National Cancer Institute Panel Research Articles

Head-to-head comparative study: evaluating three panels for MSI-PCR testing in patients with colorectal and gastric cancer

AimsDue to the lack of large clinical cohorts in the Chinese populations with colorectal cancer (CRC) and gastric cancer (GC), there is no consensus among the preferred panel for microsatellite...

Incidence and detection of high microsatellite instability in colorectal cancer in a Chinese population: a meta-analysis

The 4 most common types of DNA mutations in tumors are single-nucleotide variations, insertion-deletion, fusion, and copy number variations. This is followed by microsatellite instability (MSI), which is known to trigger the development of MSI-high (MSI-H) cancer and is responsible for 300,000 new cases of cancer per year in China. We aim to conduct a meta-analysis based on a comparison between the positive rates of the National Cancer Institute (NCI) panel (also known as 2B3D NCI panel) and mononucleotide panels for the diagnosis of MSI in the Chinese population. In the present meta-analysis, we searched the PubMed, Embase, Web of Science, CNKI, Wanfang, CQVIP, and CBM databases. MSI diagnosis studies by PCR and capillary electrophoresis were included to compare the incidence of MSI-H in colorectal cancer obtained from panels with different microsatellite markers. Egger's bias test was used to assess risk of bias. Seventeen articles were included, which used the Newcastle-Ottawa Scale (NOS) scale for quality evaluation. The NOS scores of the included documents were ≥7 points, and the quality of the documents met the requirements. The incidence of MSI-H detected by the 2B3D NCI panel was 13.5% [95% confidence interval (CI): 10.8-16.4, I2=52.321%, P=0.026, n=10 studies including 2,681 participants], the incidence of MSI-H detected by the mononucleotide panels was 10.6% (95% CI: 7.1-14.7, I2=81.147%, P=0.000, n=7 studies including 3,249 participants). This indicates that, in the Chinese population, the 2B3D NCI panel can detect 27.4% more MSI-H cancers than the mononucleotide panels, 54.7% more MSI-H cancers than the panel of 6 mononucleotides, and its sensitivity is comparable to that of Promega. The findings of the meta-analysis demonstrated that, using the 2B3D NCI panel for MSI detection can avoid the underestimation of the incidence MSI-H in colorectal cancer and can be considered the most suitable panel for MSI detection in the Chinese population. The inclusion of only published data might be a potential source of publication bias.

The interaction between BRAF mutation and microsatellite instability (MSI) status in determining survival outcomes after adjuvant 5FU based chemotherapy in stage III colon cancer.

The predictive role of biomarkers in colon cancer is still being defined. The aim of this study is to determine the interaction between BRAF mutation and microsatellite instability (MSI) status in determining survival benefit after adjuvant 5-FU based chemotherapy in stage III colon cancer. We performed a retrospective cohort study including all curatively resected stage III colon cancer cases over a 33-year period. A clinicopathological database was collated (adjuvant chemotherapy, age, gender, obstruction, perforation, tumor location, grade, mucin, nodal stage, extramural vascular, and perineural invasion). BRAF (V600E) mutation testing was performed and MSI status established by immunohistochemistry for mismatch repair proteins and molecular testing for National Cancer Institute panel markers. Patients were categorized into four groups for comparison: MSS and BRAF-ve (termed " traditional"), MSI and BRAF-ve (termed " presumed Lynch"), MSI and BRAF+ve (termed " sporadic MSI"), and MSS and BRAF+ve (termed " other BRAF"). The primary endpoint was cancer specific survival. Interaction testing was conducted to determine whether there were different responses to chemotherapy between groups. A total of 686 unselected cases met inclusion criteria and had tissue available, of which 15.7% had BRAF mutation (BRAF+ve) and 13.8% had MSI. Thirty-nine percent received chemotherapy. Overall, adjuvant chemotherapy produced a cancer specific survival benefit (HR 0.66, 95% CI, 0.49-0.88, P < 0.01). On adjusted analysis, neither BRAF nor MSI status were individually predictive of survival benefit. On adjusted analysis specifically of the chemotherapy effect in each subgroup, only patients in the presumed Lynch (HR 0.260, 95% CI, 0.09-0.80, P < 0.01) and other BRAF groups (HR 0.45, 95% CI, 0.23-0.87, P < 0.01) had a significant survival benefit from chemotherapy. On interaction testing of subgroups, adjusting for all the clinicopathological parameters, only patients in the presumed Lynch group (HR 0.277, 95% CI, 0.10-0.75, P < 0.01) gained a differentially greater benefit from chemotherapy than other groups. In this historical cohort, MSI testing is predictive of response to adjuvant chemotherapy in stage III colon cancer, but only when results are interpreted in combination with BRAF. This supports the role of routine testing for these biomarkers.

