Cytokine‐regulated urokinase‐type‐plasminogen‐activator (uPA) production by human breast fibroblasts in vitro

It has been shown that, in breast stroma, urokinasetype plasminogen activator (uPA) mRNA is predominantly expressed by myofibroblasts located at the invasive areas of the tumor. To examine which factors present in a tumor environment are candidates responsible for the induction of these uPAproducing myofibroblasts, we studied in vitro the capacity of a paired panel of normal and tumorderived human breast fibroblasts to produce uPA protein and the myofibroblast marker smoothmuscleactin (SMA) in response to various cytokines implicated in the process of tissueremodeling during malignant transformation.We found that fibroblasts produced increased amounts of uPA protein after exposure to aFGF, bFGF, EGF, PDGFBB, and IFN, were unaffected in this respect by IL6, MCSF, GMCSF and Oncostatin M, and produced decreased amounts of uPA protein after exposure to IL1, TNF, IGFI, and IGFII. None of these cytokines were able to induce a striking increase in the fraction of SMApositive fibroblasts. On the other hand, 25pM TGF₁ increased the fraction of SMApositive fibroblasts 5fold in both normal and tumortissuederived fibroblasts. Nonetheless, the normalderived fibroblasts were unaffected in their uPAproducing capacity by TGF₁, and the tumorderived fibroblasts produced decreased amounts of uPA protein after exposure to this cytokine, implying that at least in vitro the myofibroblast phenotype is not a prerequisite for the production of uPA by human breast fibroblasts. In addition, we established that the basaluPAproduction of both normal and tumorderived fibroblasts was increased by autocrinely produced bFGFlike activity, and that the basaluPAproduction of at least the normalderived fibroblasts was decreased by autocrinely produced IGFlike activity.Altogether, our data suggest an active role for fibroblasts in the process of uPAdirected breast tumor proteolysis.     

MTA1 regulates higher‐order chromatin structure and histone H1‐chromatin interaction in‐vivo

In the current study, for the first time, we found that metastasis‐associated gene 1 (MTA1) was a higher‐order chromatin structure organizer that decondenses the interphase chromatin and mitotic chromosomes. MTA1 interacts dynamically with nucleosomes during the cell cycle progression, prominently contributing to the mitotic chromatin/chromosome structure transitions at both prophase and telophase. We showed that the decondensation of interphase chromatin by MTA1 was independent of Mi‐2 chromatin remodeling activity. H1 was reported to stabilize the compact higher‐order chromatin structure through its interaction with DNA. Our data showed that MTA1 caused a reduced H1‐chromatin interaction in‐vivo. Moreover, the dynamic MTA1‐chromatin interaction in the cell cycle contributed to the periodical H1‐chromatin interaction, which in turn modulated chromatin/chromosome transitions. Although MTA1 drove a global decondensation of chromatin structure, it changed the expression of only a small proportion of genes. After MTA1 overexpression, the up‐regulated genes were distributed in clusters along with down‐regulated genes on chromosomes at parallel frequencies.     

Subtype‐dependent prognostic relevance of an interferon‐induced pathway metagene in node‐negative breast cancer

The majority of gene expression signatures developed to predict the likelihood to relapse in breast cancer (BC) patients assigns a high risk score to patients with Estrogen Receptor (ER) negative or highly proliferating tumors. We aimed to identify a signature of differentially expressed (DE) metagenes, rather than single DE genes, associated with distant metastases beyond classical risk factors.We used 105 gene expression profiles from consecutive BCs to identify metagenes whose prognostic role was defined on an independent series of 92 ESR1+/ERBB2− node‐negative BCs (42 cases developing metastases within 5 years from diagnosis and 50 cases metastasis‐free for more than 5 years, comparable for age, tumor size, ER status and surgery). Findings were validated on publicly available datasets of 684 node‐negative BCs including all the subtypes.Only a metagene containing interferon‐induced genes (IFN metagene) proved to be predictive of distant metastasis in our series of patients with ESR1+/ERBB2− tumors (P = 0.029), and such a finding was validated on 457 ESR1+/ERBB2− BCs from public datasets (P = 0.0424). Conversely, the IFN metagene was associated with a low risk of metastasis in 104 ERBB2+ tumors (P = 0.0099) whereas it did not prove to significantly affect prognosis in 123 ESR1−/ERBB2− tumors (P = 0.2235). A complex prognostic interaction was revealed in ESR1+/ERBB2− and ERBB2+ tumors when the association between the IFN metagene and a T‐cell metagene was considered.The study confirms the importance of analyzing prognostic variables separately within BC subtypes, highlights the advantages of using metagenes rather than genes, and finally identifies in node‐negative ESR1+/ERBB2− BCs, the unfavorable role of high IFN metagene expression.     

