Phase III Prospectively Randomized Trial of Perioperative 5-FU After Curative Resection for Colon Cancer: An Intergroup Trial of the ECOG-ACRIN Cancer Research Group (E1292).

BACKGROUND: Studies suggest that adjuvant chemotherapy should be initiated at the earliest possible time. The Eastern Cooperative Oncology Group (ECOG) and Intergroup evaluated the effect of perioperative fluorouracil (5-FU) on overall survival (OS) for colon cancer. PATIENTS AND METHODS: This phase III trial randomized patients to receive continuous infusional 5-FU for 7 days starting within 24 h after curative resection (arm A) or no perioperative 5-FU (arm B). Patients with Dukes' B3 and C disease received adjuvant chemotherapy per standard of care. The primary endpoint of the trial was overall survival in patients with Dukes' B3 and C disease. The secondary objective was to determine whether a week of perioperative infusion would affect survival in patients with Dukes' B2 colon cancer with no additional chemotherapy. RESULTS: From August 1993 to May 2000, 859 patients were enrolled and 855 randomized (arm A: 427; arm B: 428). The trial was terminated early due to slow accrual. The median follow-up is 15.4 years (0.03-20.3 years). Among patients with Dukes' B3 and C disease, there was no statistically significant difference in OS [median 10.3 years (95% CI 8.4, 13.2) for perioperative chemotherapy and 9.3 years (95% CI 5.7, 12.3) for no perioperative therapy, one-sided log-rank p = 0.178, HR = 0.88 (95% CI 0.66, 1.16)] or disease-free survival (DFS). For patients with Dukes' B2 disease, there was also no significant difference in OS (median 16.1 versus 12.9 years) or DFS. There was no difference between treatment arms in operative complications. One week of continuous infusion of 5-FU was tolerable; 18% of arm A patients experienced grade 3 or greater toxicity.

Immunotherapy-Based Neoadjuvant Treatment of Advanced Microsatellite Instability-High Gastric Cancer: A Case Series.

Despite the use of first-line therapies like fluoropyrimidine and platinum-based cytotoxic chemotherapy, gastric cancer (GC) continues to carry a poor prognosis. Recent subgroup analyses of first-line phase III trials have demonstrated that patients with microsatellite instability-high (MSI-H) metastatic GC derive significant improvement in survival rates when immune checkpoint inhibitors (ICIs) are combined with chemotherapy compared with chemotherapy alone. However, it remains to be seen whether the success of ICIs in the metastatic setting can be translated into earlier stages of GC with resectable disease. We report 6 cases of locally advanced, nonmetastatic MSI-H GC that all demonstrated favorable response following treatment with pembrolizumab in addition to neoadjuvant chemotherapy. With the exception of immune-related colitis in one patient, pembrolizumab was well-tolerated. To our knowledge, this is the first reported US case series of patients treated with an ICI in combination with neoadjuvant chemotherapy for advanced, nonmetastatic, resectable or unresectable MSI-H GC.

A review of nasopharyngeal swab and saliva tests for SARS-CoV-2 infection: Disease timelines, relative sensitivities, and test optimization.

Testing is an essential part of containment of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. This review summarizes studies for SARS-CoV-2 infection and testing. Nasopharyngeal samples are best at sensitivity detection, especially in early stages of disease and in asymptomatic individuals. Current swab processing involves a 100- to 1000-fold dilution of the patient sample. Future optimization of testing should focus on using smaller volumes of viral transport media and swab designs to increase comfort and increased viral adhesion.

Intergroup Randomized Phase III Study of Postoperative Oxaliplatin, 5-Fluorouracil, and Leucovorin Versus Oxaliplatin, 5-Fluorouracil, Leucovorin, and Bevacizumab for Patients with Stage II or III Rectal Cancer Receiving Preoperative Chemoradiation: A Trial of the ECOG-ACRIN Research Group (E5204).

