Statin and rottlerin small-molecule inhibitors restrict colon cancer progression and metastasis via MACC1.

MACC1 (Metastasis Associated in Colon Cancer 1) is a key driver and prognostic biomarker for cancer progression and metastasis in a large variety of solid tumor types, particularly colorectal cancer (CRC). However, no MACC1 inhibitors have been identified yet. Therefore, we aimed to target MACC1 expression using a luciferase reporter-based high-throughput screening with the ChemBioNet library of more than 30,000 compounds. The small molecules lovastatin and rottlerin emerged as the most potent MACC1 transcriptional inhibitors. They remarkably inhibited MACC1 promoter activity and expression, resulting in reduced cell motility. Lovastatin impaired the binding of the transcription factors c-Jun and Sp1 to the MACC1 promoter, thereby inhibiting MACC1 transcription. Most importantly, in CRC-xenografted mice, lovastatin and rottlerin restricted MACC1 expression and liver metastasis. This is-to the best of our knowledge-the first identification of inhibitors restricting cancer progression and metastasis via the novel target MACC1. This drug repositioning might be of therapeutic value for CRC patients.

Metastasis-associated in colon cancer 1 is an independent prognostic biomarker for survival in Klatskin tumor patients.

UNLABELLED: Curative treatment of intrahepatic cholangiocarcinoma (ICC) and hilar cholangiocarcinoma (Klatskin tumors) is limited to surgical resection or orthotopic liver transplantation. However, not all patients benefit from a surgical approach and suffer from early tumor recurrence. Response to chemotherapy is generally poor and, until today, no targeted therapy could be established. Metastasis-associated in colon cancer 1 (MACC1) is a recently discovered regulator of the hepatocyte growth factor (HGF)/Met/mitogen-activated protein kinase pathway, which induces proliferation, migration, and invasion in cell culture, as well as metastasis in mice. MACC1 expression shows a significant correlation with Met expression in colon cancer tissue and is highly prognostic for occurrence of distant metastasis and survival in colon cancer patients. Thus, we aimed to measure the expression of MACC1, Met, and HGF messenger RNA in microdissected tumor tissue and corresponding normal liver tissue of 156 patients with Klatskin tumors (n = 76) and ICC (n = 80) using real-time quantitative reverse-transcriptase polymerase chain reaction. We used immunohistochemical staining to validate the results. MACC1 expression in tumor tissue of both tumor entities was significantly higher than in corresponding normal liver tissue (P < 0.001). Klatskin tumor patients with a history of tumor recurrence had significantly higher MACC1 expression than those without tumor recurrence (P = 0.005). Uni- und multivariate survival analysis showed that Klatskin tumor patients with high MACC1 had a significantly shorter overall (OS) and disease-free survival (DFS; P = 0.001 and P < 0.001, respectively). The multivariate analysis confirmed MACC1 to be an independent factor for overall survival in Klatskin tumor patients (hazard ratio: 2.777; 95% confidence interval: 1.389-5.555; P = 0.004). CONCLUSION: Our study identified MACC1 as a highly prognostic biomarker for OS and DFS in Klatskin tumor patients. MACC1 expression could become an important diagnostic tool and might be a candidate for targeted therapy.

High MACC1 expression in combination with mutated KRAS G13 indicates poor survival of colorectal cancer patients.