Inhibition of PARP1‐dependent end‐joining contributes to Olaparib‐mediated radiosensitization in tumor cells

Poly‐ADP‐ribose‐polymerase inhibitors (PARPi) are considered to be optimal tools for specifically enhancing radiosensitivity. This effect has been shown to be replication‐dependent and more profound in HR‐deficient tumors. Here, we present a new mode of PARPi‐mediated radiosensitization which was observed in four out of six HR‐proficient tumor cell lines (responders) investigated, but not in normal cells. This effect is replication‐independent, as the radiosensitization remained unaffected following the inhibition of replication using aphidicolin. We showed that responders are radiosensitized by Olaparib because their DSB‐repair is switched to PARP1‐dependent end‐joining (PARP1‐EJ), as evident by (i) the significant increase in the number of residual γH2AX foci following irradiation with 3Gy and treatment with Olaparib, (ii) the enhanced enrichment of PARP1 at the chromatin after 3Gy and (iii) the inhibition of end‐joining activity measured by a specific reporter substrate upon Olaparib treatment. This is the first study which directly demonstrates the switch to PARP1‐EJ in tumor cells and its contribution to the response to Olaparib as a radiosensitizer, findings which could widen the scope of application of PARPi in tumor therapy.     

Metastatic bone pain palliation with 89‐Sr and 186‐Re‐HEDP in breast cancer patients

Aim. The study evaluates the therapeutic efficacy of Strontium89chloride (89Sr) and 186Re1,1hydroxyethylidene diphosphonate (186ReHEDP) in the palliation of painful bone metastases from breast cancer. Patients and methods. Fifty patients with painful multifocal bone metastases from breast cancer entered the study and were randomized into two groups according to the radiopharmaceutical used: 148 MBq 89Sr i.v. (Group A: 25 patients) and 1406 MBq 186ReHEDP i.v. (Group B: 25 patients). Pain palliation was evaluated on the basis of the Wisconsin pain test improvement at two months and response was graded as complete, partial, minimal or absent. Hematological toxicity and side effects were reported according to WHO guidelines. Results. The global response rate was 84% (21/25) for 89Sr and 92% (23/25) for 186ReHEDP, respectively. The onset of pain palliation appeared significantly earlier in Group B (p<0.0001). The duration of pain relief ranged from two months to 14 months (mean of 125 days with a median value of 120 days) in Group A and from one month to 12 months (mean of 107 days with a median value of 60 days) in Group B (p=0.39). A moderate hematological toxicity was apparent in both groups. Platelet and white blood cell counts returned to baseline levels within 12 weeks after 89Sr administration and 6 weeks after 186ReHEDP administration (p<0.01). Conclusions. Both 89Sr and 186ReHEDP are effective and safe in bone pain palliation in breast cancer with the latter showing a significantly faster onset of pain relief.     

5‐Fluorouracil preferentially sensitizes mutant KRAS non‐small cell lung carcinoma cells to TRAIL‐induced apoptosis

Mutations in the KRAS gene are very common in non–small cell lung cancer (NSCLC), but effective therapies targeting KRAS have yet to be developed. Interest in tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL), a potent inducer of cell death, has increased following the observation that TRAIL can selectively kill a wide variety of human cancer cells without killing normal cells both in vitro and in xenograft models. However, results from clinical trials of TRAIL‐based therapy are disappointingly modest at best and many have demonstrated a lack of therapeutic benefit. Current research has focused on selecting a subpopulation of cancer patients who may benefit from TRAIL‐based therapy and identifying best drugs to work with TRAIL. In the current study, we found that NSCLC cells with a KRAS mutation were highly sensitive to treatment with TRAIL and 5‐fluorouracil (5FU). Compared with other chemotherapeutic agents, 5FU displayed the highest synergy with TRAIL in inducing apoptosis in mutant KRAS NSCLC cells. We also found that, on a mechanistic level, 5FU preferentially repressed survivin expression and induced expression of TRAIL death receptor 5 to sensitize NSCLC cells to TRAIL. The combination of low‐dose 5FU and TRAIL strongly inhibited xenograft tumor growth in mice. Our results suggest that the combination of TRAIL and 5FU may be beneficial for patients with mutant KRAS NSCLC.     

