01). Western blot and immunohistochemistry assay demonstrated that coix seed extract could significantly mitigate gemcitabine-induced upregulation of ABCB1 and ABCG2 protein. The Pearson correlation analysis demonstrated that the bioluminescent pharmacokinetic parameters and pharmacodynamic index have strong association in vitro and in vivo. In conclusion coix seed extract could augment the efficacy of gemcitabine therapy in pancreatic cancer cells may at least partly due to the alteration of ABC transporter-mediated drug efflux function.

OBJECTIVE: We hypothesized that the risk of CRC in night-shift workers might be different according to insulin receptor substrates status. METHODS: Among 77,470 eligible women having night work assessed in the Nurses’ Health Study, we documented a total of 1,397 CRC cases, of which 304 or 308 had available data on IRS1 and IRS2, respectively. We used duplication method Cox proportional hazards regression analysis for competing risks to calculate HRs and 95% CIs for each CRC subtype. We measured tumor IRS1 or IRS2 expression by immunohistochemistry. RESULTS: Compared with women who never worked night-shifts, those working ≥ 15 years night-shifts had a marginal trend of increased overall risk of CRC (P(trend) = 0.06, multivariable HR = 1.20, 95% CI, 0.99 to 1.45). Longer duration of night-shift work was associated with a higher risk of IRS2-positive tumors (multivariable HR = 2.69, 95% CI 1.48 to 4.89, P(trend) = 0.001, ≥ 15 years night-shifts vs. never) but not with IRS2-negative tumors (multivariable HR = 0.90, 95% CI 0.54 to 1.51, P(trend) = 0.72, P(heterogeneity) for IRS2 = 0.008). Similarly, the corresponding multivariable HRs were 1.81 for IRS1-positive tumors (95% CI 0.94 to 3.48, P(trend) = 0.06) and 1.13 for IRS1-negative tumors (95% CI 0.71 to 1.80, P(trend) = 0.56, P(heterogeneity) for IRS1 = 0.02). CONCLUSION: Our molecular pathological epidemiology data suggest a potential role of IRS in mediating carcinogenesis induced by night-shift work. IMPACT: Although these findings need validation, rotating night-shift might increase CRC risk in women with abnormal insulin receptor pathway.

Intranasal (i.n.) administration is an efficient route for enhancing drug delivery to the brain, bypassing the blood-brain barrier (BBB) and eliminating systemic side effects. The purpose of this study was to investigate the nose-to-brain delivery efficiency of adriamycin (ADM) loaded in cholesterol-modified pullulan self-assembled nanoparticles (CHSP-SAN) via i.n. administration. The prepared nanodrugs (ADM-CHSP-SAN) were characterized as uniform size (112.8±1.02 nm), high drug loading capacity (7.65±0.58 %), and sustained release. CHSP-SAN showed good biocompatibility and low toxicity on HBMEC and C6 cells. The enhanced delivery of ADM across the BBB with CHSP-SAN was demonstrated by the reduced half maximal inhibitory concentration (IC

Background Evidence suggests that high-level physical activity may potentially reduce cancer mortality through its immune enhancement effect. We therefore hypothesized that survival benefits associated with physical activity might be stronger in colorectal carcinomas with lower immune reaction at diagnosis. Methods Using molecular pathological epidemiology databases of 470 colon and rectal carcinoma cases in the Nurses’ Health Study and the Health Professionals Follow-up Study, we assessed the prognostic association of postdiagnosis physical activity in strata of densities of CD3+ cells, CD8+ cells, CD45RO (PTPRC)+ cells, or FOXP3+ cells in tumor tissue. Cox proportional hazards regression model was used to adjust for potential confounders, including microsatellite instability, CpG island methylator phenotype, long interspersed nucleotide element-1 methylation, KRAS, BRAF, and PIK3CA mutations, and expression of CTNNB1 (beta-catenin), PTGS2 (cyclooxygenase-2), and IRS1. Results The association of postdiagnosis physical activity with colorectal cancer-specific mortality differed by CD3+ cell density (Pinteraction < .001). Multivariable-adjusted colorectal cancer-specific mortality hazard ratios for a quartile-unit increase in physical activity were 0.56 (95% confidence interval = 0.38 to 0.83) among cases with the lowest quartile of CD3+ cell density compared with 1.14 (95% confidence interval = 0.79 to 1.65) in cases with the highest quartile. We observed no differential survival association of physical activity by densities of CD8+ cells, CD45RO+ cells, or FOXP3+ cells. Conclusions The association between postdiagnosis physical activity and colorectal cancer survival appeared stronger for carcinomas with lower T cell infiltrates, suggesting an interactive effect of exercise and immunity on colorectal cancer progression.