BACKGROUND: The addition of bevacizumab to chemotherapy improved outcomes for patients with metastatic colon cancer. E5204 was designed to test whether the addition of bevacizumab to mFOLFOX6, following neoadjuvant chemoradiation and definitive surgery, could improve overall survival (OS) in patients with stage II/III adenocarcinoma of the rectum. SUBJECTS, MATERIALS, AND METHODS: Patients with stage II/III rectal cancer who had completed neoadjuvant 5-fluorouracil-based chemoradiation and had undergone complete resection were enrolled. Patients were randomized to mFOLFOX6 (Arm A) or mFOLFOX6 with bevacizumab (Arm B) administered every 2 weeks for 12 cycles. RESULTS: E5204 registered only 355 patients (17% of planned accrual goal) as it was terminated prematurely owing to poor accrual. At a median follow-up of 72 months, there was no difference in 5-year overall survival (88.3% vs. 83.7%) or 5-year disease-free survival (71.2% vs. 76.5%) between the two arms. The rate of treatment-related grade ≥ 3 adverse events (AEs) was 68.8% on Arm A and 70.7% on Arm B. Arm B had a higher proportion of patients who discontinued therapy early as a result of AEs and patient withdrawal than did Arm A (32.4% vs. 21.5%, p = .029).The most common grade 3-4 treatment-related AEs were neutropenia, leukopenia, neuropathy, diarrhea (without prior colostomy), and fatigue. CONCLUSION: At 17% of its planned accrual, E5204 did not meet its primary endpoint. The addition of bevacizumab to FOLFOX6 in the adjuvant setting did not significantly improve OS in patients with stage II/III rectal cancer. IMPLICATIONS FOR PRACTICE: At 17% of its planned accrual, E5204 was terminated early owing to poor accrual. At a median follow-up of 72 months, there was no significant difference in 5-year overall survival (88.3% vs. 83.7%) or in 5-year disease-free survival (71.2% vs. 76.5%) between the two arms. Despite significant advances in the treatment of rectal cancer, especially in improving local control rates, the risk of distant metastases and the need to further improve quality of life remain a challenge. Strategies combining novel agents with chemoradiation to improve both distant and local control are needed.

Signet ring cell colorectal cancer: genomic insights into a rare subpopulation of colorectal adenocarcinoma.

BACKGROUND: Signet ring cell carcinoma (SRCC) is a rare subtype of colorectal cancer (CRC). The aim of this study was to characterise the genomic alterations and outcomes of SRCC. METHODS: Medical records of metastatic CRC (mCRC) patients whose tumours were evaluated by NGS analysis were reviewed. SC-mCRC were classified into two groups: SRCC (>50% signet ring cells) and adenocarcinoma (AC) with SC component (≤50% signet ring cells). RESULTS: Six hundred and sixty-five mCRC patients were included. Of the 93 mCRC cases with SC features, 63 had slides for review. Of those 63 cases, 35 were confirmed SRCC, and 28 were AC with SC component. Compared with AC group, KRAS and PIK3CA mutations (mts) were found in only 11% (OR: 0.13) and 3% (OR: 0.15) of SRCC cases, respectively. In contrast to the 44% rate of APC mts in AC group, only 3% of SRCC patients had APC mts (OR = 0.04). CONCLUSIONS: SRCC has distinct molecular features, including low rates of KRAS, PIK3CA and APC mts. Further study to identify activation pathways and potential therapeutic targets are needed.

NCCN Guidelines Insights: Genetic/Familial High-Risk Assessment: Colorectal, Version 2.2019.

Identifying individuals with hereditary syndromes allows for improved cancer surveillance, risk reduction, and optimized management. Establishing criteria for assessment allows for the identification of individuals who are carriers of pathogenic genetic variants. The NCCN Guidelines for Genetic/Familial High-Risk Assessment: Colorectal provide recommendations for the assessment and management of patients with high-risk colorectal cancer syndromes. These NCCN Guidelines Insights focus on criteria for the evaluation of Lynch syndrome and considerations for use of multigene testing in the assessment of hereditary colorectal cancer syndromes.