Novel circulating microRNA signature as a potential non‐invasive multi‐marker test in ER‐positive early‐stage breast cancer: A case control study

Introduction

There are currently no highly sensitive and specific minimally invasive biomarkers for detection of early‐stage breast cancer. MicroRNAs (miRNAs) are present in the circulation and may be unique biomarkers for early diagnosis of human cancers. The aim of this study was to investigate the differential expression of miRNAs in the serum of breast cancer patients and healthy controls.

Methods

Global miRNA analysis was performed on serum from 48 patients with ER‐positive early‐stage breast cancer obtained at diagnosis (24 lymph node‐positive and 24 lymph node‐negative) and 24 age‐matched healthy controls using LNA‐based quantitative real‐time PCR (qRT‐PCR). A signature of miRNAs was subsequently validated in an independent set of 111 serum samples from 60 patients with early‐stage breast cancer and 51 healthy controls and further tested for reproducibility in 3 independent data sets from the GEO Database.

Results

A multivariable signature consisting of 9 miRNAs (miR‐15a, miR‐18a, miR‐107, miR‐133a, miR‐139‐5p, miR‐143, miR‐145, miR‐365, miR‐425) was identified that provided considerable discrimination between breast cancer patients and healthy controls. Further, the ability of the 9 miRNA signature to stratify samples from breast cancer patients and healthy controls was confirmed in the validation set (p = 0.012) with a corresponding AUC = 0.665 in the ROC‐curve analysis. No association between miRNA expression and tumor grade, tumor size, menopausal‐ or lymph node status was observed. The signature was also successfully validated in a previously published independent data set of circulating miRNAs in early‐stage breast cancer (p = 0.024).

Conclusions

We present herein a 9 miRNA signature capable of discriminating between ER‐positive breast cancer and healthy controls. Using a specific algorithm based on the 9 miRNA signature, the risk for future individuals can be predicted. Since microRNAs are highly stable in blood components, this signature might be useful in the development of a blood‐based multi‐marker test to improve early detection of breast cancer. Such a test could potentially be used as a screening tool to identify individuals who would benefit from further diagnostic assessment.     

Successful co‐treatment with LHRH‐agonist for ovarian over‐stimulation and cystic formation in premenopausal tamoxifen exposure

The present study evaluates the potential beneficial effect of cotreatment with LHRHagonist in resolving premenopausal tamoxifen's induced supraphysiological serum 17 estradiol levels and persistent ovarian cysts. Ultrasonographic and serum hormonal evaluations were performed before, during, and following three consecutive injections of long acting LHRHagonist administered to 14 premenopausal breast cancer patients treated with tamoxifen, who had supraphysiological serum 17 estradiol levels and simultaneous persistent ovarian cysts. Within 3 weeks of the first LHRHagonist injection, all patients had menopausal serum estradiol levels. Ovarian cysts completely disappeared within 2 months following the first injection. Following the discontinuation of LHRHagonist cotreatment, serum estradiol levels remained in physiological levels and the ovaries remained a normal size in 64.3% of the patients for 13.3 ± 11.5 months. 28.6% of the patients had a gradual reappearance of high serum estradiol levels and of ovarian cysts, and were, therefore, treated with a second course of LHRHagonist. Following the second course, serum estradiol levels remained in physiological levels and the ovaries remained a normal size for 8–15 months. It is concluded that short duration of cotreatment with long acting LHRHagonist administered to premenopausal breast cancer patients treated with tamoxifen, successfully resolved the tamoxifeninduced supraphysiological serum 17 estradiol levels and the ovarian cysts.affiliated with Sackler Faculty of Medicine, Tel Aviv University     

