p21 Protein Outperforms Clinico-pathological Criteria in Predicting Liver Metastases in Pancreatic Endocrine Tumors.

BACKGROUND/AIM: P21 is a cyclin-dependent kinase inhibitor regulating the cell cycle as a tumor suppressor. Using a p21 immunohistochemistry (IHC) assay, we compared tumor p21 levels with conventional clinico-pathological criteria in primary pancreatic endocrine tumor subsets with and without liver metastases. MATERIALS AND METHODS: Sections from tissue microarray (TMA) including 13 archival metastatic primary and 18 non-metastatic primary pancreatic endocrine carcinomas/tumors (MP-PECAs/NMP-PETs) were stained with a monoclonal anti-p21WAFI,CIP primary antibody. Tumor p21 IHCs were scored as the sum of intensity (0-3) and proportion scores (0-5) (Total Allred score: 0-8), and as p21% labelling index in the tumor. ROC curve analysis was used for most optimal p21 score cut-off (4 or >) and Fisher's exact test was used to compare the association among tumor p21 scores, conventional prognostic criteria, and liver metastases. RESULTS: For PET/PECA patients, mean ages were 55.6 years (27-73) and 49.3 years (28-71), M/F ratios were 7/11 and 7/6. Mean p21 labelling index (%) for MP- PECAs was 24% (range=3-63%) vs. 9% for NMP-PETs (range=1-25%) (p=0.022). The mean p21 index in MP-PECAs was significantly higher (24%) as compared to PIs (7%) (p=0.0047). Using a p21 Allred score of ≥4, high p21 IHC score had strong association with the presence of liver metastases (p-value <0.001). High tumor p21 IHC score had a 93% sensitivity, 68% specificity, 78% predictive accuracy, 66% positive, and 94% negative predictive values. CONCLUSION: In patients with primary PETs, p21 IHC is superior to conventional criteria in predicting presence or absence of liver metastases.

Evaluation of cell-free DNA approaches for multi-cancer early detection.

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.

Ras Pathway Activation and MEKi Resistance Scores Predict the Efficiency of MEKi and SRCi Combination to Induce Apoptosis in Colorectal Cancer.

Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. The RAS pathway is activated in more than 55% of CRC and has been targeted for therapeutic intervention with MEK inhibitors. Unfortunately, many patients have de novo resistance, or can develop resistance to this new class of drugs. We have hypothesized that much of this resistance may pass through SRC as a common signal transduction node, and that inhibition of SRC may suppress MEK inhibition resistance mechanisms. CRC tumors of the Consensus Molecular Subtype (CMS) 4, enriched in stem cells, are difficult to successfully treat and have been suggested to evade traditional chemotherapy agents through resistance mechanisms. Here, we evaluate targeting two pathways simultaneously to produce an effective treatment by overcoming resistance. We show that combining Trametinib (MEKi) with Dasatinib (SRCi) provides enhanced cell death in 8 of the 16 tested CRC cell lines compared to treatment with either agent alone. To be able to select sensitive cells, we simultaneously evaluated a validated 18-gene RAS pathway activation signature score along with a 13-gene MEKi resistance signature score, which we hypothesize predict tumor sensitivity to this dual targeted therapy. We found the cell lines that were sensitive to the dual treatment were predominantly CMS4 and had both a high 18-gene and a high 13-gene score, suggesting these cell lines had potential for de novo MEKi sensitivity but were subject to the rapid development of MEKi resistance. The 13-gene score is highly correlated to a score for SRC activation, suggesting resistance is dependent on SRC. Our data show that gene expression signature scores for RAS pathway activation and for MEKi resistance may be useful in determining which CRC tumors will respond to the novel drug combination of MEKi and SRCi.

An integrative gene expression signature analysis identifies CMS4 KRAS-mutated colorectal cancers sensitive to combined MEK and SRC targeted therapy.

BACKGROUND: Over half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s). METHODS: We classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi "bypass" resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154). RESULTS: Here we report a central role of SRC in mediating "bypass"-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P < 10-320), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs. CONCLUSIONS: Large human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node--an Achilles' heel--in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC.