Apoptosis and expression of apoptosis-related genes in mouse intestinal tissue after whole-body proton exposure.

Energetic protons are the most abundant particle type in space and can pose serious health risks to astronauts during long-duration missions. The health effects of proton exposure are also a concern for cancer patients undergoing radiation treatment with accelerated protons. To investigate the damage induced by energetic protons in vivo to radiosensitive organs, 6-week-old BALB/c male mice were subjected to 250 MeV proton radiation at whole-body doses of 0.1, 1, and 2 Gy. The gastrointestinal (GI) tract of each exposed animal was dissected 4 h post-irradiation, and the isolated small intestinal tissue was analyzed for histopathological and gene expression changes. Histopathologic observation of the tissue using standard hematoxylin and eosin (H&E) staining methods to screen for morphologic changes showed a marked increase in apoptotic lesions for even the lowest dose of 0.1 Gy, similar to X- or γ rays. The percentage of apoptotic cells increased dose-dependently, but the dose response appeared supralinear, indicating hypersensitivity at low doses. A significant decrease in surviving crypts and mucosal surface area, as well as in cell proliferation, was also observed in irradiated mice. Gene expression analysis of 84 genes involved in the apoptotic process showed that most of the genes affected by protons were common between the low (0.1 Gy) and high (1 and 2 Gy) doses. However, the genes that were distinctively responsive to the low or high doses suggest that high doses of protons may cause apoptosis in the small intestine by direct damage to the DNA, whereas low doses of protons may trigger apoptosis through a different stress response mechanism.

NCCN Guidelines Insights: Colorectal Cancer Screening, Version 1.2018.

The NCCN Guidelines for Colorectal Cancer (CRC) Screening outline various screening modalities as well as recommended screening strategies for individuals at average or increased-risk of developing sporadic CRC. The NCCN panel meets at least annually to review comments from reviewers within their institutions, examine relevant data, and reevaluate and update their recommendations. These NCCN Guidelines Insights summarize 2018 updates to the NCCN Guidelines, with a primary focus on modalities used to screen individuals at average-risk for CRC.

MIIP haploinsufficiency induces chromosomal instability and promotes tumour progression in colorectal cancer.

The gene encoding migration and invasion inhibitory protein (MIIP), located on 1p36.22, is a potential tumour suppressor gene in glioma. In this study, we aimed to explore the role and mechanism of action of MIIP in colorectal cancer (CRC). MIIP protein expression gradually decreased along the colorectal adenoma-carcinoma sequence and was negatively correlated with lymph node and distant metastasis in 526 colorectal tissue samples (p < 0.05 for all). Analysis of The Cancer Genome Atlas (TCGA) data showed that decreased MIIP expression was significantly associated with MIIP hemizygous deletion (p = 0.0005), which was detected in 27.7% (52/188) of CRC cases, and associated with lymph node and distant metastasis (p < 0.05 for both). We deleted one copy of the MIIP gene in HCT116 CRC cells using zinc finger nuclease technology and demonstrated that MIIP haploinsufficiency resulted in increased colony formation and cell migration and invasion, which was consistent with the results from siRNA-mediated MIIP knockdown in two CRC cell lines (p < 0.05 for all). Moreover, MIIP haploinsufficiency promoted CRC progression in vivo (p < 0.05). Genomic instability and spectral karyotyping assays demonstrated that MIIP haploinsufficiency induced chromosomal instability (CIN). Besides modulating the downstream proteins of APC/CCdc20 , securin and cyclin B1, MIIP haploinsufficiency inhibited topoisomerase II (Topo II) activity and induced chromosomal missegregation. Therefore, we report that MIIP is a novel potential tumour suppressor gene in CRC. Moreover, we characterized the MIIP gene as a novel CIN suppressor gene, through altering the stability of mitotic checkpoint proteins and disturbing Topo II activity. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical and Molecular Characteristics of Post-Colonoscopy Colorectal Cancer: A Population-based Study.