BACKGROUND: The metastasis-associated in colon cancer 1 (MACC1) gene has been identified as prognostic biomarker for colorectal cancer (CRC). Here, we aimed at the refinement of risk assessment by separate and combined survival analyses of MACC1 expression with any of the markers KRAS mutated in codon 12 (KRAS G12) or codon 13 (KRAS G13), BRAF V600 mutation and MSI status in a retrospective study of 99 CRC patients with tumors UICC staged I, II and III. FINDINGS: We showed that only high MACC1 expression (HR: 6.09, 95% CI: 2.50-14.85, P < 0.001) and KRAS G13 mutation (HR: 5.19, 95% CI: 1.06-25.45, P = 0.042) were independent prognostic markers for shorter metastasis-free survival (MFS). Accordingly, Cox regression analysis revealed that patients with high MACC1 expression and KRAS G13 mutation exhibited the worst prognosis (HR: 14.48, 95% CI: 3.37-62.18, P < 0.001). Patients were classified based on their molecular characteristics into four clusters with significant differences in MFS (P = 0.003) by using the SPSS 2-step cluster function and Kaplan-Meier survival analysis. CONCLUSION: According to our results, patients with high MACC1 expression and mutated KRAS G13 exhibited the highest risk for metachronous metastases formation. Moreover, we demonstrated that the "Traditional pathway" with an intermediate risk for metastasis formation can be further subdivided by assessing MACC1 expression into a low and high risk group with regard to MFS prognosis. This is the first report showing that identification of CRC patients at high risk for metastasis is possible by assessing MACC1 expression in combination with KRAS G13 mutation.

The course of gastric cancer following surgery is associated with genetic variations of the interleukin-1 receptor antagonist and interleukin-1ß.

BACKGROUND: Inflammation, especially the cytokine response of the IL-1 family, has been shown to influence susceptibility to gastric cancer. In addition, several other pro-inflammatory cytokines have been demonstrated to influence metastasis and resistance to chemotherapy. Therefore, genetic variations within these genes may not only affect susceptibility but also influence the outcome of gastric cancer patients. A limited number of studies showed indeed an association of IL-1ß and IL-1RN variations with survival of gastric cancer patients. However, results are inconsistent, possibly because of different patient cohorts and different therapies. METHODS: In this retrospective cohort study we genotyped 154 patients with gastric cancer for IL-1ß and IL-1RN variations. Patients had undergone pathologically proven R0 resection and had received no additional adjuvant treatment. RESULTS: We show here a protective association with disease-free survival for both heterozygous genotypes, IL-1ß SNP C-511T (rs16944) and IL-1RN VNTR. The combination of both heterozygous genotypes is the strongest predictor independent of UICC stage. CONCLUSION: Genetic variations in the IL-1ß and IL-1RN genes influence disease progression in gastric cancer. Screening for these genetic variations might help to stratify therapies for gastric cancer patients in the future.

Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial.

BACKGROUND: Previous results of the EORTC intergroup trial 40983 showed that perioperative chemotherapy with FOLFOX4 (folinic acid, fluorouracil, and oxaliplatin) increases progression-free survival (PFS) compared with surgery alone for patients with initially resectable liver metastases from colorectal cancer. Here we present overall survival data after long-term follow-up. METHODS: This randomised, controlled, parallel-group, phase 3 study recruited patients from 78 hospitals across Europe, Australia, and Hong Kong. Eligible patients aged 18-80 years who had histologically proven colorectal cancer and up to four liver metastases were randomly assigned (1:1) to either perioperative FOLFOX4 or surgery alone. Perioperative FOLFOX4 consisted of six 14-day cycles of oxaliplatin 85mg/m(2), folinic acid 200 mg/m(2) (DL form) or 100 mg/m(2) (L form) on days 1-2 plus bolus, and fluorouracil 400 mg/m(2) (bolus) and 600 mg/m(2) (continuous 22 h infusion), before and after surgery. Patients were centrally randomised by minimisation, adjusting for centre and risk score and previous adjuvant chemotherapy to primary surgery for colorectal cancer, and the trial was open label. Analysis of overall survival was by intention to treat in all randomly assigned patients. FINDINGS: Between Oct 10, 2000, and July 5, 2004, 364 patients were randomly assigned to a treatment group (182 patients in each group, of which 171 per group were eligible and 152 per group underwent resection). At a median follow-up of 8·5 years (IQR 7·6-9·5), 107 (59%) patients in the perioperative chemotherapy group had died versus 114 (63%) in the surgery-only group (HR 0·88, 95% CI 0·68-1·14; p=0·34). In all randomly assigned patients, median overall survival was 61·3 months (95% CI 51·0-83·4) in the perioperative chemotherapy group and 54·3 months (41·9-79·4) in the surgery alone group. 5-year overall survival was 51·2% (95% CI 43·6-58·3) in the perioperative chemotherapy group versus 47·8% (40·3-55·0) in the surgery-only group. Two patients in the perioperative chemotherapy group and three in the surgery-only group died from complications of protocol surgery, and one patient in the perioperative chemotherapy group died possibly as a result of toxicity of protocol treatment. INTERPRETATION: We found no difference in overall survival with the addition of perioperative chemotherapy with FOLFOX4 compared with surgery alone for patients with resectable liver metastases from colorectal cancer. However, the previously observed benefit in PFS means that perioperative chemotherapy with FOLFOX4 should remain the reference treatment for this population of patients. FUNDING: Norwegian and Swedish Cancer Societies, Cancer Research UK, Ligue Nationale Contre Cancer, US National Cancer Institute, Sanofi-Aventis.