Non‐genotoxic environmental carcinogens

Using a dataset with more than 6000 compounds, the performance of eight quantitative structure activity relationships (QSAR) models was evaluated: ACD/Tox Suite, Absorption, Distribution, Metabolism, Elimination, and Toxicity of chemical substances (ADMET) predictor, Derek, Toxicity Estimation Software Tool (T.E.S.T.), TOxicity Prediction by Komputer Assisted Technology (TOPKAT), Toxtree, CEASAR, and SARpy (SAR in python). In general, the results showed a high level of performance. To have a realistic estimate of the predictive ability, the results for chemicals inside and outside the training set for each model were considered. The effect of applicability domain tools (when available) on the prediction accuracy was also evaluated. The predictive tools included QSAR models, knowledge-based systems, and a combination of both methods. Models based on statistical QSAR methods gave better results.     

Collective migration of cancer‐associated fibroblasts is enhanced by overexpression of tight junction‐associated proteins claudin‐11 and occludin

It has been suggested that cancer‐associated fibroblasts (CAFs) positioned at the desmoplastic areas of various types of cancer are capable of executing a migratory program, characterized by accelerated motility and collective configuration. Since CAFs are reprogrammed derivatives of normal progenitors, including quiescent fibroblasts, we hypothesized that such migratory program could be context‐dependent, thus being regulated by specific paracrine signals from the adjacent cancer population. Using the traditional scratch assay setup, we showed that only specific colon cancer cell lines (i.e. HT29) were able to induce collective CAF migration. By performing quantitative proteomics (SILAC), we identified a 2.7‐fold increase of claudin‐11, a member of the tight junction apparatus, in CAFs that exerted such collectivity in their migratory pattern. Further proteomic investigations of cancer cell line secretomes revealed a specific signature, involving TGF‐β, as potential mediator of this effect. Normal colonic fibroblasts stimulated with TGF‐β exerted myofibroblastic differentiation, occludin (OCLN) and claudin‐11 (CLDN11) overexpression and cohort formation. Subsequently, inhibition of TGF‐β attenuated all the previous effects. Immunohistochemistry of the universal tight junction marker occludin in a cohort of 30 colorectal adenocarcinoma patients defined a CAF subpopulation expressing tight junctions. Overall, these data suggest that cancer cells may induce CLDN11 overexpression and subsequent collective migration of peritumoral CAFs via TGF‐β secretion.     

Interleukin‐6 drives melanoma cell motility through p38α‐MAPK‐dependent up‐regulation of WNT5A expression

Extensive research has demonstrated a tumor‐promoting role of increased WNT5A expression in malignant melanoma. However, very little light has been shed upon how WNT5A expression is up‐regulated in melanoma. A potential regulator of WNT5A expression is the pro‐inflammatory cytokine Interleukin (IL)‐6, which shares the ability of WNT5A to increase melanoma cell invasion. Here, we investigate whether IL‐6 can promote melanoma cell motility through an increased expression of WNT5A. We clearly demonstrate that the WNT5A‐antagonistic peptide Box5 could inhibit IL‐6‐induced melanoma cell migration and invasion. Furthermore, IL‐6 stimulation of the human melanoma cell lines HTB63 and A375 increased the expression of WNT5A in a dose‐dependent manner. To identify the signaling mechanism responsible for this up‐regulation, we explored the involvement of the three main signals induced by IL‐6; STAT3, Akt and ERK 1/2. Of these, only STAT3 was activated by IL‐6 in the melanoma cell lines tested. However, the STAT3 inhibitor S3I‐201 failed to inhibit IL‐6‐induced WNT5A up‐regulation in HTB63 and A375 cells. Nor did STAT3 siRNA silencing affect the expression of WNT5A. In search of an alternative signaling mechanism, we detected IL‐6‐induced activation of p38‐MAPK in HTB63 and A375 cells. The p38‐MAPK inhibitor SB203580 abolished the IL‐6‐induced WNT5A up‐regulation and blocked IL‐6‐induced melanoma cell invasion. The latter effect could be rescued by the addition of recombinant WNT5A. Notably, immunoprecipitation analysis revealed that only the p38α‐MAPK isoform was activated by IL‐6, and subsequent siRNA silencing of p38α‐MAPK abolished the IL‐6‐induced up‐regulation of WNT5A. Taken together, we demonstrate a novel link between the two melanoma pro‐metastatic agents IL‐6 and WNT5A explaining how IL‐6 can increase melanoma cell invasion and thus promote the metastatic process. This finding provides a basis for future therapeutic intervention of melanoma progression.     