Experimental evidence suggests that the let-7 family of noncoding RNAs suppresses adaptive immune responses, contributing to immune evasion by the tumor. We hypothesized that the amount of let-7a and let-7b expression in colorectal carcinoma might be associated with limited T-lymphocyte infiltrates in the tumor microenvironment and worse clinical outcome. Utilizing the molecular pathological epidemiology resources of 795 rectal and colon cancers in two U.S.-nationwide prospective cohort studies, we measured tumor-associated let-7a and let-7b expression levels by quantitative reverse-transcription PCR, and CD3+, CD8+, CD45RO (PTPRC)+, and FOXP3+ cell densities by tumor tissue microarray immunohistochemistry and computer-assisted image analysis. Logistic regression analysis and Cox proportional hazards regression were used to assess associations of let-7a (and let-7b) expression (quartile predictor variables) with T-cell densities (binary outcome variables) and mortality, respectively, controlling for tumor molecular features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Compared with cases in the lowest quartile of let-7a expression, those in the highest quartile were associated with lower densities of CD3+ [multivariate odds ratio (OR), 0.40; 95% confidence interval (CI), 0.23–0.67; Ptrend = 0.003] and CD45RO+ cells (multivariate OR, 0.31; 95% CI, 0.17–0.58; Ptrend = 0.0004), and higher colorectal cancer-specific mortality (multivariate hazard ratio, 1.82; 95% CI, 1.42–3.13; Ptrend = 0.001). In contrast, let-7b expression was not significantly associated with T-cell density or colorectal cancer prognosis. Our data support the role of let-7a in suppressing antitumor immunity in colorectal cancer and suggest let-7a as a potential target of immunotherapy. Cancer Immunol Res; 4(11); 927–35. ©2016 AACR.

The presence of Fusobacterium nucleatum (F. nucleatum) in colorectal carcinoma tissue has been associated with microsatellite instability (MSI), lower-level T-cell infiltrates, and poor clinical outcomes. Considering differences in the tumor–immune microenvironment between MSI-high and non–MSI-high carcinomas, we hypothesized that the association of F. nucleatum with immune response might differ by tumor MSI status. Using samples from 1,041 rectal and colon cancer patients within the Nurses’ Health Study and Health Professionals Follow-up Study, we measured F. nucleatum DNA in tumor tissue by a quantitative polymerase chain reaction assay. Multivariable logistic regression models were used to examine the association between F. nucleatum status and histopathologic lymphocytic reactions or density of CD3+ cells, CD8+ cells, CD45RO (PTPRC)+ cells, or FOXP3+ cells in strata of tumor MSI status. We adjusted for potential confounders, including CpG island methylator phenotype; LINE-1 methylation; and KRAS, BRAF, and PIK3CA mutations. The association of F. nucleatum with tumor-infiltrating lymphocytes (TIL) and intratumoral periglandular reaction differed by tumor MSI status (Pinteraction = 0.002). The presence of F. nucleatum was negatively associated with TIL in MSI-high tumors [multivariable odds ratio (OR), 0.45; 95% confidence interval (CI), 0.22–0.92], but positively associated with TIL in non–MSI-high tumors (multivariable OR 1.91; 95% CI, 1.12–3.25). No significant differential association was observed for peritumoral lymphocytic reaction, Crohn-like lymphoid reaction, or T-cell densities. In conclusion, the association of F. nucleatum with immune response to colorectal carcinoma differs by tumor MSI status, suggesting that F. nucleatum and MSI status interact to affect antitumor immune reactions. Cancer Immunol Res; 6(11); 1327–36. ©2018 AACR. See related Spotlight on p. 1290.