Interaction of lncRNA MIR100HG with hnRNPA2B1 facilitates m6A-dependent stabilization of TCF7L2 mRNA and colorectal cancer progression.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a process linked to metastasis and drug resistance with non-coding RNAs (ncRNAs) playing pivotal roles. We previously showed that miR-100 and miR-125b, embedded within the third intron of the ncRNA host gene MIR100HG, confer resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in colorectal cancer (CRC). However, whether the MIR100HG transcript itself has a role in cetuximab resistance or EMT is unknown. METHODS: The correlation between MIR100HG and EMT was analyzed by curating public CRC data repositories. The biological roles of MIR100HG in EMT, metastasis and cetuximab resistance in CRC were determined both in vitro and in vivo. The expression patterns of MIR100HG, hnRNPA2B1 and TCF7L2 in CRC specimens from patients who progressed on cetuximab and patients with metastatic disease were analyzed by RNAscope and immunohistochemical staining. RESULTS: The expression of MIR100HG was strongly correlated with EMT markers and acted as a positive regulator of EMT. MIR100HG sustained cetuximab resistance and facilitated invasion and metastasis in CRC cells both in vitro and in vivo. hnRNPA2B1 was identified as a binding partner of MIR100HG. Mechanistically, MIR100HG maintained mRNA stability of TCF7L2, a major transcriptional coactivator of the Wnt/ß-catenin signaling, by interacting with hnRNPA2B1. hnRNPA2B1 recognized the N6-methyladenosine (m6A) site of TCF7L2 mRNA in the presence of MIR100HG. TCF7L2, in turn, activated MIR100HG transcription, forming a feed forward regulatory loop. The MIR100HG/hnRNPA2B1/TCF7L2 axis was augmented in specimens from CRC patients who either developed local or distant metastasis or had disease progression that was associated with cetuximab resistance. CONCLUSIONS: MIR100HG and hnRNPA2B1 interact to control the transcriptional activity of Wnt signaling in CRC via regulation of TCF7L2 mRNA stability. Our findings identified MIR100HG as a potent EMT inducer in CRC that may contribute to cetuximab resistance and metastasis by activation of a MIR100HG/hnRNPA2B1/TCF7L2 feedback loop.

APC and TP53 Mutations Predict Cetuximab Sensitivity across Consensus Molecular Subtypes.

Recently, it was suggested that consensus molecular subtyping (CMS) may aide in predicting response to EGFR inhibitor (cetuximab) therapies. We recently identified that APC and TP53 as two tumor suppressor genes, when mutated, may enhance cetuximab sensitivity and may represent easily measured biomarkers in tumors or blood. Our study aimed to use APC and TP53 mutations (AP) to refine the CMS classification to better predict responses to cetuximab. In total, 433 CRC tumors were classified into CMS1-4 subtypes. The cetuximab sensitivity (CTX-S) signature scores of AP vs. non-AP tumors were determined across each of the CMS classes. Tumors harboring combined AP mutations were predominantly enriched in the CMS2 class, and to a lesser degree, in the CMS4 class. On the other hand, AP mutated CRCs had significantly higher CTX-S scores compared to non-AP CRCs across all CMS classes. Similar results were also obtained in independent TCGA tumor collections (n = 531) and in PDMR PDX/PDO/PDC models (n = 477). In addition, the in vitro cetuximab growth inhibition was preferentially associated with the CMS2 cell lines harboring A/P genotypes. In conclusion, the AP mutation signature represents a convenient biomarker that refines the CMS classification to identify CRC subpopulations predicted to be sensitive to EGFR targeted therapies.

Impact of rurality on melanoma diagnosis in Utah.

AIM: To analyze trends in Utah melanoma diagnosis and study the impact of rurality. PATIENTS & METHODS: State-wide melanoma incidence was calculated using Surveillance, Epidemiology, and End Results data (2005-2013). A subset of 5199 patients treated in an integrated healthcare system was further stratified for urban or rural residence. RESULTS: Early-stage tumors accounted for most of the increase in melanoma incidence over time. Age-adjusted melanoma incidence rate was higher in rural counties (46.7 vs 39.4). Anatomic site and stage did not differ between rural and urban patients. Rural patients were more commonly diagnosed by a local primary care provider. CONCLUSION: Rurality had an impact on melanoma diagnosis in the specialty and location of the diagnosing provider.

Telehealth Is a Sustainable Population Health Strategy to Lower Costs and Increase Quality of Health Care in Rural Utah.