Potential biomarkers of long‐term benefit from single‐agent trastuzumab or lapatinib in HER2‐positive metastatic breast cancer

In 2009 a prospective, randomized Phase II trial ({"type":"clinical-trial","attrs":{"text":"NCT00842998","term_id":"NCT00842998"}}NCT00842998) was initiated to evaluate the activity of HER2‐targeting agents without chemotherapy (CT) in HER2‐positive metastatic breast cancer (MBC) patients. The primary tumors of the patients enrolled in this study offered a unique opportunity to identify biomarkers that could predict durable clinical benefit from CT‐free anti‐HER2 therapy.Patients with HER2‐positive MBC were randomized to trastuzumab or lapatinib as first‐line therapy. CT was added to anti‐HER2 therapy in patients failing to achieve tumor regression at the 8‐week evaluation and in those progressing at any time. Expression analysis of 105 selected genes was performed from formalin‐fixed paraffin‐embedded primary tumor samples. The research‐based PAM50 intrinsic subtypes were also identified. Additionally, quantitative HER2 (H2T) and p95HER2 (p95) protein expression were evaluated by HERmark® and VeraTag® assay, respectively. Predictors of persistence on protocol (PP) were studied by Cox univariate and multivariate analysis.Nineteen patients were enrolled. Median overall survival was 43 months and median PP was 3.8 months (0.8–38.8+), with 4 patients (21.1%) persisting on single agent trastuzumab or lapatinib for longer than 12 mo (14.9–38.8 + mo). Seventeen patients were evaluable for PP. Gene expression analysis revealed that high expression of the 17q12‐21 amplicon genes HER2 and GRB7, and the PAM50 HER2‐enriched intrinsic profile, were significantly associated with longer PP. Conversely, high expression of luminal‐related genes such as PGR, MDM2 or PIK3CA, or the PAM50 luminal intrinsic profile correlated with reduced PP. Moreover, increasing H2T/p95 ratio was found to be significantly associated with longer PP (HR 0.56 per 2‐fold increase in H2T/p95, P = 0.0015).Our data suggest that patients belonging to the “HER2‐enriched” subtype and/or having high H2T/p95 protein expression ratio are exquisitely sensitive to anti‐HER2 agents. MBC patients with these tumors could be candidates for studies aimed at establishing chemotherapy‐free regimens.     

Oncogenic signaling in amphiregulin and EGFR‐expressing PTEN‐null human breast cancer

A subset of triple negative breast cancer (TNBC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) and loss of PTEN, and patients with these determinants have a poor prognosis. We used cell line models of EGFR‐positive/PTEN null TNBC to elucidate the signaling networks that drive the malignant features of these cells and cause resistance to EGFR inhibitors. In these cells, amphiregulin (AREG)‐mediated activation of EGFR results in up‐regulation of fibronectin (FN1), which is known to be a mediator of invasive capacity via interaction with integrin β1. EGFR activity in this PTEN null background also results in Wnt/beta‐catenin signaling and activation of NF‐κB. In addition, AKT is constitutively phosphorylated in these cells and is resistant to gefitinib. Expression profiling demonstrated that AREG‐activated EGFR regulates gene expression differently than EGF‐activated EGFR, and functional analysis via genome‐scale shRNA screening identified a set of genes, including PLK1 and BIRC5, that are essential for survival of SUM‐149 cells, but are uncoupled from EGFR signaling. Thus, our results demonstrate that in cells with constitutive EGFR activation and PTEN loss, critical survival genes are uncoupled from regulation by EGFR, which likely mediates resistance to EGFR inhibitors.     