Effect of Hyperthermic Intraperitoneal Chemotherapy on Cytoreductive Surgery in Gastric Cancer With Synchronous Peritoneal Metastases: The Phase III GASTRIPEC-I Trial.

PURPOSE: In patients with peritoneal metastasis (PM) from gastric cancer (GC), chemotherapy is the treatment of choice. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) are still being debated. This randomized, controlled, open-label, multicenter phase III trial (EudraCT 2006-006088-22; ClinicalTrials.gov identifier: NCT02158988) explored the impact on overall survival (OS) of HIPEC after CRS. PATIENTS AND METHODS: Adult patients with GC and histologically proven PM were randomly assigned (1:1) to perioperative chemotherapy and CRS alone (CRS-A) or CRS plus HIPEC (CRS + H). HIPEC comprised mitomycin C 15 mg/m2 and cisplatin 75 mg/m2 in 5 L of saline perfused for 60 minutes at 42°C. The primary end point was OS; secondary endpoints included progression-free survival (PFS), other distant metastasis-free survival (MFS), and safety. Analyses followed the intention-to-treat principle. RESULTS: Between March 2014 and June 2018, 105 patients were randomly assigned (53 patients to CRS-A and 52 patients to CRS + H). The trial stopped prematurely because of slow recruitment. In 55 patients, treatment stopped before CRS mainly due to disease progression/death. Median OS was the same for both groups (CRS + H, 14.9 [97.2% CI, 8.7 to 17.7] months v CRS-A, 14.9 [97.2% CI, 7.0 to 19.4] months; P = .1647). The PFS was 3.5 months (95% CI, 3.0 to 7.0) in the CRS-A group and 7.1 months (95% CI, 3.7 to 10.5; P = .047) in the CRS + H group. The CRS + H group showed better MFS (10.2 months [95% CI, 7.7 to 14.7] v CRS-A, 9.2 months [95% CI, 6.8 to 11.5]; P = .0286). The incidence of grade ≥3 adverse events (AEs) was similar between groups (CRS-A, 38.1% v CRS + H, 43.6%; P = .79). CONCLUSION: This study showed no OS difference between CRS + H and CRS-A. PFS and MFS were significantly better in the CRS + H group, which needs further exploration. HIPEC did not increase AEs.

Current status of gene therapy for cancer.