BACKGROUND & AIMS: Colonoscopy provides incomplete protection from colorectal cancer (CRC), but determinants of post-colonoscopy CRC are not well understood. We compared clinical features and molecular characteristics of CRCs diagnosed at different time intervals after a previous colonoscopy. METHODS: We performed a population-based, cross-sectional study of incident CRC cases in Denmark (2007-2011), categorized as post-colonoscopy or detected during diagnostic colonoscopy (in patients with no prior colonoscopy). We compared prevalence of proximal location and DNA mismatch repair deficiency (dMMR) in CRC tumors, relative to time since previous colonoscopy, using logistic regression and cubic splines to assess temporal variation. RESULTS: Of 10,365 incident CRCs, 725 occurred after colonoscopy examinations (7.0%). These were more often located in the proximal colon (odds ratio [OR], 2.34; 95% confidence interval [CI], 1.90-2.89) and were more likely to have dMMR (OR, 1.26; 95% CI, 1.00-1.59), but were less likely to be metastatic at presentation (OR, 0.65; 95% CI, 0.48-0.89) compared with CRCs diagnosed in patients with no prior colonoscopy. The highest proportions of proximal and/or dMMR tumors were observed in CRCs diagnosed 3-6 years after colonoscopy, but these features were still more frequent among cancers diagnosed up to 10 years after colonoscopy. The relative excess of dMMR tumors was most pronounced in distal cancers. In an analysis of 85 cases detected after colonoscopy, we found BRAF mutations in 23% of tumors and that 7% of cases had features of Lynch syndrome. Colonoscopy exams were incomplete in a higher proportion of cases diagnosed within <1 year (in 38%) than in those diagnosed within 1-10 years after colonoscopy (16%). CONCLUSIONS: In a study of incident CRC cases in Denmark, we observed that tumors found in patients who have undergone colonoscopy are more often proximal and have dMMR compared to CRCs detected in patients without previous colonoscopies. The excess of right-sided tumors and the modest independent effects of dMMR reinforce the importance of proper colonoscopic examination of the proximal large bowel.

Increased Risk of Colorectal Cancer Development Among Patients With Serrated Polyps.

BACKGROUND & AIMS: Sessile serrated adenomas/polyps (SSA/Ps) and traditional serrated adenomas (TSAs) are now distinguished from hyperplastic polyps and recognized as precursors to colorectal cancer (CRC). We studied CRC risks associated with serrated polyps. METHODS: By using Danish databases (1977-2009), we conducted a nationwide population-based, case-control study nested within individuals who had received colonoscopies (n = 272,342), and identified 2045 CRC cases and 8105 CRC-free individuals (controls). For each case and control, we identified the first colorectal polyp(s) that underwent a biopsy or were excised during or after the initial colonoscopy, and obtained tissue blocks for hyperplastic lesions. Four expert pathologists reviewed these lesions using current terminology for serrated polyps. We used logistic regression to compute odds ratios (ORs) to associate the risk of CRC with polyp type and estimated the absolute risks by multiplying the risk in patients with no polyps by these ORs. RESULTS: Seventy-nine cases and 142 controls had SSA/Ps (OR, 3.07; 95% confidence interval [CI], 2.30-4.10). SSA/Ps with cytology markers of dysplasia were associated with a particularly high OR (4.76; 95% CI, 2.59-8.73). Women with SSA/P had a higher risk for CRC than men with SSA/P (OR for women, 5.05; 95% CI, 3.05-8.37 vs OR for men, 2.18; 95% CI, 1.24-3.82); patients with SSA/P proximal to the splenic flexure had the highest risk for CRC (OR, 12.42; 95% CI, 4.88-31.58). The OR for CRC was 4.84 in the 14 cases vs 17 controls with TSAs (95% CI, 2.36-9.93), 2.51 in the 757 cases vs 1698 controls with conventional adenomas (95% CI, 2.25-2.80), and 1.30 in the 55 cases vs 235 controls with hyperplastic polyps (95% CI, 0.96-1.77). The 10-year risk for CRC was 4.4% for patients with SSA/P with dysplasia, 4.5% for patients with TSAs, and 2.3% for patients with conventional adenomas. CONCLUSION: Patients with SSA/P or TSA are at increased risk for CRC; their level of risk is similar to or higher than that of patients with conventional adenomas.