Instability of a dinucleotide repeat in the 3′‐untranslated region (UTR) of the microsomal prostaglandin E synthase‐1 (mPGES‐1) gene in microsatellite instability‐high (MSI‐H) colorectal carcinoma

DNA mismatch‐repair gene mutations, with consequent loss of functional protein expression, result in microsatellite instability (MSI). Microsatellite sequences are found in coding regions and in regulatory regions of genes (i.e., 5′‐UTRs and 3′‐UTRs). In addition to being a surrogate marker of defective mismatch repair, deletion or insertion microsatellite sequences can dysregulate gene expression in MSI‐H (microsatellite instability‐high) tumors. The microsomal prostaglandin E synthase‐1 (mPGES‐1) gene product, mPGES‐1, participates in prostaglandin E2 (PGE2) production. Moreover, mPGES‐1 is often overexpressed in human colorectal tumors, and is thought to contribute to progression of these tumors. Here we identified a dinucleotide repeat, (GT)24, in the mPGES‐1 gene 3′ untranslated region (3′‐UTR), and analyzed its mutation frequencies in MSI‐H and microsatellite stable (MSS) tumors. The (GT)24 repeat exhibited instability in all MSI‐H tumors examined (14), but not in any of the MSS tumors (13). In most cases, (GT)24 repeat instability resulted in insertion of additional GT units. We also determined mPGES‐1 mRNA levels in MSI‐H and MSS colorectal cancer cell lines. Three of four previously designated “MSI‐H” cell lines showed higher mPGES‐1 mRNA levels compared to MSS cell lines; correlations between elevated mPGES‐1 mRNA levels and microsatellite (GT)24 repeat characteristics are present for all six cell lines. Our results demonstrate that mPGES‐1 is a target gene of defective mismatch repair in human colorectal cancer, with functional consequence.     

Biomarkers for local recurrence after breast‐conservation — a nested case‐control study

Purpose: A previous cohort study of 759 women with invasive T1T2 breast cancer operated on with breastconserving surgery in Stockholm between 1976 and 1986 indicated that age <50 years, no postoperative irradiation, and nodal involvement were independent risk factors for ipsilateral breast tumor recurrences (IBTR). The aim of the current study was to analyse if selected biological markers assayed in tumor specimens from these patients could add prognostic information, thereby helping to identify groups of patients at high versus low risk of IBTR.Methods: The study was designed as a casecontrol study nested within the cohort. The cohort was stratified according to nodal status and the use of postoperative irradiation. In these four strata, the cases were those 80 women who developed IBTR between 1977 and 1994. In each stratum, women without IBTR were randomly selected as controls (n=159). Median time at risk was 12 (8–18) years. The following factors were analysed: histopathological tumor grade according to Elston–Ellis, DNA ploidy, immunohistochemical staining for apoptosis, angiogenesis, Ki67 (MIB1), cerbB2, p53, waf1, and bcl2. The prognostic role of each factor was assessed using linear logistic regression methods.Results: In univariate analyses only age <50 years was identified as a significant risk factor for IBTR, whereas none of the studied biomarkers yielded statistically significant information. However, in a multivariate model, age, MIB-1-index, and tumor grade significantly influenced the risk for IBTR: the odds-ratio (OR) for age 50 years was 0.4, 95% confidence interval (CI)=0.2–0.9; for medium or high grade tumors it was 0.4 (CI=09–0.9); and for MIB-1-index >30%, 2.1 (CI=1.0–4.4). In women 50 years, MIB-1-index >30% was associated with an OR of 3.5 (CI=1.4–8.8) compared to those who were younger. Patients 50 years with MIB-1-index 30% were thus identified as a low-risk group with an OR of 0.2 (CI=0.1–0.5). A possible high-risk group was patients <50 years with tumors showing a combination of c-erbB-2 and waf-1 immunoreactivity, with an OR of 6.7 (CI=1.3–34.7).Conclusion: Women 50 years with MIB-1-index 30% constituted a subgroup with a low risk of IBTR. This observation raises the issue whether this group of patients might be spared postoperative irradiation following breast-conserving surgery. However, due to the methodology of the study, including the large number of comparisions, the presented results warrant cautious interpretation and should be regarded as tentative.     