Molecular prediction of adjuvant cisplatin efficacy in Non-Small Cell Lung Cancer (NSCLC)-validation in two independent cohorts.

IntroductionEffective predictive biomarkers for selection of patients benefiting from adjuvant platinum-based chemotherapy in non-small cell lung cancer (NSCLC) are needed. Based on a previously validated methodology, molecular profiles of predicted sensitivity in two patient cohorts are presented.MethodsThe profiles are correlations between in vitro sensitivity to cisplatin and vinorelbine and baseline mRNA expression of the 60 cell lines in the National Cancer Institute panel. An applied clinical samples filter focused the profiles to clinically relevant genes. The profiles were tested on 1) snap-frozen tumors from 133 patients with completely resected stage 1B-2 NSCLC randomized to adjuvant cisplatin and vinorelbine (ACV, n = 71) or no adjuvant treatment (OBS, n = 62) and 2) formalin-fixed paraffin-embedded (FFPE) tumors from 95 patients with completely resected stage 1A-3B NSCLC receiving adjuvant cisplatin and vinorelbine.ResultsThe combined cisplatin and vinorelbine profiles showed: 1) univariate Hazard Ratio (HR) for sensitive versus resistant of 0.265 (95% CI:0.079–0.889, p = 0.032) in the ACV cohort and a HR of 0.28 in a multivariate model (95% CI:0.08–1.04, p = 0.0573); 2) significant prediction at 3 year survival from surgery in univariate (HR = 0.138 (95% CI:0.035–0.537), p = 0.004) and multivariate analysis (HR = 0.14 (95% CI:0.030–0.6), p = 0.0081). No discrimination was found in the OBS cohort (HR = 1.328, p = 0.60). The cisplatin predictor alone had similar figures with 1) univariate HR of 0.37 (95% CI:0.12–1.15, p = 0.09) in the ACV cohort and 2) univariate HR of 0.14 (95% CI:0.03–0.59, p = 0.0076) to three years. Functional analysis on the cisplatin profile revealed a group of upregulated genes related to RNA splicing as a part of DNA damage repair and apoptosis.ConclusionsProfiles derived from snap-frozen and FFPE NSCLC tissue were prognostic and predictive in the patients that received cisplatin and vinorelbine but not in the cohort that did not receive adjuvant treatment.

Open source machine-learning algorithms for the prediction of optimal cancer drug therapies

Precision medicine is a rapidly growing area of modern medical science and open source machine-learning codes promise to be a critical component for the successful development of standardized and automated analysis of patient data. One important goal of precision cancer medicine is the accurate prediction of optimal drug therapies from the genomic profiles of individual patient tumors. We introduce here an open source software platform that employs a highly versatile support vector machine (SVM) algorithm combined with a standard recursive feature elimination (RFE) approach to predict personalized drug responses from gene expression profiles. Drug specific models were built using gene expression and drug response data from the National Cancer Institute panel of 60 human cancer cell lines (NCI-60). The models are highly accurate in predicting the drug responsiveness of a variety of cancer cell lines including those comprising the recent NCI-DREAM Challenge. We demonstrate that predictive accuracy is optimized when the learning dataset utilizes all probe-set expression values from a diversity of cancer cell types without pre-filtering for genes generally considered to be “drivers” of cancer onset/progression. Application of our models to publically available ovarian cancer (OC) patient gene expression datasets generated predictions consistent with observed responses previously reported in the literature. By making our algorithm “open source”, we hope to facilitate its testing in a variety of cancer types and contexts leading to community-driven improvements and refinements in subsequent applications.

