BRAF V600E mutation in papillary thyroid carcinoma: it's relation to clinical features and oncologic outcomes in a single cancer centre experience.

PURPOSE: This study focuses on the oncologic influence of BRAF V600E mutations in a cohort of Middle Eastern papillary thyroid carcinoma (PTC) patients treated at a single centre. We tested the association of BRAF V600E mutation with papillary thyroid carcinoma at King Hussein Cancer Center. METHODS: Patients with histologically confirmed PTC who underwent surgical treatment between 2006 and 2015 were included in this study. Oncological outcomes, both short- and long-termed, were collected. RESULTS: A total of 128 patients (68% females) were included in this study with a mean age of 38 years (±13.8). The median follow-up period was 50 months. The BRAF V600E mutation was found in 71% of patients. The tumour size for patients with a negative BRAF V600E mutation was significantly larger in comparison to patients who tested positive for the mutation (3.47 cm vs 2.31 cm, respectively, P = 0.009). The two groups showed similar disease-free survival (DFS) rates; positive = 75% (median 43 months (0-168)) compared to 78% for the negative BRAF V600E mutation (median 38 months (3-142)) (P = 0.162, HR = 0.731) Furthermore, both groups showed similar overall survival rates, positive = 94.5% (median 56 months (0-228)) compared to 94.6% for the negative BRAF V600E mutation (median 43 months (3-157)) (P = 0.941, HR = 0.940). CONCLUSION: BRAF V600E mutation had no effect on loco-regional recurrence, distant metastasis, overall survival, or DFS. These findings may be attributed to geographic variations or reflect that BRAF V600E may only serve as an indicator of poor prognosis in high-risk group as such.

Curative intent resection for loco-regionally recurrent colon cancer: Cleveland clinic experience.

BACKGROUND: Locoregional colon cancer recurrence occurs in around 10% of patients following initial curative intent primary resection. We hypothesized oncological results can vary based on the recurrence site. Our aim was to determine outcomes for patients undergoing resection with curative intent for locally recurrent colon cancer. METHODS: Patients with locoregional recurrence after curative intent resection for colon cancer were identified (1999-2017). Demographics, operative details and outcome data were recorded. Kaplan-Meier method was used to compare survival differences. RESULTS: Fifty-two patients (mean age, 62) were included. The most common recurrence site was primary anastomosis (48%). R0 resection was obtained in 68%. Major morbidity occurred in 37%. Patients with anastomotic recurrence had a statistically significant overall survival compared to other sites (71.6 vs. 40.8 months respectively with a P value of 0.05). CONCLUSIONS: Excellent outcomes are possible for curative intent recurrent colon cancer surgery. The site of loco-regional recurrence plays a significant role in outcomes. Table of Contents Summary: Colon cancer recurrence can be treated surgically with optimal outcomes. Anastomotic recurrence is associated with improved survival.

An up-to-date predictive model for rectal cancer survivorship reflecting tumor biology and clinical factors.

INTRODUCTION: Our aim was to develop a nomogram taking into account factors such as tumor biology to predict overall and disease-free survival for patients with primary rectal adenocarcinoma undergoing curative intent surgical resection. METHODS: Patients undergoing resection for primary rectal adenocarcinoma (2007-2017) were included. Factors reflecting tumor biology and important clinical prognosticators were included in nomogram development. Prognostic factors were assessed with multivariable analysis using Cox regression. The impact of each was assessed using Kaplan Meier survival curves. RESULTS: Overall, 1688 patients (male, 61%) with a mean age of 59.8 years (±13.5) and a median follow-up of 34.8 months (range, 12-132) were included. The only significant factors affecting the overall and disease-free survival were age at diagnosis, pathological staging, regression grade, resection margin, and tumor deposits. CONCLUSION: The current model incorporates histopathological and clinical factors. It emphasizes the importance of tumor biological factors like tumor deposits in predicting overall and disease-free survival in rectal cancer. SUMMARY: Rectal cancer outcomes are associated with certain clinical and pathological factors that can be evaluated. Tumor deposits are one such factor that can affect overall and disease-free survival.

Poly(ADP-Ribose) Polymerase Inhibition Sensitizes Colorectal Cancer-Initiating Cells to Chemotherapy.