PURPOSE: A telehealth oncology practice was created to care for patients in rural communities to improve access to health care, decrease financial burdens, and save time. PATIENTS AND METHODS: Patients with cancer at Sevier Valley Hospital in Richfield, Utah, were treated with a real-time video-based telehealth program under the care of an oncologist at a tertiary medical center. Data on financial savings, travel hours, mileage avoided, carbon emissions reduced, and revenue retained by Sevier Valley Hospital were collected from 2015 to 2018. RESULTS: From 2015 to 2018, 119 patients with cancer in Richfield, Utah, were treated with telehealth for oncology visits, accounting for 1,025 patient encounters. On average, patients saved 4 hours and 40 minutes and 332 miles roundtrip per encounter. In total, patients' savings were estimated to be $333,074. Carbon emissions were reduced by approximately 150,000 kg. Of new patient referrals, 59% were for solid tumors (70 of 119 referrals; 42 metastatic and 28 nonmetastatic cancers), and 41% were hematology consultations (49 of 119 referrals; 28 classical and 21 malignant hematologic conditions). We estimate that Sevier Valley Hospital retained $3,605,500 in revenue over this 4-year period. CONCLUSION: Using a telehealth program in rural Utah, patients with cancer benefited from substantial time and monetary savings. The local medical center was able to retain revenue it would have otherwise lost to outsourcing cancer care. Recent regulatory changes to address the COVID-19 pandemic should increase the number of patients with cancer treated via telehealth nationwide.

PTPRS drives adaptive resistance to MEK/ERK inhibitors through SRC.

PTPRS is the most commonly mutated receptor tyrosine phosphatase in colorectal cancer (CRC). PTPRS has been shown to directly affect ERK and regulate its activation and nuclear localization. Here we identify that PTPRS may play a significant role in developing adaptive resistance to MEK/ERK inhibitors (MEKi/ERKi) through SRC activation. Moreover, we demonstrate a new clinical approach to averting adaptive resistance through the use of the SRC inhibitor, dasatinib. Our data suggest the potential for dasatinib to enhance the efficacy of MEKi and ERKi by preventing adaptive resistance pathways operating through SRC.

Repurposing EGFR Inhibitor Utility in Colorectal Cancer in Mutant APC and TP53 Subpopulations.

BACKGROUND: EGFR is a major therapeutic target for colorectal cancer. Currently, extended RAS/RAF testing identifies only nonresponders to EGFR inhibitors (EGFRi). We aimed to develop a mutation signature that further refines drug-sensitive subpopulations to improve EGFRi outcomes. METHODS: A prespecified, 203-gene expression signature score measuring cetuximab sensitivity (CTX-S) was validated with two independent clinical trial datasets of cetuximab-treated patients with colorectal cancer (n = 44 and n = 80) as well as an in vitro dataset of 147 cell lines. The CTX-S score was then used to decipher mutated genes that predict EGFRi sensitivity. The predictive value of the identified mutation signature was further validated by additional independent datasets. RESULTS: Here, we report the discovery of a 2-gene (APC+TP53) mutation signature that was useful in identifying EGFRi-sensitive colorectal cancer subpopulations. Mutant APC+TP53 tumors were more predominant in left- versus right-sided colorectal cancers (52% vs. 21%, P = 0.0004), in microsatellite stable (MSS) versus microsatellite instable (MSI) cases (47% vs. 2%, P < 0.0001), and in the consensus molecular subtype 2 versus others (75% vs. 37%, P < 0.0001). Moreover, mutant APC+TP53 tumors had favorable outcomes in two cetuximab-treated patient-derived tumor xenograft (PDX) datasets (P = 0.0277, n = 52; P = 0.0008, n = 98). CONCLUSIONS: Our findings suggest that the APC and TP53 combination mutation may account for the laterality of EGFRi sensitivity and provide a rationale for refining treated populations. The results also suggest addition of APC+TP53 sequencing to extended RAS/RAF testing that may directly increase the response rates of EGFRi therapy in selected patients. IMPACT: These findings, if further validated through clinical trials, could also expand the utility of EGFRi therapies that are currently underutilized.

PTPRS Regulates Colorectal Cancer RAS Pathway Activity by Inactivating Erk and Preventing Its Nuclear Translocation.

Colorectal cancer (CRC) growth and progression is frequently driven by RAS pathway activation through upstream growth factor receptor activation or through mutational activation of KRAS or BRAF. Here we describe an additional mechanism by which the RAS pathway may be modulated in CRC. PTPRS, a receptor-type protein tyrosine phosphatase, appears to regulate RAS pathway activation through ERK. PTPRS modulates ERK phosphorylation and subsequent translocation to the nucleus. Native mutations in PTPRS, present in ~10% of CRC, may reduce its phosphatase activity while increasing ERK activation and downstream transcriptional signaling.