NCCN Guidelines Insights: Genetic/Familial High-Risk Assessment: Colorectal, Version 3.2017.

The NCCN Guidelines for Genetic/Familial High-Risk Assessment: Colorectal provide recommendations for the management of patients with high-risk syndromes associated with an increased risk of colorectal cancer (CRC). The NCCN Panel for Genetic/Familial High-Risk Assessment: Colorectal meets at least annually to assess comments from reviewers within their institutions, examine relevant data, and reevaluate and update their recommendations. These NCCN Guidelines Insights focus on genes newly associated with CRC risk on multigene panels, the associated evidence, and currently recommended management strategies.

The clinical and biological significance of MIR-224 expression in colorectal cancer metastasis.

OBJECTIVE: MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis. DESIGN: We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan-Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression. RESULTS: MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=-0.44, p<0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175). CONCLUSIONS: MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.

Association of CpG island methylator phenotype and EREG/AREG methylation and expression in colorectal cancer.

BACKGROUND: High EREG and AREG expression, and left-sided primary tumours are associated with superior efficacy of anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (CRC), but a unifying explanation of these findings is lacking. METHODS: RNA-seq, gene expression arrays, and DNA methylation profiling were completed on 179 CRC tumours. Results were validated using independent The Cancer Genome Atlas data sets. An independent cohort of 198 KRAS wild-type metastatic CRC tumours was tested for CpG island methylator phenotype (CIMP) status, and progression-free survival (PFS) with the first anti-EGFR regimen was retrospectively determined. RESULTS: EREG and AREG expression was highly inversely correlated with methylation and was inversely associated with right-sided primary tumour, BRAF mutation, and CIMP-high status. Treatment of CRC cell lines with hypomethylating agents decreased methylation and increased expression of EREG. Inferior PFS with anti-EGFR therapy was associated with CIMP-high status, BRAF mutation, NRAS mutation, and right-sided primary tumour on univariate analysis. Among known BRAF/NRAS wild-type tumours, inferior PFS remained associated with CIMP-high status (median PFS 5.6 vs 9.0 mo, P=0.023). CONCLUSIONS: EREG and AREG are strongly regulated by methylation, and their expression is associated with CIMP status and primary tumour site, which may explain the association of primary tumour site and EREG/AREG expression with anti-EGFR therapy efficacy.

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer.

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Genetic/Familial High-Risk Assessment: Colorectal Version 1.2016, NCCN Clinical Practice Guidelines in Oncology.

This is a focused update highlighting the most current NCCN Guidelines for diagnosis and management of Lynch syndrome. Lynch syndrome is the most common cause of hereditary colorectal cancer, usually resulting from a germline mutation in 1 of 4 DNA mismatch repair genes (MLH1, MSH2, MSH6, or PMS2), or deletions in the EPCAM promoter. Patients with Lynch syndrome are at an increased lifetime risk, compared with the general population, for colorectal cancer, endometrial cancer, and other cancers, including of the stomach and ovary. As of 2016, the panel recommends screening all patients with colorectal cancer for Lynch syndrome and provides recommendations for surveillance for early detection and prevention of Lynch syndrome-associated cancers.

Regulation of AURKC expression by CpG island methylation in human cancer cells.

