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Genomics; Transcriptomics; Lung cancer Genómica; Transcriptómica; Cáncer de pulmón Genòmica; Transcriptòmica; Càncer de pulmó Background The Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) to better define the cancer molecular milieu based on genomics/transcriptomics from tumor and analogous normal tissue biopsies. SPRING is the first trial to assess a SIMS-based tri-therapy regimen in advanced non-small cell lung cancer (NSCLC). Methods Patients with advanced NSCLC (no EGFR, ALK, or ROS1 alterations; PD-L1 unrestricted; ≤2 prior therapy lines) received avelumab, axitinib, and palbociclib (3 + 3 dose escalation design). Results Fifteen patients were treated (five centers, four countries): six at each of dose levels 1 (DL1) and DL2; three at DL3. The most common ≥Grade 3 adverse events were neutropenia, hypertension, and fatigue. The recommended Phase II dose (RP2D) was DL1: avelumab 10 mg/kg IV q2weeks, axitinib 3 mg po bid, and palbociclib 75 mg po daily (7 days off/21 days on). Four patients (27%) achieved a partial response (PR) (progression-free survival [PFS]: 14, 24, 25 and 144+ weeks), including two after progression on pembrolizumab. Four patients attained stable disease (SD) that lasted ≥24 weeks: 24, 27, 29, and 64 weeks. At DL1 (RP2D), four of six patients (66%) achieved stable disease (SD) ≥6 months/PR (2 each). Responders included patients with no detectable PD-L1 expression and low tumor mutational burden. Conclusions Overall, eight of 15 patients (53%) achieved clinical benefit (SD ≥ 24 weeks/PR) on the avelumab, axitinib, and palbociclib combination. This triplet showed antitumor activity in NSCLC, including in tumors post-pembrolizumab progression, and was active at the RP2D, which was well tolerated. This work was supported by the ARC Foundation for Cancer Research, Villejuif, France and Worldwide Innovative Network (WIN) Association––WIN Consortium, Villejuif, France, sponsor of the study. WIN was responsible for the study design, collection, analysis, and interpretation of data as well as writing of the report. The study drugs were provided by Pfizer, as part of an alliance between Merck KGaA, Darmstadt, Germany and Pfizer. Funded in part by National Cancer Institute grant P30 CA023100 and the Joan and Irwin Jacobs Fund philanthropic fund (RK).

The expanding targeted therapy landscape requires combinatorial biomarkers for patient stratification and treatment selection. This requires simultaneous exploration of multiple genes of relevant networks to account for the complexity of mechanisms that govern drug sensitivity and predict clinical outcomes. We present the algorithm, Digital Display Precision Predictor (DDPP), aiming to identify transcriptomic predictors of treatment outcome. For example, 17 and 13 key genes were derived from the literature by their association with MTOR and angiogenesis pathways, respectively, and their expression in tumor versus normal tissues was associated with the progression-free survival (PFS) of patients treated with everolimus or axitinib (respectively) using DDPP. A specific eight-gene set best correlated with PFS in six patients treated with everolimus: AKT2, TSC1, FKB-12, TSC2, RPTOR, RHEB, PIK3CA, and PIK3CB (r = 0.99, p = 5.67E−05). A two-gene set best correlated with PFS in five patients treated with axitinib: KIT and KITLG (r = 0.99, p = 4.68E−04). Leave-one-out experiments demonstrated significant concordance between observed and DDPP-predicted PFS (r = 0.9, p = 0.015) for patients treated with everolimus. Notwithstanding the small cohort and pending further prospective validation, the prototype of DDPP offers the potential to transform patients’ treatment selection with a tumor- and treatment-agnostic predictor of outcomes (duration of PFS).

Background Recent studies have aimed to identify gene mutation profiles to explain the cause of TNBC therapy limitations. Methods The purpose of our study was to use Next Generation Sequencing (NGS) of 46 genes with a well-defined role in cancer in a cohort of TNBC patients in order to identify novel markers that could lead to the development of strategic, adjuvant, gene-targeted therapies. Results A total of 118 gene mutations in 35 genes, 75 mutations in BRCA1 and 92 mutations in BRCA2 were identified. The clinical assessment of the identified mutations showed 27 to be possibly damaging and 59 to be damaging. TP53, KDR, PIK3CA (rs3729687), ATM, AKT1 and KIT were among the most frequently mutated genes in our TNBC cohort. The SNP AKT1 (rs3730358) was suggested to modify the risk of breast cancer. SNP PIK3CA (rs3729687) is a damaging mutation that we found to be correlated with the prognosis of TNBC. The survival curve analysis showed that the presence of AKT1, TP53, KDR, KIT, BRCA1 and BRCA2 mutations is correlated with a poor prognosis. Conclusion We show a strong association between TNBC and mutations in BRCA1/2 genes and the poor outcome of these patients. Moreover, we identified several other unknown mutations putatively associated with the poor prognosis of TNBC tumors. We also discovered novel mutations never before associated with breast cancer that could putatively account for the poor prognosis of the TNBC tumors.

Critical processes such as growth, invasion, and metastasis of cancer cells are sustained via bidirectional cell-to-cell communication in tissue complex environments. Such communication involves the secretion of soluble factors by stromal cells and/or cancer cells within the tumor microenvironment (TME). Both stromal and cancer cells have been shown to export bilayer nanoparticles: encapsulated regulatory molecules that contribute to cell-to-cell communication. These nanoparticles are known as extracellular vesicles (EVs) being classified into exosomes, microvesicles, and apoptotic bodies. EVs carry a vast repertoire of molecules such as oncoproteins and oncopeptides, DNA fragments from parental to target cells, RNA species (mRNAs, microRNAs, and long non-coding RNA), and lipids, initiating phenotypic changes in TME. According to their specific cargo, EVs have crucial roles in several early and late processes associated with tumor development and metastasis. Emerging evidence suggests that EVs are being investigated for their implication in early cancer detection, monitoring cancer progression and chemotherapeutic response, and more relevant, the development of novel targeted therapeutics. In this study, we provide a comprehensive understanding of the biophysical properties and physiological functions of EVs, their implications in TME, and highlight the applicability of EVs for the development of cancer diagnostics and therapeutics.

Simple Summary Cytopathology cannot distinguish benign from malignant follicular lesions in 20–30% of cases. These indeterminate cases includes the so-called follicular neoplasms (FNs) according to The Bethesda System for Reporting Thyroid Cytopathology. Frozen samples from 66 classic follicular adenomas (cFAs) and carcinomas (cFTCs) studied by array-comparative genomic hybridization identified three specific alterations of cFTCs (losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X) confirmed by fluorescent in situ hybridization (FISH) in a second independent series of 60 touch preparations from frozen samples of cFAs and cFTCs. In a third independent set of 27 cases of already stained pre-operative fine-needle aspiration cytology samples diagnosed as FNs and histologically verified, FISH analysis using these three markers identified half of cFTCs. Specificity of our assay for identifying cFTCs is higher than 98% which might be comparable with BRAF600E testing in cases of suspicion of classic papillary thyroid carcinomas. Abstract Although fine-needle aspiration cytology (FNAC) is helpful in determining whether thyroid nodules are benign or malignant, this distinction remains a cytological challenge in follicular neoplasms. Identification of genomic alterations in cytological specimens with direct and routine techniques would therefore have great clinical value. A series of 153 cases consisting of 72 and 81 histopathologically confirmed classic follicular adenomas (cFAs) and classic follicular thyroid carcinomas (cFTCs), respectively, was studied by means of different molecular techniques in three different cohorts of patients (pts). In the first cohort (training set) of 66 pts, three specific alterations characterized by array comparative genomic hybridization (aCGH) were exclusively found in half of cFTCs. These structural abnormalities corresponded to losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X. The second independent cohort (validation set) of 60 pts confirmed these data on touch preparations of frozen follicular neoplasms by triple DNA fluorescent in situ hybridization using selected commercially available probes. The third cohort, consisting of 27 archived cytological samples from an equal number of pts that had been obtained for preoperative FNAC and morphologically classified as and histologically verified to be follicular neoplasms, confirmed our previous findings and showed the feasibility of the DNA FISH (DNA fluorescent in situ hybridization) assay. All together, these data suggest that our triple DNA FISH diagnostic assay may detect 50% of cFTCs with a specificity higher than 98% and be useful as a low-cost adjunct to cytomorphology to help further classify follicular neoplasms on already routinely stained cytological specimens.