PURPOSE OF REVIEW: In recent years, remarkable progress has been made in the development of cancer gene therapy into an applicable treatment modality for immunogene, suicide, gene correction and oncolytic therapies. New exciting developments for gene suppression or miRNA therapies are under way. The efforts are focused on more efficient and specific attack at known and novel targets, improvement of vector delivery and therapeutic efficacy. In this review, promising and new gene therapy approaches and clinical studies are briefly discussed to highlight important future directions of preclinical and clinical efforts. RECENT FINDINGS: Apart from progress for vector development and even more important, improvements for suicide, T-cell-based, oncolytic virus therapies were achieved. In addition, new emerging therapies are successfully developed, which are particularly promising for siRNA-based technologies applied to gene suppression therapy. Novel approaches, such as transcription factor ODN-based decoy, complement the spectrum of current cancer gene therapy. SUMMARY: In summary, cancer gene therapy has made remarkable progress in the improvement/refinement of existing strategies and delivery systems. The field is moving toward a therapeutic option, which will also be applicable for the treatment of disseminated metastases. Furthermore, numerous new approaches are about to be translated in clinical trials.

Loss of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) induces apoptotic processes in pancreatic carcinoma cells.

Early invasive growth and metastasis are features of pancreatic cancer that rely on its resistance to anoikis, an apoptosis program activated on loss of matrix anchorage. How anoikis is regulated is unclear. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-kinase (GNE) was silenced, or p16 was overexpressed, in human pancreatic carcinoma cells. Gene expression profiling, enzymatic assays, Western blotting, and cell cycle analysis were conducted. Silencing of GNE, the key enzyme of sialic acid biosynthesis, sensitizes pancreatic cancer cells to anoikis. Accordingly, we observed a loss of GNE enzyme activity in cells, which became anoikis susceptible after transfection with the tumor suppressor p16. Similarly, studies of another cell line with low GNE activity revealed strong anoikis susceptibility, confirming the association of low GNE activity and anoikis susceptibility. Gene expression profiling demonstrated that the loss of GNE triggered the transcriptional activation of the ATF4-ATF3-CHOP pathway, leading to apoptosis in the framework of the unfolded protein response. In silico analysis showed that GNE up-regulation occurred predominantly in pancreatic cancer but also in other malignancies. Delineation of GNE-dependent signaling pathways may provide targets that control anchorage dependence and/or restore drug efficacy, which is of utmost relevance for the treatment of pancreatic cancer.

Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway-dependent gene signatures in colorectal cancer cells.

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

Performance of the cobas EZH2 mutation test on clinical samples from non-Hodgkin lymphoma patients.

OBJECTIVE: To present the technical verification and clinical validation of the companion diagnostic assay, cobas® EZH2 Mutation Test (cobas EZH2 Test), targeting gain-of-function EZH2 mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). The focus is on patient clinical samples proving that the test met the performance criteria required for FDA approval of a companion diagnostic test. DESIGN: Epizyme, Inc., Eisai Co., Ltd., and Roche Molecular Systems, Inc., collaborated to develop the cobas EZH2 Test on an RT-PCR platform. The assay design needed to detect the gain-of-function EZH2 mutations found in FL and DLBCL indications. Thus, the test was optimized for investigational purposes in a clinical trial setting. Part of its technical verification included testing of patient tumor samples with a documented diagnosis of FL and DLBCL procured from commercial vendors, and the clinical validation used patient samples from the Epizyme clinical study. Both the technical performance verification method correlation study (104 clinical commercially acquired samples) and the clinical validation accuracy study (341 patient samples from the therapeutic study) used next-generation sequencing as a reference method to establish true vs. false results by cobas EZH2 Test. The reproducibility study used a 15-member panel of DNA samples with varying EZH2 mutation status from procured clinical FL and DLBCL patient samples under multiple variables. RESULTS: Single and rare, infrequent double EZH2 mutations were detected in FL and DLBCL samples. Agreements between results from cobas EZH2 and sequencing were >98% from commercial clinical samples and from the therapeutic study clinical samples. The reproducibility study obtained 178 to 180 valid results for each panel member, with an overall invalid rate of 0.37%. The agreement for each per panel member was 100%. CONCLUSION: cobas EZH2 Test data demonstrated that the test is reliable and will perform well in a commercial customer environment.

GIPC1 regulates MACC1-driven metastasis.