Design, synthesis and molecular docking of novel structural hybrids of substituted isatin based pyrazoline and thiadiazoline as antitumor agents

Cancer, which is considered to be the world’s most serious illness cause 8.2 million deaths and this rate may double by 2030. We herein report a new series of 3-(2-(p-substituted)-2-((5-phenyl-1,3,4-thiadiazol-2-yl)imino)-2-(p-substituted)ethylidene)indolin-2-one (15–19) and 5-substituted-5′-substituted phenyl-2′,4′-dihydrospiro[indoline-3,3′-pyrazol]-2-one derivatives (20–24) as potent anticancer agents. These compounds were evaluated for in vitro antitumor activity against the National Cancer Institute panel of 60 cancer cell lines. Among all the synthesized compounds, two compounds 15 and 16 showed remarkable antitumor activity with GI50 (MG-MID) values of 0.65 & 0.72 µM, respectively against Non-small cell lung cancer. To gain insight for mode of binding with Epidermal Growth Factor Receptor kinase enzyme, these compounds were further subjected to docking studies.

Microsatellite instability and survival in gastric cancer: A systematic review and meta-analysis.

Microsatellite instability (MSI) is associated with the prognosis in several cancers and is used for determination of the chemotherapy regimen in stage II colon cancer in the National Comprehensive Cancer Network guideline. However, the association between MSI and the prognosis of gastric cancer remains unclear. PubMed database was searched until January 2014 using MeSH terms and key words to identify the studies evaluating MSI and prognosis of gastric cancer and the references were manually searched. The main outcome was the overall survival rate and the subordinate outcome was the association between high-frequency MSI (MSI-H) and clinicopathological characteristics. Eight studies met the inclusion criteria and the majority of data were collected retrospectively. There were 1,976 patients, 431 of which were MSI-H patients, with a range of 11.68-33.82%. Four studies used the National Cancer Institute panel to define MSI-H, the other four had microsatellite markers ranging 2-11. Significant associations were found in three studies and the overall summary estimate was hazard ratio, 0.63 (95% confidence interval, 0.52-0.77), with no evidence of inter-study heterogeneity (I2=0.0%). MSI-H patients were identified to have a tendency to have less lymph node (LN) metastasis, superficial tumor invasion and to be intestinal type. In conclusion, MSI-H gastric cancers have an improved prognosis, accompanied with reduced risk of LN metastasis, tumor invasion and mortality.

Copy number variations are progressively associated with the pathogenesis of colorectal cancer in ulcerative colitis.

To evaluate the association of known copy number variations (CNVs) in ulcerative colitis (UC) progressing to colorectal cancer. Microsatellite instability analysis using the National Cancer Institute's panel of markers, and CNV association studies using Agilent 2 × 105 k arrays were done in tissue samples from four patient groups with UC: those at low risk (LR) or high risk of developing colorectal cancer, those with premalignant dysplastic lesions, and those with colitis-associated colorectal cancer (CAC). DNA from tissue samples of these groups were independently hybridized on arrays and analyzed. The data obtained were further subjected to downstream bioinformatics enrichment analysis to examine the correlation with CAC progression. Microarray analysis highlighted a progressive increase in the total number of CNVs [LR (n = 178) vs CAC (n = 958), 5.3-fold], gains and losses [LR (n = 37 and 141) vs CAC (n = 495 and 463), 13.4- and 3.3-fold, respectively], size [LR (964.2 kb) vs CAC (10540 kb), 10.9-fold] and the number of genes in such regions [LR (n = 119) vs CAC (n = 455), 3.8-fold]. Chromosome-wise analysis of CNVs also showed an increase in the number of CNVs across each chromosome. There were 38 genes common to all four groups in the study; 13 of these were common to cancer genes from the Genetic Disease Association dataset. The gene set enrichment analysis and ontology analysis highlighted many cancer-associated genes. All the samples in the different groups were microsatellite stable. Increasing numbers of CNVs are associated with the progression of UC to CAC, and warrant further detailed exploration.

NCI group urges the medical community to address the increasing problem of cancer overdiagnosis and overtreatment

NCI group urges the medical community to address the increasing problem of cancer overdiagnosis and overtreatment