9581 Background: The Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) algorithm in order to predict treatment response by comparing tumor and normal tissue biopsies on both genomic and transcriptomic platforms. SPRING is the first trial to assess a SIMS-based tri-therapy regimen in advanced non-small cell lung cancer (NSCLC). Methods: Patients with advanced NSCLC (no EGFR or ALK alterations; no ROS1 alterations if tested; PD-L1 unrestricted; ≤2 prior therapy lines) were treated with avelumab, axitinib, and palbociclib (3+3 dose escalation design). Tumor and normal endobronchial mucosal biopsies were obtained on all patients for retrospective SIMS algorithm validation. Results: Fifteen patients were treated: 6 at dose level 1 (DL1); 6, dose level 2 (DL2); 3, dose level 3 (DL3). Three dose-limiting toxicities (DLTs) at least possibly drug-related occurred: 1 DLT at DL2 (Grade 3 (G3) infusion reaction); 2 patients with DLTs at DL3 (1 with G3 hand/foot syndrome and G3 fatigue and 1 with G5 respiratory failure). Among 14 evaluable patients, the partial response (PR) rate was 28.6% (4/14 patients including 2/6 patients at DL1; two PRs in patients who failed prior pembrolizumab; two PRs in patients with PD-L1 < 1%). The maximum tolerated dose was avelumab 10 mg/kg IV q2weeks, axitinib 5 mg PO BID continuous, palbociclib 75 mg PO daily on days 8-28 of a 28 day cycle (DL2). DL2 was above the recommended phase II dose (RP2D), since 5/6 patients treated at DL2 required later treatment delays and/or dose reductions, mostly due to neutropenia. To further evaluate DL1, 3 patients were added to this cohort (total of 6). Since no DLTs were seen at DL1, and 5 of 6 patients did not require dose reduction, DL1 (avelumab 10 mg/kg IV q2weeks, axitinib 3 mg PO BID continuous, palbociclib 75 mg PO daily on days 8-28 of a 28 day cycle) is the RP2D. Conclusions: The RP2D was determined to be dose level 1. This triplet showed antitumor activity in patients with NSCLC, including those progressing on prior pembrolizumab. SIMS algorithm correlates of response are being assessed. Clinical trial information: NCT03386929 .

Despite of remarkable progress made in the head and neck cancer (HNC) therapy, the survival rate of this metastatic disease remain low. Tailoring the appropriate therapy to patients is a major challenge and highlights the unmet need to have a good preclinical model that will predict clinical response. Hence, we developed an accurate and time efficient drug screening method of tumor ex vivo analysis (TEVA) system, which can predict patient-specific drug responses. In this study, we generated six patient derived xenografts (PDXs) which were utilized for TEVA. Briefly, PDXs were cut into 2 × 2 × 2 mm3 explants and treated with clinically relevant drugs for 24 h. Tumor cell proliferation and death were evaluated by immunohistochemistry and TEVA score was calculated. Ex vivo and in vivo drug efficacy studies were performed on four PDXs and three drugs side-by-side to explore correlation between TEVA and PDX treatment in vivo. Efficacy of drug combinations was also ventured. Optimization of the culture timings dictated 24 h to be the time frame to detect drug responses and drug penetrates 2 × 2 × 2 mm3 explants as signaling pathways were significantly altered. Tumor responses to drugs in TEVA, significantly corresponds with the drug efficacy in mice. Overall, this low cost, robust, relatively simple and efficient 3D tissue-based method, employing material from one PDX, can bypass the necessity of drug validation in immune-incompetent PDX-bearing mice. Our data provides a potential rationale for utilizing TEVA to predict tumor response to targeted and chemo therapies when multiple targets are proposed.

mAb-based blocking of the immune checkpoints involving the CTLA4-B7 and PD1-PDL1 inhibitory axes enhance T-cell–based adaptive immune responses in patients with cancer. We show here that antitumor responses by natural killer (NK) cells can be enhanced by a checkpoint-blocking mAb, 14-25-9, which we developed against proliferating cell nuclear antigen (PCNA). PCNA is expressed on the surface of cancer cells and acts as an inhibitory ligand for the NK-cell receptor, NKp44-isoform1. We tested for cytoplasmic- and membrane-associated PCNA by FACS- and ImageStream-based staining of cell lines and IHC of human cancer formalin fixed, paraffin embedded tissues. The mAb, 14-25-9, inhibited binding of chimeric NKp44 receptor to PCNA and mostly stained the cytoplasm and membrane of tumor cells, whereas commercial antibody (clone PC10) stained nuclear PCNA. NK functions were measured using ELISA-based IFNγ secretion assays and FACS-based killing assays. The NK92-NKp44-1 cell line and primary human NK cells showed increased IFNγ release upon coincubation with mAb 14-25-9 and various solid tumor cell lines and leukemias. Treatment with 14-25-9 also increased NK cytotoxic activity. In vivo efficacy was evaluated on patient-derived xenografts (PDX)-bearing NSG mice. In PDX-bearing mice, intravenous administration of mAb 14-25-9 increased degranulation (CD107a expression) of intratumorally injected patient autologous or allogeneic NK cells, as well as inhibited tumor growth when treated long term. Our study describes a mAb against the NKp44-PCNA innate immune checkpoint that can enhance NK-cell antitumor activity both in vitro and in vivo.

Purpose The impact of a personalized cancer treatment strategy (ie, matching patients with drugs based on specific biomarkers) is still a matter of debate. Methods We reviewed phase II single-agent studies (570 studies; 32,149 patients) published between January 1, 2010, and December 31, 2012 (PubMed search). Response rate (RR), progression-free survival (PFS), and overall survival (OS) were compared for arms that used a personalized strategy versus those that did not. Results Multivariable analysis (both weighted multiple linear regression and random effects meta-regression) demonstrated that the personalized approach, compared with a nonpersonalized approach, consistently and independently correlated with higher median RR (31% v 10.5%, respectively; P < .001) and prolonged median PFS (5.9 v 2.7 months, respectively; P < .001) and OS (13.7 v 8.9 months, respectively; P < .001). Nonpersonalized targeted arms had poorer outcomes compared with either personalized targeted therapy or cytotoxics, with median RR of 4%, 30%, and 11.9%, respectively; median PFS of 2.6, 6.9, and 3.3 months, respectively (all P < .001); and median OS of 8.7, 15.9, and 9.4 months, respectively (all P < .05). Personalized arms using a genomic biomarker had higher median RR and prolonged median PFS and OS (all P ≤ .05) compared with personalized arms using a protein biomarker. A personalized strategy was associated with a lower treatment-related death rate than a nonpersonalized strategy (median, 1.5% v 2.3%, respectively; P < .001). Conclusion Comprehensive analysis of phase II, single-agent arms revealed that, across malignancies, a personalized strategy was an independent predictor of better outcomes and fewer toxic deaths. In addition, nonpersonalized targeted therapies were associated with significantly poorer outcomes than cytotoxic agents, which in turn were worse than personalized targeted therapy.