Background: Identification of cancer metastasis-relevant molecular networks is desired to provide the basis for understanding and developing intervention strategies. Here we address the role of GIPC1 in the process of MACC1-driven metastasis. MACC1 is a prognostic indicator for patient metastasis formation and metastasis-free survival. MACC1 controls gene transcription, promotes motility, invasion and proliferation of colon cancer cells in vitro, and causes tumor growth and metastasis in mice. Methods: By using yeast-two-hybrid assay, mass spectrometry, co-immunoprecipitation and peptide array we analyzed GIPC1 protein binding partners, by using the MACC1 gene promoter and chromatin immunoprecipitation and electrophoretic mobility shift assay we probed for GIPC1 as transcription factor. We employed GIPC1/MACC1-manipulated cell lines for in vitro and in vivo analyses, and we probed the GIPC1/MACC1 impact using human primary colorectal cancer (CRC) tissue. Results: We identified MACC1 and its paralogue SH3BP4 as protein binding partners of the protein GIPC1, and we also demonstrated the binding of GIPC1 as transcription factor to the MACC1 promoter (TSS to -60 bp). GIPC1 knockdown reduced endogenous, but not CMV promoter-driven MACC1 expression, and diminished MACC1-induced cell migration and invasion. GIPC1 suppression reduced tumor growth and metastasis in mice intrasplenically transplanted with MACC1-overexpressing CRC cells. In human primary CRC specimens, GIPC1 correlates with MACC1 expression and is of prognostic value for metastasis formation and metastasis-free survival. Combination of MACC1 and GIPC1 expression improved patient survival prognosis, whereas SH3BP4 expression did not show any prognostic value. Conclusions: We identified an important, dual function of GIPC1 - as protein interaction partner and as transcription factor of MACC1 - for tumor progression and cancer metastasis.

SNPs in the coding region of the metastasis-inducing gene MACC1 and clinical outcome in colorectal cancer.

BACKGROUND: Colorectal cancer is one of the main cancers in the Western world. About 90% of the deaths arise from formation of distant metastasis. The expression of the newly identified gene metastasis associated in colon cancer 1 (MACC1) is a prognostic indicator for colon cancer metastasis. Here, we analyzed for the first time the impact of single nucleotide polymorphisms (SNPs) in the coding region of MACC1 for clinical outcome of colorectal cancer patients. Additionally, we screened met proto-oncogene (Met), the transcriptional target gene of MACC1, for mutations. METHODS: We sequenced the coding exons of MACC1 in 154 colorectal tumors (stages I, II and III) and the crucial exons of Met in 60 colorectal tumors (stages I, II and III). We analyzed the association of MACC1 polymorphisms with clinical data, including metachronous metastasis, UICC stages, tumor invasion, lymph node metastasis and patients' survival (n = 154, stages I, II and III). Furthermore, we performed biological assays in order to evaluate the functional impact of MACC1 SNPs on the motility of colorectal cancer cells. RESULTS: We genotyped three MACC1 SNPs in the coding region. Thirteen % of the tumors had the genotype cg (rs4721888, L31V), 48% a ct genotype (rs975263, S515L) and 84% a gc or cc genotype (rs3735615, R804T). We found no association of these SNPs with clinicopathological parameters or with patients' survival, when analyzing the entire patients' cohort. An increased risk for a shorter metastasis-free survival of patients with a ct genotype (rs975263) was observed in younger colon cancer patients with stage I or II (P = 0.041, n = 18). In cell culture, MACC1 SNPs did not affect MACC1-induced cell motility and proliferation. CONCLUSION: In summary, the identification of coding MACC1 SNPs in primary colorectal tumors does not improve the prediction for metastasis formation or for patients' survival compared to MACC1 expression analysis alone. The ct genotype (rs975263) might be associated with a reduced survival for younger colon cancer patients in early stages. However, further studies with larger sample sizes are needed.