Suppression of AKT Anti-Apoptotic Signaling by a Novel Drug Candidate Results in Growth Arrest and Apoptosis of Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is the third most common cause of cancer fatalities worldwide, with limited treatment options and five year survival rates of between <5 and 15%. To address this medical need, we conducted a screen of a drug-like small molecule library for HCC-selective cytotoxins. We report here the identification of a disubstituted aminothiazole termed HBF-0079, with remarkable selective toxicity for HCC-derived cell lines versus non-HCC liver lines and most other cancer lines. HBF-0079 caused irreversible growth arrest and apoptosis of the HCC lines Huh7, Hep3B, HepaRG as well as the hepatoblastoma line HepG2, with CC50 values from ∼0.7−7.7 µM, while more than 45 µM was needed to achieve CC50 values for the immortalized normal hepatocyte lines THLE-2 and PH5CH. Of the sixty cancer lines from the National Cancer Institute panel, only five exhibited >50% growth inhibition by HBF-0079. In Huh7 cells, HBF-0079 induced cell cycle arrest in G1 and concomitant apoptosis, and its effects were irreversible after removal of the compound. These observations corroborate a loss of AKT phosphorylation at the mTORC2-targeted residue S473, with concurrent loss of phosphorylation of the mTORC1 targets SK6 and 4EBP1 in Huh7 but not PH5CH cells. Finally, growth of Hep3B-derived tumors in a murine xenograft model was significantly repressed by the compound through either systemic or intratumoral administration of formulated HBF-0079. The potential for development of this drug candidate is discussed.

Abstract 1872: Identification of novel single-nucleotide polymorphisms associated with anticancer drug effects using the SNPChip384 platform

Abstract The identification of genetic factors associated with anticancer drug efficacy is an important challenge for optimising chemotherapy and developing personalised treatments. One of the difficulties lies in the absence of preclinical models for identifying reliable tracks that could be translated to the clinics. We have used in vitro models to identify relationships between the presence of a given polymorphic variation and the cytotoxicity of anticancer drugs: the National Cancer Institute (NCI) and the Japanese Foundation for Cancer Research (JFCR) cell line panels, which had been used for the primary screening of thousands of potential anticancer drugs. In order to discover new SNPs of potential interest, high-throughput screening could be an option, but genome-wide studies cannot be used because of the relatively small number of cell lines (105) and the high false-discovery rate that would result. As an intermediate option, we have established a tool (SNPChip384) allowing the simultaneous determination of 384 SNPs in a single DNA sample. The SNPs have been chosen among DNA repair genes, phase I and II drug metabolism genes, drug transport genes and genes involved in oncogenesis, signal transduction and oxidative stress. Genotyping of the 60 cell lines from the NCI panel and the 45 cell lines from the JFCR panel was carried out using the SNPChip384. The concordance between this multiplexed approach and the individual previous determinations was around 95% (0-3 discordances in the 60 cell lines of the NCI panel and 0-2 discordances in the 45 cell lines of the JFCR panel). We first confirmed the significant relationships we had already observed and published concerning MDM2, MTHFR, CYP1B1, ATIC and ERCC2 gene polymorphisms. In addition, we identified novel SNPs involved in the in vitro cytotoxicity of anticancer drugs. The NCI panel was used for establishing these associations and the JFCR panel for validating them independently. The Benjamini-Hochberg correction for multiple testing was used in order to minimize the false-discovery rate. In the NCI panel, ten gene polymorphisms successfully passed this stringent filter (the p values indicated are those obtained after correction): AHR R554K was significantly associated with irofulven cytotoxicity (p=0.0001); APC Y486Y with SN-38 cytotoxicity (p=0.007); ATP7B K832R with carmustine, busulfan and melphalan cytotoxicity (p=0.004 to 0.036); BRCA1 E1038G with geldanamycin cytotoxicity (p=0.01); CASP9 Q221R with the cytotoxicity of a taxol analogue (p=0.03); CDC25C R70C with vinblastine cytotoxicity (p=0.04); CDH1 with spiromustine cytotoxicity (p=0.02); CYP2E1 F421F with irofulven cytotoxicity (p=0.007); PARP4 G1280R with thioguanine cytotoxicity (p=0.04); TLR4 D299G with doxorubicin cytotoxicity (p=0.03). These associations open new tracks for identifying novel determinants of anticancer drug activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1872. doi:1538-7445.AM2012-1872

Does Neoadjuvant Therapy Alter KRAS and/or MSI Results in Rectal Adenocarcinoma Testing?