Bevacizumab is one of the most widely used antiangiogenic drugs in oncology, but the overall beneficial effects of this VEGF-A targeting agent are relatively modest, in part due to the lack of a biomarker to select patients most likely to respond favorably. Several molecular aberrations in cancer influence angiogenesis, including mutations in the tumor suppressor gene TP53, which occur frequently in many human malignancies. In this study, we present a multiple regression analysis of transcriptomic data in 123 patients with non–small cell lung cancer (NSCLC) showing that TP53 mutations are associated with higher VEGF-A expression (P = 0.006). This association was interesting given a recent retrospective study showing longer progression-free survival in patients with diverse tumors who receive bevacizumab, if tumors harbor mutant TP53 instead of wild-type TP53. Thus, our current findings linking TP53 mutation with VEGF-A upregulation offered a mechanistic explanation for why patients exhibit improved outcomes after bevacizumab treatment when their tumors harbor mutant TP53 versus wild-type TP53. Overall, this work warrants further evaluation of TP53 as a ready biomarker to predict bevacizumab response in NSCLC and possibly other tumor types. Cancer Res; 75(7); 1187–90. ©2015 AACR.

Suppressor of cytokine signaling (SOCS) 1 is an inducible negative regulator of cytokine signaling but its role in human cancer is not completely established. Here we report that, while SOCS1 is expressed in normal colonic epithelium and colon adenocarcinomas, its level decreases during progression of colon adenocarcinomas, the lowest level being found in the most aggressive stage and least differentiated carcinomas. Forced expression of SOCS1 in metastatic colorectal SW620 cells reverses many characteristics of Epithelial–Mesenchymal Transition (EMT), as highlighted by the disappearance of the transcription factor ZEB1 and the mesenchymal form of p120ctn and the re-expression of E-cadherin. Furthermore, miRNA profiling indicated that SOCS1 also up-regulates the expression of the mir-200 family of miRNAs, which can promote the mesenchymal–epithelial transition and reduce tumor cell migration. Accordingly, overexpression of SOCS1 induced cell morphology changes and dramatically reduced tumor cell invasion in vitro . When injected in nude mice, SOCS1-expressing SW620 cells induced metastases in a smaller number of animals than parental SW620 cells, and did not generate any adrenal gland or bone metastasis. Overall, our results suggest that SOCS1 controls metastatic progression of colorectal tumors by preventing the mesenchymal–epithelial transition (MET), including E-cadherin expression. This pathway may be associated with survival to colorectal cancer by reducing the capacity of generating metastases.

Background: NSCLC is the leading cause of cancer death. Recently, much progress has been made to identify and target specific oncogenic drivers such as EGFR mutations and ALK translocations. Unfortunately, the majority of NSCLCs do not have a single driver, but instead harbor complex systems of molecular alterations. The advent of immunooncology has opened newer avenues for treating these patients, but still, many tumors are resistant. Therefore, selection of precise therapies targeting the appropriate pathways in these tumors is an important area of research. In previously published work, the Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) algorithm to match patients to a targeted drug triplet. The SIMS algorithm is based on the hypothesis that the use of both genomic as well as transcriptomic data is important in selecting an ideal tri-therapy regimen. Since the transcriptome varies widely between different types of normal tissue, the relative level of transcription of any given gene in a tumor must be normalized against the expected level of transcription of that gene in the analogous normal tissue. The SPRING trial is the first trial assessing a rational, SIMS-based tri-therapy regimen in advanced NSCLC. The drugs utilized for SPRING–avelumab (anti-PDL1), axitinib (VEGFR inhibitor) and palbociclib (CDK4/6 inhibitor)–were chosen because the SIMS genomic/transcriptomic analysis demonstrated that ~10% of NSCLCs have a combination of these targets. Methods: Patients with locally advanced or metastatic NSCLC without EGFR or ALK alterations are being enrolled and treated with avelumab, palbociclib, and axitinib after informed consent. During phase I, any prior treatment is allowed. SPRING will assess the safety of the tri-therapy combination and determine the recommended phase II dose (RP2D) (3+3 dose escalation). Once the RP2D is found, the study will proceed directly to the phase II portion, which will permit only previously untreated patients in the metastatic setting. A biopsy of both tumor and normal tissue (by endobronchial mucosal biopsy) is obtained on all patients, both of which are analysed in a central genomics and transcriptomics platform. The SIMS algorithm is being validated retrospectively by determining the degree of genomic and transcriptomic matching and correlating with outcome. Results: The phase I study is open in USA, Israel, Spain and Luxembourg and has enrolled 12 patients to date and has progressed to cohort 3. The phase II portion is expected to open later in 2019. This research is supported by the EMD Serono/Pfizer alliance and ARC Foundation for cancer research. Citation Format: Benjamin Solomon, Enriqueta Felip, Jair Bar, Guy Berchem, Lyudmila Bazhenova, Pierre Saintigny, Nicolas Girard, Raed Sulaiman, Catherine Bresson, Fanny Wunder, J Jack Lee, Jacques Raynaud, Eitan Rubin, Brandon Young, Vladimir Lazar, Amir Onn, Brian Leyland Jones, Razelle Kurzrock. Survival Prolongation by Rationale INnovative Genomics (SPRING): An international WIN Consortium Phase I/II proof-of-concept study to explore the safety and efficacy of a tri-therapy approach using avelumab, palbociclib and axitinib in advanced/metastatic non-small cell lung cancer (NSCLC) with integrated genomic and transcriptomic correlates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT223.

The spliceosome is a large ribonucleoprotein complex that guides pre-mRNA splicing in eukaryotic cells. Here, we determine whether the spliceosome could constitute an attractive therapeutic target in cancer. Analysis of gene expression arrays from lung, breast, and ovarian cancers datasets revealed that several genes encoding components of the core spliceosome composed of a heteroheptameric Sm complex were overexpressed in malignant disease as compared with benign lesions and could also define a subset of highly aggressive breast cancers. siRNA-mediated depletion of SmE (SNRPE) or SmD1 (SNRPD1) led to a marked reduction of cell viability in breast, lung, and melanoma cancer cell lines, whereas it had little effect on the survival of the nonmalignant MCF-10A breast epithelial cells. SNRPE or SNRPD1 depletion did not lead to apoptotic cell death but autophagy, another form of cell death. Indeed, induction of autophagy was revealed by cytoplasmic accumulation of autophagic vacuoles and by an increase in both LC3 (MAP1LC3A) protein conversion and the amount of acidic autophagic vacuoles. Knockdown of SNRPE dramatically decreased mTOR mRNA and protein levels and was accompanied by a deregulation of the mTOR pathway, which, in part, explains the SNRPE-dependent induction of autophagy. These findings provide a rational to develop new therapeutic agents targeting spliceosome core components in oncology. Cancer Res; 73(7); 2247–58. ©2013 AACR.

Triple-negative breast cancer (TNBC) is a heterogeneous group of tumours characterised by lack of expression of oestrogen-, progesterone- and human epidermal growth factor receptors. TNBC, which represents approximately 15% of all mammary tumours, has a poor prognosis because of an aggressive behaviour and the lack of specific treatment. Accordingly, TNBC has become a major focus of research into breast cancer and is now classified into several molecular subtypes, each with a different prognosis. Pathological angiogenesis occurs at a late stage in the proliferation of TNBC and is associated with invasion and metastasis; there is an association with metabolic syndrome. Semaphorins are a versatile family of proteins with multiple roles in angiogenesis, tumour growth and metastasis and may represent a clinically useful focus for therapeutic targeting in this type of breast cancer. Another important field of investigation into the control of pathological angiogenesis is related to the expression of noncoding RNA (ncRNA) – these molecules can be considered as a therapeutic target or as a biomarker. Several molecular agents for intervening in the activity of different signalling pathways are being explored in TNBC, but none has so far proved effective in clinical trials and the disease continues to pose a defining challenge for clinical management as well as innovative cancer research.