Integrative marker analysis allows risk assessment for metastasis in stage II colon cancer.

OBJECTIVES: Individualized risk assessment in patients with UICC stage II colon cancer based on a panel of molecular genetic alterations. BACKGROUND: Risk assessment in patients with colon cancer and localized disease (UICC stage II) is not sufficiently reliable. Development of metachronous metastasis is assumed to be governed largely by individual tumor genetics. METHODS: Fresh frozen tissue from 232 patients (T3-4, N0, M0) with complete tumor resection and a median follow-up of 97 months was analyzed for microsatellite stability, KRAS exon 2, and BRAF exon 15 mutations. Gene expression of the WNT-pathway surrogate marker osteopontin and the metastasis-associated genes SASH1 and MACC1 was determined for 179 patients. The results were correlated with metachronous distant metastasis risk (n = 22 patients). RESULTS: Mutations of KRAS were detected in 30% patients, mutations of BRAF in 15% patients, and microsatellite instability in 26% patients. Risk of recurrence was associated with KRAS mutation (P = 0.033), microsatellite stable tumors (P = 0.015), decreased expression of SASH1 (P = 0.049), and increased expression of MACC1 (P < 0.001). MACC1 was the only independent parameter for recurrence prediction (hazard ratio: 6.2; 95% confidence interval: 2.4-16; P < 0.001). Integrative 2-step cluster analysis allocated patients into 4 groups, according to their tumor genetics. KRAS mutation, BRAF wild type, microsatellite stability, and high MACC1 expression defined the group with the highest risk of recurrence (16%, 7 of 43), whereas BRAF wild type, microsatellite instability, and low MACC1 expression defined the group with the lowest risk (4%, 1 of 26). CONCLUSIONS: MACC1 expression predicts development of metastases, outperforming microsatellite stability status, as well as KRAS/BRAF mutation status.

Predictive factors for the benefit of perioperative FOLFOX for resectable liver metastasis in colorectal cancer patients (EORTC Intergroup Trial 40983).

OBJECTIVE: In EORTC study 40983, perioperative FOLFOX increased progression-free survival (PFS) compared with surgery alone for patients with initially 1 to 4 resectable liver metastases from colorectal cancer (CRC). We conducted an exploratory retrospective analysis to identify baseline factors possibly predictive for a benefit of perioperative FOLFOX on PFS. METHODS: The analysis was based on 237 events from 342 eligible patients. Cox proportional hazards regression models with a significance level of 0.1 were used to build up univariate and multivariate models. RESULTS: After adjustment for identified prognostic factors, moderately (5.1-30 ng/mL) and highly (>30 ng/mL) elevated carcinoembryonic antigen (CEA) serum levels were both predictive for the benefit of perioperative chemotherapy (interaction P = 0.07; hazard ratio [HR] = 0.58 and HR = 0.52 for treatment benefit). For patients with moderately or highly elevated CEA (>5 ng/mL), the 3-year PFS was 35% with perioperative chemotherapy compared to 20% with surgery alone. Performance status (PS) 0 and BMI lower than 30 were also predictive for the benefit of perioperative chemotherapy (interaction P = 0.04 and P = 0.02). However, the number of patients with PS 1 and BMI 30 or higher were limited. The benefit of perioperative therapy was not influenced by the number of metastatic lesions (1 vs 2-4, interaction HR = 0.98). CONCLUSIONS: Perioperative FOLFOX seems to benefit in particular patients with resectable liver metastases from CRC when CEA is elevated and when PS is unaffected, regardless of the number of metastatic lesions.ClinicalTrials.gov number NCT00006479.

Discovery of a novel tumour metastasis-promoting gene, NVM-1.