Whole Genome Sequencing Analysis of a Stage IV Colon Cancer Patient with a 10-Year Disease-Free Survival following Systemic Chemotherapy/Bevacizumab.

It is rare that stage IV colon cancer is cured with chemotherapy. Here we report the long-term survival of a patient who presented with highly advanced disease characterized by a papillary architecture as well as porta hepatis lymph nodes but responded extremely well to FOLFIRI/bevacizumab. His original tumor underwent comprehensive genomic testing that included whole genome DNA sequencing, targeted sequencing, and RNA sequencing. These genetic results suggest the patient's tumor harbored mutations in APC, KRAS, and TP53 as well as in PIK3CB. Moreover, the RNA-seq data suggested that the tumor belonged to the consensus molecular subtype 4, the "inflamed, immune phenotype," with increased angiogenesis. Deep sequencing of highly responsive cancers may yield molecular insights into mechanisms underpinning a remarkable response.

lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via Wnt/ß-catenin signaling.

De novo and acquired resistance, which are largely attributed to genetic alterations, are barriers to effective anti-epidermal-growth-factor-receptor (EGFR) therapy. To generate cetuximab-resistant cells, we exposed cetuximab-sensitive colorectal cancer cells to cetuximab in three-dimensional culture. Using whole-exome sequencing and transcriptional profiling, we found that the long non-coding RNA MIR100HG and two embedded microRNAs, miR-100 and miR-125b, were overexpressed in the absence of known genetic events linked to cetuximab resistance. MIR100HG, miR-100 and miR-125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100 and miR-125b coordinately repressed five Wnt/ß-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. Our results describe a double-negative feedback loop between MIR100HG and the transcription factor GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These findings identify a clinically actionable, epigenetic cause of cetuximab resistance.

Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer.

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

Adaptation of a RAS pathway activation signature from FF to FFPE tissues in colorectal cancer.

BACKGROUND: The KRAS gene is mutated in about 40 % of colorectal cancer (CRC) cases, which has been clinically validated as a predictive mutational marker of intrinsic resistance to anti-EGFR inhibitor (EGFRi) therapy. Since nearly 60 % of patients with a wild type KRAS fail to respond to EGFRi combination therapies, there is a need to develop more reliable molecular signatures to better predict response. Here we address the challenge of adapting a gene expression signature predictive of RAS pathway activation, created using fresh frozen (FF) tissues, for use with more widely available formalin fixed paraffin-embedded (FFPE) tissues. METHODS: In this study, we evaluated the translation of an 18-gene RAS pathway signature score from FF to FFPE in 54 CRC cases, using a head-to-head comparison of five technology platforms. FFPE-based technologies included the Affymetrix GeneChip (Affy), NanoString nCounter™ (NanoS), Illumina whole genome RNASeq (RNA-Acc), Illumina targeted RNASeq (t-RNA), and Illumina stranded Total RNA-rRNA-depletion (rRNA). RESULTS: Using Affy_FF as the "gold" standard, initial analysis of the 18-gene RAS scores on all 54 samples shows varying pairwise Spearman correlations, with (1) Affy_FFPE (r = 0.233, p = 0.090); (2) NanoS_FFPE (r = 0.608, p < 0.0001); (3) RNA-Acc_FFPE (r = 0.175, p = 0.21); (4) t-RNA_FFPE (r = -0.237, p = 0.085); (5) and t-RNA (r = -0.012, p = 0.93). These results suggest that only NanoString has successful FF to FFPE translation. The subsequent removal of identified "problematic" samples (n = 15) and genes (n = 2) further improves the correlations of Affy_FF with three of the five technologies: Affy_FFPE (r = 0.672, p < 0.0001); NanoS_FFPE (r = 0.738, p < 0.0001); and RNA-Acc_FFPE (r = 0.483, p = 0.002). CONCLUSIONS: Of the five technology platforms tested, NanoString technology provides a more faithful translation of the RAS pathway gene expression signature from FF to FFPE than the Affymetrix GeneChip and multiple RNASeq technologies. Moreover, NanoString was the most forgiving technology in the analysis of samples with presumably poor RNA quality. Using this approach, the RAS signature score may now be reasonably applied to FFPE clinical samples.

A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC.

Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC.

Adenomatous polyposis coli (APC) regulates miR17-92 cluster through ß-catenin pathway in colorectal cancer.