Colorectal cancer (CRC) remains a leading killer in the U.S. with resistance to treatment as the largest hurdle to cure. Colorectal cancer-initiating cells (CICs) are a self-renewing tumor population that contribute to tumor relapse. Here, we report that patient-derived CICs display relative chemoresistance compared with differentiated progeny. In contrast, conventional cell lines failed model therapeutic resistance. CICs preferentially repaired chemotherapy-induced DNA breaks, prompting us to interrogate DNA damage pathways against which pharmacologic inhibitors have been developed. We found that CICs critically depended on the key single-strand break repair mediator, poly(ADP-ribose) polymerase (PARP), to survive treatment with standard-of-care chemotherapy. Small molecule PARP inhibitors (PARPi) sensitized CICs to chemotherapy and reduced chemotherapy-treated CIC viability, self-renewal, and DNA damage repair. Although PARPi monotherapy failed to kill CICs, combined PARPi therapy with chemotherapy attenuated tumor growth in vivo. Clinical significance of PARPi for CRC patients was supported by elevated PARP levels in colorectal tumors compared with normal colon, with further increases in metastases. Collectively, our results suggest that PARP inhibition serves as a point of fragility for CICs by augmenting therapeutic efficacy of chemotherapy. Stem Cells 2019;37:42-53.

Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase.

Tumors adapt their phenotypes during growth and in response to therapies through dynamic changes in cellular processes. Connexin proteins enable such dynamic changes during development, and their dysregulation leads to disease states. The gap junction communication channels formed by connexins have been reported to exhibit tumor-suppressive functions, including in triple-negative breast cancer (TNBC). However, we find that connexin 26 (Cx26) is elevated in self-renewing cancer stem cells (CSCs) and is necessary and sufficient for their maintenance. Cx26 promotes CSC self-renewal by forming a signaling complex with the pluripotency transcription factor NANOG and focal adhesion kinase (FAK), resulting in NANOG stabilization and FAK activation. This FAK/NANOG-containing complex is not formed in mammary epithelial or luminal breast cancer cells. These findings challenge the paradigm that connexins are tumor suppressors in TNBC and reveal a unique function for Cx26 in regulating the core self-renewal signaling that controls CSC maintenance.

Inclusion of the Mesentery in Ileocolic Resection for Crohn's Disease is Associated With Reduced Surgical Recurrence.

BACKGROUND AND AIMS: Inclusion of the mesentery during resection for colorectal cancer is associated with improved outcomes but has yet to be evaluated in Crohn's disease. This study aimed to determine the rate of surgical recurrence after inclusion of mesentery during ileocolic resection for Crohn's disease. METHODS: Surgical recurrence rates were compared between two cohorts. Cohort A [n = 30] underwent conventional ileocolic resection where the mesentery was divided flush with the intestine. Cohort B [n = 34] underwent resection which included excision of the mesentery. The relationship between mesenteric disease severity and surgical recurrence was determined in a separate cohort [n = 94]. A mesenteric disease activity index was developed to quantify disease severity. This was correlated with the Crohn's disease activity index and the fibrocyte percentage in circulating white cells. RESULTS: Cumulative reoperation rates were 40% and 2.9% in cohorts A and B [P = 0.003], respectively. Surgical technique was an independent determinant of outcome [P = 0.007]. Length of resected intestine was shorter in cohort B, whilst lymph node yield was higher [12.25 ± 13 versus 2.4 ± 2.9, P = 0.002]. Advanced mesenteric disease predicted increased surgical recurrence [Hazard Ratio 4.7, 95% Confidence Interval: 1.71-13.01, P = 0.003]. The mesenteric disease activity index correlated with the mucosal disease activity index [r = 0.76, p < 0.0001] and the Crohn's disease activity index [r = 0.70, p < 0.0001]. The mesenteric disease activity index was significantly worse in smokers and correlated with increases in circulating fibrocytes. CONCLUSIONS: Inclusion of mesentery in ileocolic resection for Crohn's disease is associated with reduced recurrence requiring reoperation.

New Advances and Challenges of Targeting Cancer Stem Cells.

The second International Cancer Stem Cell Conference in Cleveland, Ohio, on September 20-23, 2016, convened 330 attendees from academic, industrial, and clinical organizations. It featured a debate on the concepts and challenges of the cancer stem cells (CSC) as well as CSC-centered scientific sessions on clinical trials, genetics and epigenetics, tumor microenvironment, immune suppression, metastasis, therapeutic resistance, and emerging novel concepts. The conference hosted 35 renowned speakers, 100 posters, 20 short talks, and a preconference workshop. The reported advances of CSC research and therapies fostered new collaborations across national and international borders, and inspired the next generation's young scientists. Cancer Res; 77(19); 5222-7. ©2017 AACR.

CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors.

Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and non-CSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

Glioblastoma Cancer Stem Cells Evade Innate Immune Suppression of Self-Renewal through Reduced TLR4 Expression.