We have previously reported that over-expression of a panel of 119 genes correlates with the metastatic potential of pancreatic carcinoma cells. We sought to identify and functionally characterize candidate tumour metastasis promoting genes among this library using a secondary phenotype-assisted screen. Here we report the discovery of the metastasis-promoting function of a hitherto not characterized gene located on chromosome 14 (ORF138), which we have named 'novel metastasis-promoting gene 1' (NVM-1). The NVM-1 transcript is extensively alternatively spliced, is expressed endogenously in a number of different tissues, and is strongly over-expressed at the protein level in a variety of human tumour types. Importantly, NVM-1 expression stimulates the migratory and invasive behaviour of tumour cells and promotes metastasis formation in experimental animals in vivo. Up-regulation of FMNL2 and MT1E and down-regulation of TIMP4 and MHC-I is observed as a consequence of NVM-1 expression. Together these data identify NVM-1 as a gene that is functionally involved in tumour metastasis, and suggest that NVM-1 may constitute a promising therapeutic target for inhibition of tumour metastasis.

Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis.

Inappropriate transcriptional repression involving histone deacetylases (HDACs) is a prominent cause for the development of leukemia. We now identify faulty expression of a specific mediator of transcriptional repression in a solid tumor. Loss of the adenomatosis polyposis coli (APC) tumor suppressor induces HDAC2 expression depending on the Wnt pathway and c-Myc. Increased HDAC2 expression is found in the majority of human colon cancer explants, as well as in intestinal mucosa and polyps of APC-deficient mice. HDAC2 is required for, and sufficient on its own to prevent, apoptosis of colonic cancer cells. Interference with HDAC2 by valproic acid largely diminishes adenoma formation in APC(min) mice. These findings point toward HDAC2 as a particularly relevant potential target in cancer therapy.

Agile design and development of a high throughput cobas SARS-CoV-2 RT-PCR diagnostic test.

Diagnostic testing is essential for management of the COVID-19 pandemic. An agile assay design methodology, optimized for the cobas® 6800/8800 system, was used to develop a dual-target, qualitative SARS-CoV-2 RT-PCR test using commercially available reagents and existing sample processing and thermocycling profiles. The limit of detection was 30-52 copies/mL for USA-WA1/2020. Assay sensitivity was confirmed for SARS-CoV-2 variants Alpha, Beta, Gamma, Delta and Kappa. The coefficients of variation of the cycle threshold number (Ct) were between 1.1 and 2.2%. There was no difference in Ct using nasopharyngeal compared to oropharyngeal swabs in universal transport medium (UTM). A small increase in Ct was observed with specimens collected in cobas PCR medium compared to UTM. In silico analysis indicated that the dual-target test is capable of detecting all >1,800,000 SARS-CoV-2 sequences in the GISAID database. Our agile assay design approach facilitated rapid development and deployment of this SARS-CoV-2 RT-PCR test.

Identification of early molecular markers for breast cancer.

BACKGROUND: The ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma. Since DCIS is a curable disease, it would be highly desirable to identify molecular markers that allow early detection. Mice transgenic for the WAP-SV40 early genome region were used as a model for DCIS development. Gene expression profiling was carried out on DCIS-bearing mice and control animals. Additionally, a set of human DCIS and invasive mammary tumors were analyzed in a similar fashion. Enhanced expression of these marker genes in human and murine samples was validated by quantitative RT-PCR. Besides, marker gene expression was also validated by immunohistochemistry of human samples. Furthermore in silico analyses using an online microarray database were performed. RESULTS: In DCIS-mice seven genes were identified that were significantly up-regulated in DCIS: DEPDC1, NUSAP1, EXO1, RRM2, FOXM1, MUC1 and SPP1. A similar up-regulation of homologues of the murine genes was observed in human DCIS samples. Enhanced expression of these genes in DCIS and IDC (invasive ductal carcinoma) was validated by quantitative RT-PCR and immunohistochemistry. CONCLUSIONS: By comparing murine markers for the ductal carcinoma in situ (DCIS) of the mammary gland with genes up-regulated in human DCIS-samples we were able to identify a set of genes which might allow early detection of DCIS and invasive carcinomas in the future. The similarities between gene expression in DCIS and invasive carcinomas in our data suggest that the early detection and treatment of DCIS is of utmost relevance for the survival of patients who are at high risk of developing breast carcinomas.