AURKC, a member of the Aurora kinase gene family, is highly expressed in testis but is either moderately expressed or repressed in most somatic cells. Varying expression of AURKC has been observed in human cancers, but the underlying mechanisms of differential expression have been investigated only to a limited extent. We investigated the role of promoter CpG methylation in the regulation of AURKC gene expression in human cancer cells, in relation to a recently reported AURKC transcription repressor PLZF/ZBTB16, implicated in transformation and tumorigenesis. AURKC and PLZF/ZBTB16 expression profiles were investigated in reference to CpG methylation status on the AURKC promoter experimentally, and also in The Cancer Genome Atlas (TCGA) dataset involving multiple cancer types. AURKC promoter showed dense to moderate hypermethylation correlating with low to moderate expression of the gene in normal somatic cells and cancer cell lines, while testis with high expression revealed marked hypo-methylation. Treatment with the demethylating agent, 5-aza-dC, but not the histone deacetylase (HDAC) inhibitor, TSA, led to elevated expression in cancer cell lines, indicating that promoter DNA methylation negatively regulates AURKC expression. High expression of PLZF in PLZF-transfected cells treated with 5-aza-dC only partially repressed expression of AURKC despite 5-aza-dC also inducing elevated PLZF expression. Analyses of the TCGA data showed differential expression of AURKC in multiple cancer types and stronger correlation of AURKC expression with CpG methylation compared to PLZF levels. These findings demonstrate that differential promoter CpG methylation is an important mechanism regulating AURKC expression in cancer cells.

Colorectal Cancer Screening, Version 1.2015.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Colorectal Cancer Screening provide recommendations for selecting individuals for colorectal cancer screening, and for evaluation and follow-up of colon polyps. These NCCN Guidelines Insights summarize major discussion points of the 2015 NCCN Colorectal Cancer Screening panel meeting. Major discussion topics this year were the state of evidence for CT colonography and stool DNA testing, bowel preparation procedures for colonoscopy, and guidelines for patients with a positive family history of colorectal cancer.

Cellular location and expression of Na+, K+ -ATPase α subunits affect the anti-proliferative activity of oleandrin.

The purpose of this study was to investigate whether intracellular distribution of Na(+), K(+) -ATPase α3 subunit, a receptor for cardiac glycosides including oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellular distribution of Na(+), K(+) -ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na(+), K(+) -ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na(+), K(+) -ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy.

MLH1-silenced and non-silenced subgroups of hypermutated colorectal carcinomas have distinct mutational landscapes.

Approximately 15% of colorectal carcinomas (CRCs) exhibit a hypermutated genotype accompanied by high levels of microsatellite instability (MSI-H) and defects in DNA mismatch repair. These tumours, unlike the majority of colorectal carcinomas, are often diploid, exhibit frequent epigenetic silencing of the MLH1 DNA mismatch repair gene, and have a better clinical prognosis. As an adjunct study to The Cancer Genome Atlas consortium that recently analysed 224 colorectal cancers by whole exome sequencing, we compared the 35 CRCs (15.6%) with a hypermutated genotype to those with a non-hypermutated genotype. We found that 22 (63%) of the hypermutated CRCs exhibited transcriptional silencing of the MLH1 gene, a high frequency of BRAF V600E gene mutations, and infrequent APC and KRAS mutations, a mutational pattern significantly different from their non-hypermutated counterparts. However, the remaining 13 (37%) hypermutated CRCs lacked MLH1 silencing, contained tumours with the highest mutation rates ('ultramutated' CRCs), and exhibited higher incidences of APC and KRAS mutations, but infrequent BRAF mutations. These patterns were confirmed in an independent validation set of 250 exome-sequenced CRCs. Analysis of mRNA and microRNA expression signatures revealed that hypermutated CRCs with MLH1 silencing had greatly reduced levels of WNT signalling and increased BRAF signalling relative to non-hypermutated CRCs. Our findings suggest that hypermutated CRCs include one subgroup with fundamentally different pathways to malignancy than the majority of CRCs. Examination of MLH1 expression status and frequencies of APC, KRAS, and BRAF mutation in CRC may provide a useful diagnostic tool that could supplement the standard microsatellite instability assays and influence therapeutic decisions.