To our knowledge, the genotoxic effects of neoadjuvant chemoradiation therapy on molecular diagnostic testing results are unknown. However, if neoadjuvant treatments were to alter molecular test results, clinical decision-making could be misled. This raises questions about the appropriateness of using posttreatment tumor for testing. To address this, rectal adenocarcinomas both before and after neoadjuvant treatment were evaluated for alterations in KRAS and microsatellite instability (MSI) testing. Neoadjuvant chemoradiation therapy is common in this tumor type, and alterations in these 2 tests would significantly impact management. A total of 17 rectal adenocarcinoma patients with available pretreatment and posttreatment tumor were studied. MSI testing used the revised National Cancer Institute panel of 5 mononucleotide microsatellite repeats, comparing cancers with matched normal control tissues. KRAS codon 12-point and 13-point mutations were examined by polymerase chain reaction amplification and bidirectional sequencing. MSI and KRAS results were unchanged comparing rectal cancer tissue before and after chemoradiotherapy in all 17 patients (P=1.000; 95% CI: 0.3969-2.520). All 17 tumors (100%) were microsatellite stable. KRAS testing identified 12 (72%) wild-type tumors and 5 (28%) codon 12 or 13 mutant tumors with identical KRAS point mutations before and after treatment. The identified MSI and KRAS mutational prevalences parallel those reported in the rectal cancer literature. Neoadjuvant therapy did not alter KRAS codon 12 or 13 or MSI results in rectal adenocarcinoma, providing evidence that either pretreatment biopsy or posttreatment resection tissues are appropriate for testing.

Assessment of the beneficial loci and prognostic implications of microsatellite instability in gastric carcinoma

Microsatellite instability (MSI) is a hyper-mutable phenotype caused by the loss of DNA mismatch repair activity, and plays a crucial role in gastric carcinogenesis. To clarify the role of genetic instability in relation to clinicopathological variables, and to identify predictive MSI markers that facilitate the early detection and improve the classification of gastric carcinomas (GCs), 13 microsatellite (MS) loci, including the National Cancer Institute (NCI) panel of MS markers (D2S123, D5S346, D17S250, BAT25 and BAT26) and 8 dinucleotide repeats (D3S1260, D5S107, D5S409, D17S261, D17S520, D17S855, D18S34 and D18S61) were studied in GC patients. MSI was found in 88.2% (30/34) of GC cases and the number of high-frequency MSI (MSI-H, 23.5%, 8/34), low-frequency MSI (64.7%, 22/34), and stable MSI (11.8%, 4/34), was calculated. Among the MS loci analyzed, D18S34 and D17S261 (15/34, 44.1%) exhibited the highest frequency of MSI, followed by D2S123 (14/34, 41.2%), D5S107, D5S346, D5S409 and D18S61 (12/34, 35.3%) (MSI>35%). MSI-H was particularly prevalent in older patients and was mainly found in the antrum and poorly differentiated tumors. Furthermore, MSI-H was significantly associated with lymph node involvement cases in females. One notable finding in this analysis was that the markers, D17S250 and D17S520, exhibited a significantly higher percentage of MSI in advanced gastric carcinomas than in early gastric carcinomas (P=0.046 and 0.046, respectively), and the D17S520 and BAT26 loci represented significant different correlations between the tumor stages (P=0.038 and 0.042, respectively). This study indicates that the novel markers, D18S34 and D17S261, perform more favorably than the NCI panel for the detection of MSI, and the D17S520 locus presents a potential target for predicting the clinical impact of GC. These novel MS loci may prove to be beneficial and independent tools for the construction of a comprehensive genetic classification for GC cases.

Cytochrome P450 1B1 Gene Polymorphisms as Predictors of Anticancer Drug Activity: Studies with In vitro Models

Cytochrome P450 1B1 (CYP1B1) is found in tumor tissue and is suspected to play a role in oncogenesis and drug resistance. CYP1B1 gene polymorphisms have been associated with the risk of developing lung and other cancers. They may be associated with tumor response to anticancer drugs. We have determined 4 frequent nonsynonymous gene polymorphisms of CYP1B1 in the human tumor cell lines panels of the National Cancer Institute (NCI) and the Japanese Foundation for Cancer Research (JFCR): rs10012 (R48G), rs1056827 (A119S), rs1056836 (L432V), and rs1800440 (N453S). Numerous anticancer drugs have been tested against these panels that offer the opportunity to detect associations between gene polymorphisms and drug sensitivity. CYP1B1 single nucleotide polymorphisms were in marked linkage disequilibrium. The L432V allelic variants were significantly associated with reduced sensitivity to DNA-interacting anticancer agents, alkylators, camptothecins, topoisomerase II inhibitors, and some antimetabolites. For instance, in the NCI panel, cell lines homozygous for the V432 allele were globally 2-fold resistant to alkylating agents (P = 5 × 10(-10)) and 4.5-fold to camptothecins (P = 6.6 × 10(-9)) than cell lines homozygous for the L432 allele. Similar features were exhibited by the JFCR panel. Cell lines homozygous for the V432 allele were globally less sensitive to DNA-interfering drugs than cell lines having at least 1 common allele. There was no significant association between mRNA expression of CYP1B1 and CYP1B1 genotype, and no significant association between CYP1B1 mRNA expression and drug cytotoxicity. These observations open the way to clinical studies exploring the role of CYP1B1 gene polymorphisms for predicting tumor sensitivity to chemotherapy.