Germline variants in double-strand DNA damage repair (dsDDR) genes (e.g., BRCA1/2) predispose to pancreatic adenocarcinoma (PDAC) and may predict sensitivity to platinum-based chemotherapy and poly(ADP) ribose polymerase (PARP) inhibitors. We sought to determine the prevalence and significance of germline cancer susceptibility gene variants in PDAC with paired somatic and survival analyses. Using a customized next-generation sequencing panel, germline/somatic DNA was analyzed from 289 patients with resected PDAC ascertained without preselection for high-risk features (e.g., young age, personal/family history). All identified variants were assessed for pathogenicity. Outcomes were analyzed using multivariable-adjusted Cox proportional hazards regression. We found that 28/289 (9.7%; 95% confidence interval [CI] 6.5–13.7%) patients carried pathogenic/likely pathogenic germline variants, including 21 (7.3%) dsDDR gene variants (3 BRCA1, 4 BRCA2, 14 other dsDDR genes [ATM, BRIP1, CHEK2, NBN, PALB2, RAD50, RAD51C]), 3 Lynch syndrome, and 4 other genes (APC p.I1307K, CDKN2A, TP53). Somatic sequencing and immunohistochemistry identified second hits in the tumor in 12/27 (44.4%) patients with germline variants (1 failed sequencing). Compared with noncarriers, patients with germline dsDDR gene variants had superior overall survival (hazard ratio [HR] 0.54; 95% CI 0.30–0.99; P = 0.05). Nearly 10% of PDAC patients harbor germline variants, although the majority lack somatic second hits, the therapeutic significance of which warrants further study.

Inhibitors targeting the PDCD1 (programmed cell death 1, PD-1) immune checkpoint pathway have revolutionized cancer treatment strategies. The TIME (Tumor Immunity in the MicroEnvironment) classification based on tumor CD274 (PDCD1 ligand 1, PD-L1) expression and tumor-infiltrating lymphocytes (TIL) has been proposed to predict response to immunotherapy. It remains to be determined clinical, pathological, and molecular features of TIME subtypes of colorectal cancer. Using 812 colon and rectal carcinoma cases from the Nurses' Health Study and Health Professionals Follow-up Study, we examined the association of tumor characteristics and survival outcomes with four TIME subtypes (TIME 1, CD274(low)/TIL(absent); TIME 2, CD274(high)/TIL(present); TIME 3, CD274(low)/TIL(present); and TIME 4, CD274(high)/TIL(absent)). In survival analyses, Cox proportional hazards models were adjusted for potential confounders, including microsatellite instability (MSI) status, CpG island methylator phenotype (CIMP) status, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutation status. TIME subtypes 1, 2, 3 and 4 had 218 (27%), 117 (14%), 103 (13%), and 374 (46%) colorectal cancer cases, respectively. Compared with TIL-absent subtypes (TIME 1 and 4), TIL-present subtypes (TIME 2 and 3) were associated with high-level MSI, high-degree CIMP, BRAF mutation, and higher amounts of neoantigens (p < 0.001). TIME subtypes were not significantly associated with colorectal cancer-specific or overall survival. In conclusion, TIL-present TIME subtypes of colorectal cancer are associated with high levels of MSI and neoantigen load, supporting better responsiveness to cancer immunotherapy. Further studies examining tumor molecular alterations and additional factors in the tumor microenvironment may inform development of immunoprevention and immunotherapy strategies.

Evidence indicates a complex link between microbiota, tumor characteristics, and host immunity in the tumor microenvironment. In experimental studies, bifidobacteria appear to modulate intestinal epithelial cell differentiation. Accumulating evidence suggests that bifidobacteria may enhance the antitumor immunity and efficacy of immunotherapy. We hypothesized that the amount of bifidobacteria in colorectal carcinoma tissue might be associated with tumor differentiation and higher immune response to colorectal cancer. Using a molecular pathologic epidemiology database of 1313 rectal and colon cancers, we measured the amount of Bifidobacterium DNA in carcinoma tissue by a quantitative PCR assay. The multivariable regression model was used to adjust for potential confounders, including microsatellite instability status, CpG island methylator phenotype, long-interspersed nucleotide element-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Intratumor bifidobacteria were detected in 393 cases (30%). The amount of bifidobacteria was associated with the extent of signet ring cells (P = 0.002). Compared with Bifidobacterium-negative cases, multivariable odd ratios for the extent of signet ring cells were 1.29 (95% CI, 0.74–2.24) for Bifidobacterium-low cases and 1.87 (95% CI, 1.16–3.02) for Bifidobacterium-high cases (P(trend) = 0.01). The association between intratumor bifidobacteria and signet ring cells suggests a possible role of bifidobacteria in determining distinct tumor characteristics or as an indicator of dysfunctional mucosal barrier in colorectal cancer.