Background Resectable non-small cell lung cancer (NSCLC) treatment options most often consist of surgical resection along with adjuvant chemotherapy (ACT). The benefit of ACT however is modest and is accompanied by important side effects. Objective One central quest in the field is therefore the identification of a predictive marker of the response to ACT. Methods We applied an unbiased approach based on high content analysis of expression data generated from a discovery patient cohort. Results We identified MMS19, a component of the cytoplasmic Iron-Sulfur Assembly (CIA) machinery important for the Nucleotide Excision Repair (NER) pathway as a pivotal gene for cisplatin toxicity. We then confirmed the association between MMS19 expression and the response to Cisplatin treatment in a panel of NSCLC cell lines. Finally we validated these pre-clinical data in a subgroup of JBR.10 trial patients through a hypothesis-driven analysis, and showed that MMS19 levels associated with ACT benefit. Conclusions We therefore propose the expression level of MMS19 as a candidate predictive marker of ACT benefit in resected NSCLC patients.

The impact of a biomarker-based (personalized) cancer treatment strategy in the setting of phase 1 clinical trials was analyzed.To compare patient outcomes in phase 1 studies that used a biomarker selection strategy with those that did not.PubMed search of phase 1 cancer drug trials (January 1, 2011, through December 31, 2013).Studies included trials that evaluated single agents, and reported efficacy end points (at least response rate [RR]).Data were extracted independently by 2 investigators.Response rate and progression-free survival (PFS) were compared for arms that used a personalized strategy (biomarker selection) vs those that did not. Overall survival was not analyzed owing to insufficient data.A total of 346 studies published in the designated 3-year time period were included in the analysis. Multivariable analysis (meta-regression and weighted multiple regression models) demonstrated that the personalized approach independently correlated with a significantly higher median RR (30.6% [95% CI, 25.0%-36.9%] vs 4.9% [95% CI, 4.2%-5.7%]; P .001) and a longer median PFS (5.7 [95% CI, 2.6-13.8] vs 2.95 [95% CI, 2.3-3.7] months; P .001). Targeted therapy arms that used a biomarker-based selection strategy (n = 57 trials) were associated with statistically improved RR compared with targeted therapy arms (n = 177 arms) that did not (31.1% [95% CI, 25.4%-37.4%] vs 5.1% [95% CI, 4.3%-6.0%]; P .001). Nonpersonalized targeted arms had outcomes comparable with those that tested a cytotoxic agent (median RR, 5.1% [95% CI, 4.3%-6.0%] vs 4.7% [95% CI, 3.6%-6.2%]; P = .63; respectively; median PFS, 3.3 [95% CI, 2.6-4.0] months vs 2.5 [95% CI, 2.0-3.7] months; P = .22). Personalized arms using a "genomic (DNA) biomarker" had higher median RR than those using a "protein biomarker" (42.0% [95% CI, 33.7%-50.9%] vs 22.4% [95% CI, 15.6%-30.9%]; P = .001). The median treatment-related mortality was not statistically different for arms that used a personalized strategy vs not (1.89% [95% CI, 1.36%-2.61%] vs 2.27% [95% CI, 1.97%-2.62%]; P = .31).In this meta-analysis, most phase 1 trials of targeted agents did not use a biomarker-based selection strategy. However, use of a biomarker-based approach was associated with significantly improved outcomes (RR and PFS). Response rates were significantly higher with genomic vs protein biomarkers. Studies that used targeted agents without a biomarker had negligible response rates.

Background: However hormone receptor–positive, early-stage breast cancer is a disease with a long natural history and improved survival with 5-year adjuvant endocrine treatments. Yet late recurrence remains an important issue in adjuvant therapy. Predictors of late recurrence are not yet well characterized. Method: A total of 252 breast primary tumors were selected at the Netherlands Cancer Institute from retrospective series of ER+, HER2− breast cancer patients with a follow-up of at least 10 years. Gene expression analysis was performed using Agilent 4×44K microarrays. In order to identify genes associated to late survival differences, we used the survdiff function implemented in the R package survival and we set the parameter rho to −1 to give weight to the later part of the survival curve. The survdiff function was applied to each probe individually for DMFS time considering the probe as a covariate dichotomized into 2 groups (above and below the median expression across all samples). The parameter “strata” was used to stratify the 140 patients based on additional clinico-pathological parameters (Grade, Diameter, Lymph node status and MammaPrint), in order to find genes that add prognostic value to those parameters already known. This approach uses the distant-metastasis free survival (DMFS) time as a continuous variable. Results: After univariate analysis, MammaPrint, diameter, lymph node status and grade were significantly associated to late DMFS differences (Chi-square test p-values equal to 0.016, 0.004, In order to independently validate the prognostic power of these two genes, we tested their performance in the validation set of treated patients (n = 112) and in three publicly available datasets. In all datasets, the CH25H gene confirmed to be significantly associated to metastasis-free survival time in all tested series. Conclusions: These results might indicate that CH25H is an independent marker of late metastatic relapses. CH25H catalyzes the formation of 25-hydroxycholesterol from cholesterol, leading to repress cholesterol biosynthetic enzymes. In the last years, it is emerging that lipid metabolism plays an important role in breast cancer development and progression. Taken together, these findings make the CH25H gene a potential target for late metastasis control in breast cancer. These results warrant further prospective investigation and functional characterization of CH25H in this setting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-09-05.

Breast cancer includes high number of molecular entities targetable by specific agents. In this study, array CGH and PIK3CA/AKT1 mutations were used to drive patients into targeted therapy. A prospective molecular analysis was offered to metastatic breast cancer patients for whom samples were collected prospectively or retrospectively either from frozen or paraffin-embedded tissue. Analyses were performed using array CGH (Agilent platform) and PIK3CA (exon 10 and 21) and AKT1 mutations were explored by standard Sanger sequencing. One hundred and eight patients were included. Good quality CGH was obtained in 79% cases and was better for frozen samples. Genomic alterations were identified in 50% of patients including 11 PIK3CA and 8 AKT1 mutations. Eighteen treatments (17 patients) were administered according to their molecular profile with evidence of activity in nine. Reasons for not providing a genomic-driven treatment included absence of progressive disease (38%), investigator's choice (9%), rapid PD (19%), and no drug access (21%). Array CGH correctly identified Her2 status in 97% cases; failures were related to low % of tumour cells. Our study showed that array CGH is feasible in the context of daily practice and, in combination with PIK3CA/AKT1 mutations, identifies a significant number of actionable molecular alterations that allow driving patients into specific targeted agents.

Keeping Cancer Cells At Bay Cancer cells are often aneuploid; that is, they have an abnormal number of chromosomes. But to what extent this contributes to the tumorigenic phenotype is not clear. Senovilla et al. (p. 1678 ; see the Perspective by Zanetti and Mahadevan ) found that tetraploidization of cancer cells can cause them to become immunogenic and thus aid in their clearance from the body by the immune system. Cells with excess chromosomes put stress on the endoplasmic reticulum, which leads to movement of the protein calreticulin to the cell surface. Calreticulin exposure in turn caused recognition of cancer cells in mice by the host immune system. Thus, the immune system appears to serve a protective role in eliminating hyperploid cells that must be overcome to allow unrestricted growth of cancer cells.