Cucurbitacin B inhibits STAT3 and the Raf/MEK/ERK pathway in leukemia cell line K562

Cucurbitacin B is a natural anti-cancer compound found in Cucurbitaceae. Although the anti-cancer activity of cucurbitacin B in human leukemia cells has been reported, the underlining mechanism is still unclear. To clarify its anti-cancer activity and the mechanism of action, five different leukemia cell lines (CCRF-CEM, K562, MOLT-4, RPMI-8226 and SR) of the National Cancer Institute panel were treated with cucurbitacin B. Leukemia cell growth was inhibited by cucurbitacin B with GI 50 ranged from 15.6 nM to 35.3 nM and the growth inhibition effect was attributable to G2/M phase arrest and apoptosis. Western blotting analysis of cell signaling molecules indicated that cucurbitacin B inhibits STAT3 activation and the Raf/MEK/ERK pathway in the K562 cells.

Confirmation of deficient mismatch repair (dMMR) as a predictive marker for lack of benefit from 5-FU based chemotherapy in stage II and III colon cancer (CC): A pooled molecular reanalysis of randomized chemotherapy trials

4008 Background: Patients (pts) with CC demonstrating high-frequency MSI (MSI-H) have a stage-independent improved survival compared to pts with microsatellite- stable (MSS) tumors. We have previously published that MSI status is a predictive marker for lack of response to 5-FU-based chemotherapy (rx) (Ribic, NEJM 2003). dMMR by immunohistochemisty for MMR proteins is an almost perfect predictor for MSI status. We sought to confirm the value of dMMR as a predictor of survival benefit from adjuvant rx in stage II & III CC pts in an independent dataset drawn from randomized clinical trials. Methods: MSI or IHC analyses were performed on tumors from pts enrolled in trials conducted by the NCCTG (N=135), GIVIO (N=183), and ECOG (N=23) that have not been used in previous MSI or dMMR analyses. All trials randomized pts with stage II and III CC to either 5-FU based rx (either 5-FU + levamisole or 5-FU + leucovorin, n=166)) or no post-surgical rx (n=175). XX microsatellite loci from the National Cancer Institute panel were used to amplify genomic DNA and determine MSI status (GIVIO); IHC testing for hMLH1 and hMSH2 was used for the NCCTG and ECOG trials. Patients with MSI-H tumors or negative IHC staining were classified as dMMR;, the remainder were considered to have proficient MMR (pMMR). Median follow-up on living pts was 6.4 years with a primary outcome of overall survival (OS). Results: 341 tissue specimens were examined, of which 47 (13.8%, 20 treated, 27 untreated) exhibited dMMR. Adjuvant rx had a significant beneficial effect on OS (HR = 0.69, p= 0.047) and DFS (HR = 0.59, p = 0.004) in pts with pMMR tumors. However, pts with dMMR tumors receiving 5-FU rx had no trend toward improved OS (HR = 1.26, p = 0.68) or DFS (HR=1.41, p = 0.53) compared to those randomized to no rx. Results were maintained in multivariate models adjusted for stage and age. Conclusions: Stratification of pts according to MMR status provides a more tailored approach to the use of adjuvant rx in CC. Our data suggest that in a patient being considered for 5-FU alone rx (i.e a stage II pt), MMR status should be assessed and considered in rx decision making. No significant financial relationships to disclose.

Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate.

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol®). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.