Significance of TP53 mutations as predictive markers of adjuvant cisplatin‐based chemotherapy in completely resected non‐small‐cell lung cancer

Adjuvant cisplatin‐based chemotherapy only marginally improves survival in patients with completely resected non‐small‐cell lung cancer (NSCLC). We have evaluated the predictive value of mutations in TP53, encoding the tumour suppressor p53, in the International Adjuvant Lung Cancer Trial (IALT), a randomized trial of adjuvant cisplatin‐based chemotherapy against observation. TP53 (exons 4–8) was sequenced in 524 archived specimens of IALT patients with a median follow‐up of 7.5 years. Predictive analyses were based on Cox models adjusted for clinical and pathological variables. P‐values ≤0.01 were considered as significant. Mutations were detected in 221 patients (42%) and had no predictive value for the effect of chemotherapy (interaction between TP53 and treatment: p = 0.17 for Overall Survival (OS); p = 0.06 for Disease‐Free Interval, (DFS)). However, among patients with mutations, outcome appeared worse in treatment compared to observation arms (HR for OS = 1.36 (95% CI [0.97–1.31), p = 0.08; DFS = 1.40 (95% CI [1.01–1.95]), p = 0.04). When grouping mutations into classes according to predicted effects on protein structure, the tendency towards worse outcomes was restricted to “structure” mutations affecting residues of the hydrophobic core that are not located at the p53 protein‐DNA interface (HR for death in this class vs wild‐type T53 = 1.66; 95% CI [1.10–2.52], p = 0.02). Overall, TP53 mutations are not significant predictors of outcome in this trial of cisplatin‐based chemotherapy, although a specific class of structural mutations may be associated with a tendency towards worse outcomes upon treatment.     

Multi‐marker analysis of circulating cell‐free DNA toward personalized medicine for colorectal cancer

Development of a Q‐PCR‐based assay for the high‐performance analysis of circulating cell‐free DNA (ccfDNA) requires good knowledge of its structure and size.In this work, we present the first visual determination of ccfDNA by Atomic Force Microscopy (AFM) on plasma samples from colorectal cancer (CRC) patients and healthy donors. In addition to the examination of fragment size distribution profile as performed by Q‐PCR, this analysis confirms that ccfDNA is highly fragmented and that more than 80% of ccfDNA fragments in CRC plasma are below 145 bp. We adapted an Allele‐Specific Blocker (ASB) Q‐PCR to small ccfDNA fragments to determine simultaneously the total ccfDNA concentration, the presence of point mutation, the proportion of mutated allele, and a ccfDNA integrity index. The data validated analytically these four parameters in 124 CRC clinical samples and 71 healthy individuals. The multi‐marker method, termed Intplex, enables sensitive and specific non‐invasive analysis of tumor ccfDNA, which has great potential in terms of cost, quality control, and easy implementation in every clinical center laboratory.     

Volitinib,a potent and highly selective c‐Met inhibitor,effectively blocks c‐Met signaling and growth in c‐MET amplified gastric cancer patient‐derived tumor xenograft models

Purpose

To investigate the incidence of cMET gene copy number changes and protein overexpression in Chinese gastric cancer (GC) and to preclinically test the hypothesis that the novel, potent and selective cMET small‐molecule inhibitor volitinib, will deliver potent anti‐tumor activity in cMET‐dysregulated GC patient‐derived tumor xenograft (PDX) models.

Experimental design

A range of assays were used and included; in vitro cell line panel screening and pharmacodynamic (PD) analysis, cMET fluorescence in‐situ hybridization (FISH) and immunohistochemical (IHC) tissue microarray (TMA) analysis of Chinese GC (n = 170), and anti‐tumor efficacy testing and PD analysis of gastric PDX models using volitinib.

Results

The incidence of cMET gene amplification and protein overexpression within Chinese patient GC tumors was 6% and 13%, respectively. Volitinib displayed a highly selective profile across a gastric cell line panel, potently inhibiting cell growth only in those lines with dysregulated cMET (EC50 values 0.6 nM/L–12.5 nM/L). Volitinib treatment led to pharmacodynamic modulation of cMET signaling and potent tumor stasis in 3/3 cMET‐dysregulated GC PDX models, but had negligible activity in a GC control model.

Conclusions

This study provides an assessment of tumor cMET gene copy number changes and protein overexpression incidence in a cohort of Chinese GC patients. To our knowledge, this is the first study to demonstrate anti‐tumor efficacy in a panel of cMET‐dysregulated gastric cancer PDX models, using a novel selective cMET‐inhibitor (volitinib). Thus, the translational science presented here provides strong rationale for the investigation of volitinib as a therapeutic option for patients with GC tumors harboring amplified cMET.