Background: The rapid collection of diverse genome-scale data raises the urgent need to integrate and utilise these resources for biological discovery or biomedical applications. For example, diverse transcriptomic and gene copy number variation data are currently collected for various cancers, but relatively few current methods are capable to utilise the emerging information. Methods: We developed and tested a data-integration method to identify gene networks that drive the biology of breast cancer clinical subtypes. The method simultaneously overlays gene expression and gene copy number data on protein–protein interaction, transcriptional-regulatory and signalling networks by identifying coincident genomic and transcriptional disturbances in local network neighborhoods. Results: We identified distinct driver-networks for each of the three common clinical breast cancer subtypes: oestrogen receptor (ER)+, human epidermal growth factor receptor 2 (HER2)+, and triple receptor-negative breast cancers (TNBC) from patient and cell line data sets. Driver-networks inferred from independent datasets were significantly reproducible. We also confirmed the functional relevance of a subset of randomly selected driver-network members for TNBC in gene knockdown experiments in vitro. We found that TNBC driver-network members genes have increased functional specificity to TNBC cell lines and higher functional sensitivity compared with genes selected by differential expression alone. Conclusion: Clinical subtype-specific driver-networks identified through data integration are reproducible and functionally important.

Background Previous reports have described the use of microarrays to assess the molecular classification of human breast cancers and defined new subgroups based on gene expression that are relevant to patient management through their ability to predict metastatic relapse and survival relapse. However, different mechanisms may be associated with the development of early and late distant metastases. With the hypothesis that tumors may lead to early or distant metastases based on their intrinsic biological initial features, we aimed at defining molecular profiles for several subgroups of patients based on their outcome over time. Material and methods Breast primary tumors were selected from retrospective series of patients with frozen material available. These series include patients of all ages, LN- and LN+; Estrogen or Progesteron-receptor positive, Her2-negative, no adjuvant treatment, with a follow-up of more than 10 years (y) for the control group or distant metastatic relapse as first event (DM) for the study group (n=144). Patients tumors were classified in 4 groups: no relapse at 10 y (M0), DM before 3 y (M0-3, n=30), DM between 3 and 7 y (M3-7), DM after 7 y (M7+). Samples were collected in 2 different institutions (NKI series for identifying the signature and IGR series for validation). Gene expression analysis of breast tumor samples was performed using custom-made Agilent 44K high-density microarrays and hybridized against the Mammaprint® reference pool (MRP). Tumors were also assessed for their Mammaprint® status, wound-healing signature status and their intrinsic subtypes based on the Blueprint® signature. Moreover, we identified the pathway-level activities of the patient groups using PARADIGM. Results and Discussion For the NKI series, A subset of 144 samples was included based on the selection criteria: 57 M0, 31 M0-3, 25 M3-7, 31 M7+. None of the 3 previously mentioned signatures correctly identified M0 vs. M7+ patients. In order to identify a predictive signature of late relapse (after 7y) we considered M0 and M7+ MammaPrint-Low Risk patients and we split them in a training (n=41) and in a test (n=23) sets. A 73-gene signature was able to classify M7+ patients with 75% of sensitivity and 66% of specificity on the test set. DM after 7yr showed significant activation of pathway related to inflammatory response and angiogenesis. Detailed results and validation results on the independent IGR series will be presented at the meeting. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S1-6.

Purpose: Excision repair cross-complementation group 1 (ERCC1) is a protein involved in repair of DNA platinum adducts and stalled DNA replication forks. We and others have previously shown the influence of ERCC1 expression upon survival rates and benefit of cisplatin-based chemotherapy in patients with resected non–small-cell lung cancer (NSCLC). However, little is known about the molecular characteristics of ERCC1-positive and ERCC1-negative tumors. Experimental Design: We took advantage of a cohort of 91 patients with resected NSCLC, for which we had matched frozen and paraffin-embedded samples to explore the comparative molecular portraits of ERCC1-positive and ERCC1-negative tumors of NSCLC. We carried out a global molecular analysis including assessment of ERCC1 expression levels by using both immunohistochemistry (IHC) and quantitative reverse transcriptase PCR (qRT-PCR), genomic instability, global gene and miRNA expression, and sequencing of selected key genes involved in lung carcinogenesis. Results: ERCC1 protein and mRNA expression were significantly correlated. However, we observed several cases with clear discrepancies. We noted that ERCC1-negative tumors had a higher rate of genomic abnormalities versus ERCC1-positive tumors. ERCC1-positive tumors seemed to share a common DNA damage response (DDR) phenotype with the overexpression of seven genes linked to DDR. The miRNA expression analysis identified miR-375 as significantly underexpressed in ERCC1-positive tumors. Conclusions: Our data show inconsistencies in ERCC1 expression between IHC and qRT-PCR readouts. Furthermore, ERCC1 status is not linked to specific mutational patterns or frequencies. Finally, ERCC1-negative tumors have a high rate of genomic aberrations that could consequently influence prognosis in patients with resected NSCLC. Clin Cancer Res; 17(17); 5562–72. ©2011 AACR.

Background and aim: Angiotropism and extravascular migratory metastasis (EVMM) are an important alternative means of melanoma spread. In EVMM, melanoma cells migrate along the external surfaces of vascular channels to distant sites and demonstrate microscopic angiotropism, i.e. melanoma cells arrayed along the external surfaces of vascular endothelium. Pertinent to EVMM are the origin of melanocytes from the neural crest (NC) and strong analogies of EVMM with NC cell migration. Our aim is to elucidate the molecular mechanisms underlying angiotropism and EVMM. Methods: Frozen primary melanomas, previously utilised for gene expression profiling, were analysed for angiotropism as a differential marker. From the results of this new microarray analysis, we sought to identify genes which were directly relevant to the basic mechanisms underlying EVMM. Results: Among 66 melanomas from patients who developed metastases or remained disease-free at 4 years of follow-up, 26 melanomas were angiotropic while 35 were not, and five were equivocal. The new microarray analysis identified 128 genes differentially expressed in angiotropic versus non-angiotropic melanomas. Among these 128 genes, 15 genes were potentially directly involved in EVMM, based their respective expressions in the NC (seven genes), in other malignant tumours of NC origin (three genes), in cell motility and/or migration (four genes) and in neurotropism (one gene). Conclusion: The detection of these 15 genes provides additional support for the importance of angiotropism and the mechanism of EVMM in melanoma. Ongoing studies on this new profile of 15 genes may potentially identify new targets for controlling melanoma metastasis. (C) 2011 Elsevier Ltd. All rights reserved.

12011Background: Precision medicine has focused mainly on matching drugs to tumor DNA alterations. However, not all individuals have tractable genomic alterations.We initiated the WINTHER trial to ...

A transcriptional signature of the pan–cyclin-dependent kinase (Cdk) inhibitor PHA-793887 was evaluated as a potential pharmacodynamic and/or response biomarker in tumor and skin biopsies from patients treated in a phase I clinical study. We first analyzed the expression of a number of known E2F-dependent genes that were predicted to be modulated after Cdk2 and Cdk4 inhibition in xenograft tumor and skin samples of mice treated with the compound. This panel of 58 selected genes was then analyzed in biopsies from seven patients treated with PHA-793887 in a phase I dose escalation clinical trial in solid tumors. Quantitative real-time PCR or microarray analyses were done in paired skin and tumor biopsies obtained at baseline and at cycle 1. Analysis by quantitative real-time PCR of the signature in skin biopsies of patients treated at three different doses showed significant transcriptional downregulation with a dose-response correlation. These data show that PHA-793887 modulates genes involved in cell cycle regulation and proliferation in a clinical setting. The observed changes are consistent with its mechanism of action and correlate with target modulation in skin and with clinical benefit in tumors. Mol Cancer Ther; 9(5); 1265–73. ©2010 AACR.