Tumors contain hostile inflammatory signals generated by aberrant proliferation, necrosis, and hypoxia. These signals are sensed and acted upon acutely by the Toll-like receptors (TLRs) to halt proliferation and activate an immune response. Despite the presence of TLR ligands within the microenvironment, tumors progress, and the mechanisms that permit this growth remain largely unknown. We report that self-renewing cancer stem cells (CSCs) in glioblastoma have low TLR4 expression that allows them to survive by disregarding inflammatory signals. Non-CSCs express high levels of TLR4 and respond to ligands. TLR4 signaling suppresses CSC properties by reducing retinoblastoma binding protein 5 (RBBP5), which is elevated in CSCs. RBBP5 activates core stem cell transcription factors, is necessary and sufficient for self-renewal, and is suppressed by TLR4 overexpression in CSCs. Our findings provide a mechanism through which CSCs persist in hostile environments because of an inability to respond to inflammatory signals.

Cancer Stem Cell-Secreted Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Suppressor Cell Function and Facilitates Glioblastoma Immune Evasion.

Shifting the balance away from tumor-mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid-derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune-suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self-renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5-flurouracil (5-FU) in a low-dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient-derived CSCs but not nonstem tumor cells selectively drove MDSC-mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune-suppressive enzyme arginase-1 in MDSCs in a CXCR2-dependent manner, whereas blocking MIF reduced arginase-1 production. Similarly to 5-FU, targeting tumor-derived MIF conferred a survival advantage to tumor-bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self-renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026-2039.

Reporter Systems to Study Cancer Stem Cells.

Cancer stem cells have been identified in primary tumors, patient derived xenografts, and established cancer cell lines. The development of reporters has enabled investigators to rapidly enrich for these cells and more importantly track these cells in real time. Here we describe the current state of the reporter field and their use and limitations in multiple cancers.

Preferential Iron Trafficking Characterizes Glioblastoma Stem-like Cells.

Glioblastomas display hierarchies with self-renewing cancer stem-like cells (CSCs). RNA sequencing and enhancer mapping revealed regulatory programs unique to CSCs causing upregulation of the iron transporter transferrin, the top differentially expressed gene compared with tissue-specific progenitors. Direct interrogation of iron uptake demonstrated that CSCs potently extract iron from the microenvironment more effectively than other tumor cells. Systematic interrogation of iron flux determined that CSCs preferentially require transferrin receptor and ferritin, two core iron regulators, to propagate and form tumors in vivo. Depleting ferritin disrupted CSC mitotic progression, through the STAT3-FoxM1 regulatory axis, revealing an iron-regulated CSC pathway. Iron is a unique, primordial metal fundamental for earliest life forms, on which CSCs have an epigenetically programmed, targetable dependence.

Differential connexin function enhances self-renewal in glioblastoma.

The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin 43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.

Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.

Advanced cancers display cellular heterogeneity driven by self-renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self-renewal and tumor initiation. To overcome these barriers in triple-negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well-established pluripotency gene NANOG. NANOG-GFP+ cells gave rise to both GFP+ and GFP(-) cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self-renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well-established CSC markers CD24(-) /CD44(+) , CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG-GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule-A (JAM-A). JAM-A was highly expressed in GFP+ cells and patient-derived xenograft ALDH+ CSCs compared with the GFP(-) and ALDH(-) cells, respectively. Depletion of JAM-A compromised self-renewal, whereas JAM-A overexpression induced self-renewal in GFP(-) cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non-CSCs in TNBC that can be used to identify CSC-specific targets for the development of future therapeutic strategies.

Chemotherapy activates cancer-associated fibroblasts to maintain colorectal cancer-initiating cells by IL-17A.

Many solid cancers display cellular hierarchies with self-renewing, tumorigenic stemlike cells, or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies, they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs, we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays, but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines, including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion, and targeting IL-17A signaling impaired CIC growth. Notably, IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer.

Treating symptomatic adhesions to the sigmoid colon: colectomy improves quality of life.

INTRODUCTION: When severe chronic pelvic pain, constipation, and bloating are due to dense pelvic adhesions fixing the sigmoid loops, patients may be offered the option of sigmoid colectomy. This study examines the effectiveness of colectomy in the treatment of such patients. METHODS: Patients were identified from a surgical database, demographic data were abstracted, and charts were reviewed. Patients were interviewed postoperatively. Primary end points were morbidity, length of stay, change in bowel habit, and patient satisfaction. RESULTS: There were 46 patients (44 women) with a mean age of 54.7 years (±14.3). Forty-three had a history of prior pelvic surgery (93%), including 30 (65%) with hysterectomy. One quarter had been diagnosed with irritable bowel syndrome. Surgery revealed adhesive tethering of the sigmoid colon in 43 patients (94%). Mean length of stay was 6.5 days (±2.5), and complications occurred in eight (17.3%) patients. There were no deaths, and no patients required a stoma. Follow-up in 37 patients after mean of 7.2(±5.0) years showed significant reductions in abdominal pain and bloating postoperatively, with normalization of bowel function and increase in quality of life. CONCLUSION: When severe lower abdominal pain and bloating in women who have had pelvic surgery are reproduced by colonoscopy, and there is an obvious kink in the bowel, sigmoid colectomy is worth considering.