Breast cancer: early- and late-fluorescence near-infrared imaging with indocyanine green--a preliminary study.

PURPOSE: To assess early- and late-fluorescence near-infrared imaging, corresponding to the vascular (early-fluorescence) and extravascular (late-fluorescence) phases of indocyanine green (ICG) enhancement, for breast cancer detection and benign versus malignant breast lesion differentiation. MATERIALS AND METHODS: The study was approved by the ethical review board; all participants provided written informed consent. Twenty women with 21 breast lesions were examined with near-infrared imaging before, during, and after intravenous injection of ICG. Absorption and fluorescence projection mammograms were recorded simultaneously on a prototype near-infrared imaging unit. Two blinded readers independently assessed the images and assigned visibility scores to lesions seen on the absorption and absorption-corrected fluorescence mammograms. Imaging results were compared with histopathologic findings. Lesion contrast and diameter on the fluorescence mammograms were measured, and Cohen κ, Mann-Whitney U, and Spearman ρ tests were conducted. RESULTS: The absorption-corrected fluorescence ratio mammograms showed high contrast (contrast value range, 0.25-0.64) between tumors and surrounding breast tissue. Malignant lesions were correctly defined in 11 (reader 1) and 12 (reader 2) of 13 cases, and benign lesions were correctly defined in six (reader 1) and five (reader 2) of eight cases with late-fluorescence imaging. Lesion visibility scores for malignant and benign lesions were significantly different on the fluorescence ratio mammograms (P = .003) but not on the absorption mammograms (P = .206). Mean sensitivity and specificity reached 92% ± 8 (standard error of mean) and 75% ± 16, respectively, for fluorescence ratio imaging compared with 100% ± 0 and 25% ± 16, respectively, for conventional mammography alone. CONCLUSION: Preliminary data suggest that early- and late-fluorescence ratio imaging after ICG administration can be used to distinguish malignant from benign breast lesions.

A colorectal cancer expression profile that includes transforming growth factor beta inhibitor BAMBI predicts metastatic potential.

BACKGROUND & AIMS: Much is known about the genes and mutations that cause colorectal cancer (CRC), yet only a few have been associated with CRC metastasis. We performed expression-profiling experiments to identify genetic markers of risk and to elucidate the molecular mechanisms of CRC metastasis. METHODS: We compared gene expression patterns between metastatic and nonmetastatic stage-matched human colorectal carcinomas by microarray analysis. Correlations between BAMBI and metastasis-free survival were examined by quantitative real-time polymerase chain reaction (PCR) using an independent set of human colon carcinomas. Human colon cancer cell lines were analyzed for BAMBI regulation, cell motility, and experimental metastasis. RESULTS: We established a signature of 115 genes that differentiated metastatic from nonmetastatic primary tumors. Among these, the transforming growth factor (TGF) beta inhibitor BAMBI was highly expressed in approximately half of metastatic primary tumors and metastases but not in nonmetastatic tumors. BAMBI is a target of canonical Wnt signaling that involves the beta-catenin coactivator BCL9-2. We observed an inverse correlation between level of BAMBI expression and metastasis-free survival time of patients. BAMBI inhibits TGF-beta signaling and increases migration in colon cancer cells. In mice, overexpression of BAMBI caused colon cancer cells to form tumors that metastasized more frequently to liver and lymph nodes than control cancer cells. CONCLUSIONS: BAMBI regulates CRC metastasis by connecting the Wnt/beta-catenin and TGF-beta-signaling pathways. The metastatic expression signature we describe, along with BAMBI levels, can be used in prognosis. Developmental signaling pathways appear to act in hierarchies and cooperate in tumor cell migration, invasion, and metastasis.