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non–small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27Kip1 as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC. Cancer Res; 70(5); 1793–803

Pazopanib and lapatinib are two tyrosine kinase inhibitors that have been designed to inhibit the VEGF tyrosine kinase receptors 1, 2 and 3 (pazopanib), and the HER1 and HER2 receptors in a dual manner (lapatinib). Pazopanib has also been reported to mediate inhibitory effect on a selected panel of additional tyrosine kinases such as PDGFR and c-kit. Here, we report that pazopanib and lapatinib act synergistically to induce apoptosis of A549 non-small-cell lung cancer cells. Systematic assessment of the kinome revealed that both pazopanib and lapatinib inhibited dozens of different tyrosine kinases and that their combination could suppress the activity of some tyrosine kinases (such as c-Met) that were not or only partially affected by either of the two agents alone. We also found that pazopanib and lapatinib induced selective changes in the transcriptome of A549 cells, some of which were specific for the combination of both agents. Analysis of a panel of unrelated human carcinoma cell lines revealed a signature of 52 genes whose up- or downregulation reflected the combined action of pazopanib and lapatinib. Indeed, pazopanib and lapatinib exerted synergistic cytotoxic effects on several distinct non-small-cell lung cancer cells as well as on unrelated carcinomas. Altogether, these results support the contention that combinations of tyrosine kinase inhibitors should be evaluated for synergistic antitumor effects. Such combinations may lead to a 'collapse' of pro-survival signal transduction pathways that leads to apoptotic cell death.

Purpose: We used high-resolution oligonucleotide comparative genomic hybridization (CGH) arrays and matching gene expression array data to identify dysregulated genes and to classify breast cancers according to gene copy number anomalies. Experimental Design: DNA was extracted from 106 pretreatment fine needle aspirations of stage II-III breast cancers that received preoperative chemotherapy. CGH was done using Agilent Human 4 × 44K arrays. Gene expression data generated with Affymetrix U133A gene chips was also available on 103 patients. All P values were adjusted for multiple comparisons. Results: The average number of copy number abnormalities in individual tumors was 76 (range 1-318). Eleven and 37 distinct minimal common regions were gained or lost in >20% of samples, respectively. Several potential therapeutic targets were identified, including FGFR1 that showed high-level amplification in 10% of cases. Close correlation between DNA copy number and mRNA expression levels was detected. Nonnegative matrix factorization (NMF) clustering of DNA copy number aberrations revealed three distinct molecular classes in this data set. NMF class I was characterized by a high rate of triple-negative cancers (64%) and gains of 6p21. VEGFA, E2F3, and NOTCH4 were also gained in 29% to 34% of triple-negative tumors. A gain of ERBB2 gene was observed in 52% of NMF class II and class III was characterized by a high rate of estrogen receptor–positive tumors (73%) and a low rate of pathologic complete response to preoperative chemotherapy (3%). Conclusion: The present study identified dysregulated genes that could classify breast cancer and may represent novel therapeutic targets for molecular subsets of cancers.

We have identified a gene-profile signature for human primary malignant melanoma associated with metastasis to distant sites and poor prognosis. We analyse the differential gene expression by looking at whole biological pathways rather than individual genes. Among the most significant pathways associated with progression to metastasis, we found the DNA replication (P=10(-14)) and the DNA repair pathways (P=10(-16)). We concentrated our analysis on DNA repair and found that 48 genes of this category, among a list of 234 genes, are associated with metastatic progression. These genes belong essentially to the pathways allowing recovery of stalled replication forks due to spontaneous blockage or induced DNA lesions. Because almost all these differentially expressed repair genes were overexpressed in primary tumors with bad prognosis, we speculate that primary melanoma cells that will metastasize try to replicate in a fast and error-free mode. In contrast to the progression from melanocytes to primary melanoma, genetic stability appears to be necessary for a melanoma cell to give rise to distant metastasis. This overexpression of repair genes explains nicely the extraordinary resistance of metastatic melanoma to chemo- and radio-therapy. Our results may open a new avenue for the discovery of drugs active on human metastatic melanoma.

Non–small cell lung cancer (NSCLC) with activating mutations in the epidermal growth factor receptor (EGFR) responds to EGFR tyrosine kinase inhibitors such as erlotinib. However, secondary somatic EGFR mutations (e.g., T790M) confer resistance to erlotinib. BMS-690514, a novel panHER/vascular endothelial growth factor receptor (VEGFR) inhibitor described here, exerted antiproliferative and proapoptotic effects on NSCLC cell lines, with prominent efficacy on H1975 cells expressing the T790M mutation. In this model, BMS-690514 induced a G1 cell cycle arrest, as well as ultrastructural hallmarks of apoptosis, mitochondrial release of cytochrome c, and activation of caspases involved in the intrinsic (e.g., caspase-2, caspase-3, caspase-7, and caspase-9), but not in the extrinsic (e.g., caspase-8), pathway. Caspase inhibition conferred partial protection against BMS-690514 cytotoxicity, pointing to the involvement of both caspase-dependent and caspase-independent effector mechanisms. Transcriptome analyses revealed the up-regulation of proapoptotic (e.g., Bim, Puma) and cell cycle inhibitory (e.g., p27Kip1, p57Kip2) factors, as well as the down-regulation of antiapoptotic (e.g., Mcl1), heat shock (e.g., HSP40, HSP70, HSP90), and cell cycle promoting [e.g., cyclins B1, D1, and D3; cyclin-dependent kinase 1 (CDK1); MCM family proteins; proliferating cell nuclear antigen (PCNA)] proteins. BMS-690514–induced death of H1975 cells was modified in a unique fashion by a panel of small interfering RNAs targeting apoptosis modulators. Down-regulation of components of the nuclear factor-κB survival pathway (e.g., p65, Nemo/IKKγ, TAB2) sensitized cells to BMS-690514, whereas knockdown of proapoptotic factors (e.g., Puma, Bax, Bak, caspase-2, etc.) and DNA damage–related proteins (e.g., ERCC1, hTERT) exerted cytoprotective effects. BMS-690514 is a new pan-HER/VEGFR inhibitor that may become an alternative to erlotinib for the treatment of NSCLC. [Cancer Res 2007;67(13):6253–62]

Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) gamma1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARgamma1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARgamma target genes were up-regulated in PAX8-PPARgamma-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARgamma expression profile presented a putative PPARgamma-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARgamma and as members of novel molecular pathways involved in the development of thyroid carcinomas.

Background: Irofulven is a novel alkylating agent with promising clinical activity, particularly toward ovarian and hormone-refractory prostate cancers. To facilitate additional clinical development, we have aimed to identify biological markers associated with sensitivity to the compound. Methods: Fibroblasts derived from patients with xeroderma pigmentosum or Cockayne’s syndrome along with a panel of 20 human cancer cell lines (eight different tumor types) were examined to establish the importance of nucleotide excision repair proteins in the sensitivity to irofulven. Results: Human cells deficient in nucleotide excision repair are up to 30-fold more sensitive to the cytotoxic effects of irofulven compared with repair-proficient controls, clearly indicating that nucleotide excision repair plays a crucial role in the sensitivity to the drug. Interestingly, our results show that irofulven-induced lesions are recognized by transcription-coupled repair but not by global genome repair. Another unique feature is the pronounced sensitivity of XPD and XPB helicase-deficient cells to the drug. Comparison of the IC50 values for irofulven, cisplatin, and ecteinascidin 743 with the expression levels of ERCC1, XPD, and XPG genes in different solid tumor cell lines shows no correlation between the expression levels of any of the three nucleotide excision repair proteins and the sensitivity to ecteinascidin 743. In contrast, expression of the XPG endonuclease was correlated with the cytotoxicity for irofulven and, to a lesser degree, for cisplatin. Importantly, XPG expression was also correlated with cellular nucleotide excision repair activity. Conclusions: Increasing evidence indicates that compromised nucleotide excision repair activity is frequent in several solid tumor types. The results presented here suggest that XPG expression in such tumors may be a useful marker to predict their sensitivity to irofulven.