Risk factors for delayed postpolypectomy bleeding: how to minimize your patients' risk.

PURPOSE: Secondary bleeding after colonoscopic polypectomy is a serious complication. Most studies show polyp size, location, and shape to be important risk factors but other factors may allow refinement of risk. The aim of this study is to look for other factors associated with delayed postpolypectomy bleeding. METHODS: This case-matched study compares patients who developed postpolypectomy bleeding with those who underwent uncomplicated polypectomy. Matching was performed for known risk factors: polyp size, location and shape, with a ratio of three controls to one subject. RESULTS: Postpolypectomy bleeding occurred in 19/494 patients with polyps >2.0 cm diameter (3.8%) and 11/4161 patients with polyps <2.0 cm (0.3%). There was a median interval of 3 days (range 0-9 days) from polypectomy to hemorrhage. Twenty-five patients were readmitted to this institution. Ten (40.0%) were transfused and 19 (76.0%) were re-colonoscoped, with 13 needing either cautery (n = 7) or adrenaline injection (n = 6). Ninety patients were selected as controls, matched for polyp size, location, and shape. Mean age at polypectomy was 69.9 ± 9.2 years for patients and 64.9 ± 12.2 for controls (p = 0.042); 63.3% subjects were male, compared to 47.8% of controls (p = 0.140). Univariate analysis showed that older age, piecemeal polypectomy, need for additional sedation, concurrent diverticulosis and intraprocedural bleeding were significantly associated with increased risk of delayed bleeding. The associations between delayed bleeding and additional sedation, concurrent diverticulosis and intraprocedural bleeding were confirmed by multivariate logistic regression analysis. CONCLUSION: Difficult colonoscopy and intraprocedural bleeding identify patients with a particularly high risk of secondary postpolypectomy bleeding. Preventive measures should be considered in such cases.

Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

High-throughput arrays identify distinct genetic profiles associated with lymph node involvement in rectal cancer.

BACKGROUND: Preoperative clinical diagnosis of lymph node involvement guides treatment decisions for rectal cancer. Unfortunately, clinical staging still suffers from a lack of accuracy. OBJECTIVE: The aim of this study was to evaluate objective genetic differences in primary rectal cancers with and without associated lymph node metastasis. DESIGN: cDNA microarrays were generated from fresh-frozen tumors. Normalized data underwent global unsupervised hierarchical clustering analysis, and discriminating genes were mapped. Top discriminating genes were compared between stage II and III rectal cancers by use of an empirical Bayes 2 group t test with the Statistical Analysis of Microarrays and the Reproducibility-Optimized Test Statistic software separately to guide data reduction and deal with the difficulties of simultaneous statistical inference. Ingenuity Pathways Analysis software was used to analyze discriminating genes in terms of function and biological processes. PATIENTS: Fifty-five patients with stage II and 22 patients with stage III rectal adenocarcinomas not treated with chemoradiation were included. RESULTS: Two major unsupervised clusters emerged representing stage II and III cancers. In 1 cluster, 11 of 12 patients (92%) had stage III cancer; in the other cluster, 54 of 65 patients (83%) had stage II (p < 0.001). Five significantly differentially expressed genes characterized the stage III cluster: interleukin-8, 3-hydroxy-3-methylglutaryl coenzyme A synthase, carbonic anhydrase, ubiquitin, and cystatin (all p < 0.05). Of the 12 patients with differential expression of the 5 marker genes, only one had stage II cancer. Fifty-four of 55 stage II patients clustered with alternative expression patterns of the predictor genes. Differentially expressed genes are related to cancer-associated processes, pathways, and networks. LIMITATIONS: The identified gene signatures have not yet been validated in independent patient populations. CONCLUSIONS: Distinct gene expression signatures from primary rectal adenocarcinomas can help differentiate the presence or absence of lymph node metastases. These data are informative, and validation of this gene signature may provide a novel approach for more appropriate individualized treatment selection.

Epigenomic enhancer profiling defines a signature of colon cancer.

Cancer is characterized by gene expression aberrations. Studies have largely focused on coding sequences and promoters, even though distal regulatory elements play a central role in controlling transcription patterns. We used the histone mark H3K4me1 to analyze gain and loss of enhancer activity genome-wide in primary colon cancer lines relative to normal colon crypts. We identified thousands of variant enhancer loci (VELs) that comprise a signature that is robustly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. We propose that reproducible changes in the epigenome at enhancer elements drive a specific transcriptional program to promote colon carcinogenesis.