Adenomatous polyposis coli (APC) mutation is the most common genetic change in sporadic colorectal cancer (CRC). Although deregulations of miRNAs have been frequently reported in this malignancy, APC-regulated miRNAs have not been extensively documented. Here, by using an APC-inducible cell line and array analysis, we identified a total of 26 deregulated miRNAs. Among them, members of miR-17-92 cluster were dramatically inhibited by APC and induced by enforced expression of ß-catenin. Furthermore, we demonstrate that activated ß-catenin resulted from APC loss binds to and activates the miR-17-92 promoter. Notably, enforced expression of miR-19a overrides APC tumor suppressor activity, and knockdown of miR-19a in cancer cells with compromised APC function reduced their aggressive features in vitro. Finally, we observed that expression of miR-19a significantly correlates with ß-catenin levels in colorectal cancer specimens, and it is associated to the aggressive stage of tumor progression. Thus, our study reveals that miR-17-92 cluster is directly regulated by APC/ß-catenin pathway and could be a potential therapeutic target in colon cancers with aberrant APC/ß-catenin signaling.

A Composite Gene Expression Signature Optimizes Prediction of Colorectal Cancer Metastasis and Outcome.

PURPOSE: We previously found that an epithelial-to-mesenchymal transition (EMT)-based gene expression signature was highly correlated with the first principal component (PC1) of 326 colorectal cancer tumors and was prognostic. This study was designed to improve these signatures for better prediction of metastasis and outcome. EXPERIMENTAL DESIGN: A total of 468 colorectal cancer tumors including all stages (I-IV) and metastatic lesions were used to develop a new prognostic score (ΔPC1.EMT) by subtracting the EMT signature score from its correlated PC1 signature score. The score was validated on six other independent datasets with a total of 3,697 tumors. RESULTS: ΔPC1.EMT was found to be far more predictive of metastasis and outcome than its parent scores. It performed well in stages I to III, among microsatellite instability subtypes, and across multiple mutation-based subclasses, demonstrating a refined capacity to predict distant metastatic potential even in tumors with a "good" prognosis. For example, in the PETACC-3 clinical trial dataset, it predicted worse overall survival in an adjusted multivariable model for stage III patients (HR standardized by interquartile range [IQR] = 1.50; 95% confidence interval, 1.25-1.81; P = 0.000016, N = 644). The improved performance of ΔPC1.EMT was related to its propensity to identify epithelial-like subpopulations as well as mesenchymal-like subpopulations. Biologically, the signature was correlated positively with RAS signaling but negatively with mitochondrial metabolism. ΔPC1.EMT was a "best of assessed" prognostic score when compared with 10 other known prognostic signatures. CONCLUSIONS: The study developed a prognostic signature score with a propensity to detect non-EMT features, including epithelial cancer stem cell-related properties, thereby improving its potential to predict metastasis and poorer outcome in stage I-III patients.

Suppression of Reserve MCM Complexes Chemosensitizes to Gemcitabine and 5-Fluorouracil.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is very difficult to treat with conventional chemotherapeutic regimens. Gemcitabine and 5-fluorouracil are used in the management of PDAC and act by indirectly blocking replicative forks. However, these drugs are not highly effective at suppressing disease progression, indicating a need for the development of innovative therapeutic approaches. Recent studies indicate that suppression of the MCM helicase may provide a novel means to sensitize cancer cells to chemotherapeutic agents that inhibit replicative fork progression. Mammalian cells assemble more MCM complexes on DNA than are required to start S-phase. The excess MCM complexes function as backup initiation sites under conditions of replicative stress. The current study provides definitive evidence that cosuppression of the excess/backup MCM complexes sensitizes PDAC tumor lines to both gemcitabine and 5-FU, leading to increased loss of proliferative capacity compared with drugs alone. This occurs because reduced MCM levels prevent efficient recovery of DNA replication in tumor cells exposed to drug. PDAC tumor cells are more sensitive to MCM loss in the presence of gemcitabine than are nontumor, immortalized epithelial cells. Similarly, colon tumor cells are rendered less viable when cosuppression of MCM complexes occurs during exposure to the crosslinking agent oxaliplatin or topoisomerase inhibitor etoposide. IMPLICATIONS: These studies demonstrate that suppressing the backup complement of MCM complexes provides an effective sensitizing approach with the potential to increase the therapeutic index of drugs used in the clinical management of PDAC and other cancers.