In order to assess the effect of in vitro models on the expression of key genes known to be implicated in the development or progression of cancer, we quantified by real-time quantitative PCR the expression of 28 key genes in three bladder cancer tissue specimens and in their derived cell lines, studied either as one-dimensional single cell suspensions, two-dimensional monolayers or three-dimensional spheroids. Global analysis of gene expression profiles showed that in vitro models had a dramatic impact upon gene expression. Remarkably, quantitative differences in gene expression of 2–63-fold were observed in 24 out of 28 genes among the cell models. In addition, we observed that the in vitro model which most closely mimicked in vivo mRNA phenotype varied with both the gene and the patient. These results provide evidence that mRNA expression databases based on cancer cell lines, which are studied to provide a rationale for selection of therapy on the basis of molecular characteristics of a patient's tumour, must be carefully interpreted. British Journal of Cancer (2002) 86, 1283–1289. DOI: 10.1038/sj/bjc/6600239 www.bjcancer.com © 2002 Cancer Research UK

TPS11625 Background: Today, personalized cancer medicine implies matching the patient’s tumor genomic characteristics with molecularly and immune targeted agents. Although there are an increasing number of DNA aberrations that can now be matched to a cognate therapy, some patients do not display such druggable oncogene drivers. Methods: WINTHER is an open non-randomized study involving 6 cancer centers in France, Spain, Israel, Canada and USA applying genomic and also transcriptomic assays to guide treatment decisions. The novelty of the WINTHER approach lies in the use of tumor and matched normal tissue biopsies together and an algorithm for predicting efficacy of therapies. The aim is to provide a rational therapeutic choice for all of the patients enrolled in the study whether or not they harbor actionable DNA alterations. The study endpoint is the comparison of the progression-free-survival (PFS) under the WINTHER selected therapy to the PFS of the last therapeutic line. Patients included have refractory metastatic cancer of any histological type, with at least one prior therapeutic regimen and performance status of 0 to 1. Patients who have received a matched treatment based on a molecular anomaly as their immediate prior therapy were excluded. After consent, patients undergo a tumor and histologically-matched normal tissue biopsy. Extracted DNA and RNA of both tumor and normal from frozen tissues at the local center under common standard operating procedures are sent to centralized laboratories for omics investigations. DNA is investigated at Foundation Medicine Inc. and RNA at Gustave Roussy using Agilent technology. For RNA, the WINTHER algorithm is applied on the differential RNA expression data between tumor and normal tissues and establishes the list of drugs with the presumed higher score of efficacy for each patient. Patients with actionable genomic events enter in ARM A, and patients without any druggable anomaly of the DNA enter in ARM B and are treated using the WINTHER algorithm RNA-based treatment decision tool. To date, the trial has recruited 303 patients. Clinical trial information: NCT01856296.

e23151 Background: Evolution of tumor occurs in two phases, pretreatment phase, and posttreatment phase.Tumorigenic history and path of tumorigenic evolution determine the response of tumor cells to antitumor drug and thus the outcome of treatment. Carcinomas from the same organ-site with similar genomic alterations in different patients may vary in their tumorigenic history and their diverse paths of tumorigenic evolution which cause them respond differently to the same antitumor drugs. Uniqueness of tumorigenic history and paths of tumorigenic evolution in individual patient makes it ideal to test drugs, N-of-1. Here we present our experience with 3D organotypic ex vivo culture of tumor tissue from post-operated tumor samples. Methods: Informed consent was obtained from patients ( NCT02470715). The investigation was approved by IRB of the Avera Cancer Institute and the Western IRB. Surgically resected tissue was obtained from patients with different tumors of different organ sites including ovarian, breast, lung, endometrium, and cervix at the Avera Cancer Institute, SF, SD. Tissues from patients were collected in two formats, paired samples (tumor and tumor-adjacent normal) and unpaired samples. Results: As extracellular matrix lost in cell cultures, provide important signals for cell survival, differentiation and drug resistance we have established a protocol to culture slices in the 3D format. We have established 3D slice cultures (3x3 mm) of tumor-derived tissues which can be maintained ex vivo for at least three days. Tumor cells / normal cells from different tumors/tumor-adjacent normal tissues from day zero (non-cultured) were used to compare day1, day2, and day3 cultures. At the end of the culture period, slices were embedded in paraffin for histological analysis following H&E staining as well as IHC-staining for proliferation index (Ki-67), cellular proliferation signals (pS6RP), apoptosis (activated caspase 3), or tumor angiogenesis (CD31). Ki67 staining was characteristically different in cultured normal and cultured tumor tissues. Conclusions: Here, we show that tumor-derived tissues survive in 3D Matrigel culture for a minimum of 6 hours and maximum up to 3 days. Clinical trial information: NCT02470715.

Purpose Focal and temporal tumour heterogeneity can represent a major challenge for biology-guided therapies. This study proposes to investigative molecular discrepancies between primary colorectal cancer (CRC) samples and matched metastases. Experimental design Surgical samples from primary and matched metastatic tissues from 13 CRC patients along with their adjacent normal tissue were evaluated. A mutational analysis was performed using a targeted Next Generation Sequencing assay (Foundation Medicine) with a focus on known recurrent somatic mutations as surrogate of key oncogenic events. Gene expression analysis was also performed to investigate transcriptional discrepancies. Results Among the 26 samples, 191 mutations were identified including mutations in APC (13 pts), TP53 (11 pts), and KRAS (7 pts). Global concordance rate for mutations was 78% between primary and metastatic tumours and raised to 90% for 12 known recurrent mutations in CRC. Differential gene expression analysis revealed a low number of significantly variant transcripts between primary and metastatic tumours once the tissue effect was taken into account. Only two pathways (ST_ADRENERGIC, PID_REELINPATHWAY) were differentially up-regulated in metastases among 17 variant pathways. A common profile in primary and metastatic tumours revealed conserved pathways mostly involved in cell cycle regulation. Only two pathways were significantly down regulated compared to normal control, including regulation of autophagy (KEGG_REGULATION_OF_AUTOPHAGY). Conclusion These results suggest that profiles of primary tumour can identify key alterations present in matched CRC metastases at first metastatic progression. Gene expression analysis identified mainly conserved pathways between primary tumour and matched liver metastases.

The classic model of tumor suppression implies that malignant transformation requires full “two-hit” inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a “continuum” model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the “continuum” model of tumor suppression. Cancer Res; 74(22); 6554–64. ©2014 AACR.

International audience; Purpose We designed a gene profiling experiment to identify genes involved in secondary drug resistance in mantle cell lymphomas (MCL). Experimental Design We obtained paired tissue samples collected from the same patients before treatment and after relapse or progression. Variations in gene expression between the 2 samples were estimated for 5 patients. For each gene, the mean variation was estimated for patients with a refractory primary tumor and for responders who developed secondary drug resistance. Nine genes of interest were selected on the basis of the magnitude and statistical significance of the variation of expression in responders and non-responders. Results BMP7 was the only one with significantly increased expression at relapse in patients who developed secondary resistance. Validation of BMP7 as a key gene involved in secondary resistance was performed using cultures of cell line. Incubation of BMP7 with MCL cell lines increased their resistance to bortezomib and cytarabine, while inhibition of BMP7 expression by siRNA correlated with increased cell death linked to drug application. Conclusion Variations in gene expression after treatment point out BMP7 as a key gene involved in secondary resistance in mantle cell lymphoma.