Microsatellite Instability Markers for Identifying Early-Onset Colorectal Cancers Caused by Germ-Line Mutations in DNA Mismatch Repair Genes

Microsatellite instability (MSI) testing of colorectal cancer tumors is used as a screening tool to identify patients most likely to be mismatch repair (MMR) gene mutation carriers. We wanted to examine which microsatellite markers currently used to detect MSI best predict early-onset colorectal cancer caused by germ-line mutations in MMR genes. Invasive primary tumors from a population-based sample of 107 cases of colorectal cancer diagnosed before age 45 years and tested for germ-line mutations in MLH1, MSH2, MSH6, and PMS2 and MMR protein expression were screened for MSI using the National Cancer Institute panel and an expanded 10-microsatellite marker panel. The National Cancer Institute five-marker panel system scored 31 (29%) as (NCI)MSI-High, 13 (12%) as (NCI)MSI-Low, and 63 (59%) as (NCI)MS-Stable. The 10-marker panel classified 18 (17%) as (10)MSI-High, 17 (16%) as (10)MSI-Low, and 72 (67%) as (10)MS-Stable. Of the 26 cancers that lacked the expression of at least one MMR gene, 24 (92%) were positive for some level of MSI (using either microsatellite panel). The mononucleotide repeats Bat26, Bat40, and Myb were unstable in all (10)MSI-High cancers and all MLH1 and MSH2 mutation carriers (100% sensitive). Bat40 and Bat25 were unstable in all tumors of MSH6 mutation carriers (100% sensitive). Bat40 was unstable in all MMR gene mutation carriers (100% sensitive). By incorporating seven mononucleotide repeats markers into the 10-marker panel, we were able to distinguish the carriers of MSH6 mutations (all scored (10)MSI-Low) from the MLH1 and MSH2 mutation carriers (all scored (10)MSI-High). In early-onset colorectal cancer, a microsatellite panel containing a high proportion of mononuclear repeats can distinguish between tumors caused by MLH1 and MSH2 mutations from those caused by MSH6 mutations.

Identification of gene polymorphisms of human DNA topoisomerase I in the National Cancer Institute panel of human tumour cell lines

Topoisomerase 1 (Top1), a nuclear enzyme involved in DNA relaxation, is the target of several anticancer drugs. TOP1 mutations occur in camptothecin-resistant tumour cell lines. We explored, in the NCI panel of 60 human tumour cell lines, whether polymorphic variations in the TOP1 gene could explain differences in drug sensitivity. The 21 exons of the gene were fully studied as well as five intronic domains that had previously been shown to harbour single nucleotide polymorphisms (SNPs) or mutations. PCR products covering the whole exonic sequences or the relevant intronic domains were subjected to denaturing high-performance liquid chromatography. Nucleotide variations were then determined by sequencing. Discrimination between intronic common and variant homozygous samples was performed using a restriction fragment length polymorphism technique. Only one exonic mutation was detected, at the heterozygous state; it occurs in exon 19 of a colon cancer cell line (HCT-15) and consists of a G>A transition at position 75, resulting in a Met675Ile change. The intronic sequences studied harboured the SNPs expected with allelic frequencies between 20 and 40%. Three major haplotypes, generating 92% of the 10 genotypes encountered, were defined as containing none of the intronic SNPs, or three of them, or all of them. No significant relationship was evidenced between Top1 expression and the TOP1 polymorphisms studied. However, when comparing the cytotoxicity of 138 drugs as a function of the genotypes, several drug groups, namely Top1 inhibitors, antifolates and taxanes, had significantly different IC50s as a function of the distribution of the intronic SNPs of the TOP1 gene.

Detection of microsatellite instability in endometrial cancer: advantages of a panel of five mononucleotide repeats over the National Cancer Institute panel of markers

The aim of this study was to find the optimal set of microsatellite markers for diagnosis of the microsatellite instability (MSI) phenotype in endometrial cancers. We compared the sensitivity, specificity and ease of use of a reference panel of five markers originally recommended by the National Cancer Institute (NCI) for colorectal cancer and a panel of five quasi-monomorphic mononucleotide repeat markers (pentaplex PCR system). We used these panels for establishing the MSI status of a series of 80 sporadic endometrial adenocarcinomas by comparing the allelic profiles of the markers between tumor and matching germline DNA. Both panels detected the same subset of 21 out of 80 (26%) endometrial MSI carcinomas. However, in the MSI cases, the mean instability of the five mononucleotide repeats was 96.1% as compared with a mean instability of 69.8% for the three dinucleotide repeats of the NCI panel, indicating a superiority of mononucleotide repeats over dinucleotide repeats in detecting MSI. The fact that the two panels of markers detect the same set of MSI tumors is due to the presence of two mononucleotide repeats within the NCI panel. As demonstrated previously in gastric and colon MSI cases, the pentaplex PCR reaction using mononucleotide repeats is thus an easier and more sensitive method than the NCI panel, for the screening of MSI status in endometrial tumors.