In this study, we have analyzed human primary lung adenocarcinoma tumors using global mass spectrometry to elucidate the biological mechanisms behind relapse post surgery. In total, we identified over 3000 proteins with high confidence. Supervised multivariate analysis was used to select 132 proteins separating the prognostic groups. Based on in-depth bioinformatics analysis, we hypothesized that the tumors with poor prognosis had a higher glycolytic activity and HIF activation. By measuring the bioenergetic cellular index of the tumors, we could detect a higher dependency of glycolysis among the tumors with poor prognosis. Further, we could also detect an up-regulation of HIF1α mRNA expression in tumors with early relapse. Finally, we selected three proteins that were upregulated in the poor prognosis group (cathepsin D, ENO1, and VDAC1) to confirm that the proteins indeed originated from the tumor and not from a stromal or inflammatory component. Overall, these findings show how in-depth analysis of clinical material can lead to an increased understanding of the molecular mechanisms behind tumor progression.

Introduction Breast cancer risk of recurrence is known to span 20 years, yet existing prognostic signatures are best at predicting early recurrences (≤5 years). There is a critical need to identify those patients at risk of late-relapse (>5 years), in order to select potential candidates for further treatment and to identify molecular targets for such treatment. Methods A total of 252 breast primary tumors were selected at the Netherlands Cancer Institute from a retrospective series of ER+, HER2− breast cancer patients with a follow-up of at least 10 years. Gene expression analysis was performed using Agilent 4x44K microarrays. Patients were classified in 3 groups: no relapse (M0); relapse before 5 years (M0-5) or after 5 years (M5-15). We assessed the correlation of clinico-pathological variables with late Distant Metastases (DM). We divided the patient series into a training set of untreated patients ( n = 140) and a test set of treated patients ( n = 112), to investigate whether a gene-signature or single genes could be identified for predicting late DM. Pathway level late DM correlates were identified using PARADIGM and DAVID. Results Of the clinico-pathologic variables tested, only lymph node status associated with late DM. A 241-gene signature developed on the NKI training set was able to classify M5-15 patients in the test set with a sensitivity of 77% and a specificity of 33% (AUC 0.654). This signature showed enrichment in genes involved in immune response and extracellular matrix. An alternative analysis of individual genes identified CH25H as an independent predictor of distant metastasis in our patient series. Conclusions We identified a gene signature for late metastasis in breast cancer. Our data are consistent with a model in which suppressed anti-tumoral immunity enables dormant tumor cells to re-enter the cell cycle to form metastases in response to extrinsic events in the microenvironment.

Purpose Characterization of the genomic changes that drive an individual patient's disease is critical in management of many cancers. In patients with non–small-cell lung cancer (NSCLC), obtaining tumor samples of sufficient size for genomic profiling on recurrence is often challenging. We undertook this study to compare genomic alterations identified in archived primary tumors from patients with NSCLC with those identified in metachronous or synchronous metastases. Patients and Methods Primary and matched metastatic tumor pairs from 15 patients were analyzed by using a targeted next-generation sequencing assay in a Clinical Laboratory Improvement Amendments laboratory. Genomic libraries were captured for 3,230 exons in 182 cancer-related genes plus 37 introns from 14 genes often rearranged in cancer and sequenced to high coverage. Results Among 30 tumors, 311 genomic alterations were identified of which 63 were known recurrent (32 in primary tumor, 31 in metastasis) and 248 were nonrecurrent (likely passenger). TP53 mutations were the most frequently observed recurrent alterations (12 patients). Tumors harbored two or more (maximum four) recurrent alterations in 10 patients. Comparative analysis of recurrent alterations between primary tumor and matched metastasis revealed a concordance rate of 94% compared with 63% for likely passenger alterations. Conclusion This high concordance suggests that for the purposes of genomic profiling, use of archived primary tumor can identify the key recurrent somatic alterations present in matched NSCLC metastases and may provide much of the relevant genomic information required to guide treatment on recurrence.

Starting from a clinical observation, a small subset of breast cancer patients presenting retinoic acid receptor alpha (RARA) amplification and overexpression was identified. Retinoic acid reduced cell viability of RARAamplified cell lines through interferon regulatory factor (IRF)-1 and caspase (CASP)-1 activation and RARAnonamplified cells were most likely resistant to all-trans-retinoic acid (ATRA). This study suggests that RARA-amplified breast cancers could be sensitive to retinoic acid. Background: Molecular segmentation of breast cancer allows identification of small groups of patients who present high sensitivity to targeted agents. A patient, with chemo- and trastuzumab-resistant HER2-overexpressing breast cancer, who presented concomitant acute promyelocytic leukemia, showed a response in her breast lesions to retinoic acid, arsenic, and aracytin. We therefore investigated whether RARA gene amplification could be associated with sensitivity to retinoic acid derivatives in breast cancers. Materials and Methods: Array comparative genomic hybridization and gene expression arrays were used to characterize RARA amplifications and expression in 103 breast cancer samples. In vitro activity of ATRA was characterized in T47D, SKBR3, and BT474 cell lines. Results: Retinoic acid receptor alpha was gained or amplified in 27% of HER2-positive and 13% of HER2-negative breast cancer samples. Retinoic acid receptor alpha can be coamplified with HER2. Retinoic acid receptor alpha copy number changes could be correlated with messenger RNA expression. All-trans-retinoic acid reduced cell viability of RARAamplified, but not RARA-normal, cell lines through apoptosis. Gene expression arrays showed that ATRA-induced apoptosis in RARA-amplified cell lines was related to an increase in CASP1 and IRF1. Conclusion: The results of this study suggest that breast cancers exhibiting RARA amplifications could be sensitive to retinoic acid. A phase II trial will evaluate this hypothesis in the clinical setting.

Background Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. Methods Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. Results At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. Conclusions Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human tumors. There are mainly point mutations that lead to single amino acid substitutions. The mutated proteins have a longer half-life than wild-type p53 and accumulate in the nucleus of tumor cells. Anti-p53 antibodies have been found in sera of patients with several types of cancers including breast cancer. This report describes a T cell immune response in three patients with breast tumors who had mutated p53 gene and accumulated p53 protein. All showed a humoral response to p53 protein and the T cells of these patients recognized the wild-type p53 protein and proliferated in response to it. The data reported here are relevant to the immune processes leading to autoimmunity and have a bearing on anti-p53 vaccine development in tumor immunology.

11520Background: Our goal was to compare patient outcomes between phase I studies of single agents that employed a personalized approach (defined as a biomarker-based selection strategy) versus tho...

11502Background: Harmonizing informatics in the era of next generation sequencing (NGS) is a precision medicine priority as unconnected data silos unacceptably stall advancement of genomic medicine...

It is clear from epidemiological studies that excess iron is associated with increased risk of colorectal cancer; however, questions regarding the mechanism of how iron increases cancer risk, the source of the excess iron (circulating or luminal), and whether iron reduction represents a potential therapeutic option remain unanswered. In this study, we show that after Apc deletion, the cellular iron acquisition proteins TfR1 and DMT1 are rapidly induced. Conversely, restoration of APC reduces cellular iron due to repression of these proteins. To test the functional importance of these findings, we performed in vivo investigations of the impact of iron levels on intestinal tumorigenesis. Strikingly, depletion of luminal (but not systemic) iron strongly suppressed murine intestinal tumorigenesis, whereas increased luminal iron strongly promoted tumorigenesis. Taken together, our data definitively delineate iron as a potent modifier of intestinal tumorigenesis and have important implications for dietary iron supplementation in patients at high risk of colorectal cancer.