B. Job

An immunosurveillance mechanism controls cancer cell ploidy

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. Polyploid cancer cells trigger an immune response owing to proteins aberrantly exposed on their outer surfaces. Cancer cells accommodate multiple genetic and epigenetic alterations that initially activate intrinsic (cell-autonomous) and extrinsic (immune-mediated) oncosuppressive mechanisms. Only once these barriers to oncogenesis have been overcome can malignant growth proceed unrestrained. Tetraploidization can contribute to oncogenesis because hyperploid cells are genomically unstable. We report that hyperploid cancer cells become immunogenic because of a constitutive endoplasmic reticulum stress response resulting in the aberrant cell surface exposure of calreticulin. Hyperploid, calreticulin-exposing cancer cells readily proliferated in immunodeficient mice and conserved their increased DNA content. In contrast, hyperploid cells injected into immunocompetent mice generated tumors only after a delay, and such tumors exhibited reduced DNA content, endoplasmic reticulum stress, and calreticulin exposure. Our results unveil an immunosurveillance system that imposes immunoselection against hyperploidy in carcinogen- and oncogene-induced cancers.

Expression of miR-487b and miR-410 encoded by 14q32.31 locus is a prognostic marker in neuroblastoma

Background:Combination of age at diagnosis, stage and MYCN amplification stratifies neuroblastoma into low-risk and high-risk. We aimed to establish whether a microRNA (miRNA) signature could be associated with prognosis in both groups.Methods:Microarray expression profiling of human miRNAs and quantitative reverse-transcriptase PCR of selected miRNAs were performed on a preliminary cohort of 13 patients. Results were validated on an independent cohort of 214 patients. The relationship between miRNA expression and the overall or disease-free survival was analysed on the total cohort of 227 patients using the log-rank test and the multivariable Cox proportional hazard model.Results:A total of 15 of 17 miRNAs that discriminated high-risk from low-risk neuroblastoma belonged to the imprinted human 14q32.31 miRNA cluster and two, miR-487b and miR-410, were significantly downregulated in the high-risk group. Multivariable analyses showed miR-487b expression as associated with overall survival and disease-free survival in the whole cohort, independently of clinical covariates. Moreover, miR-487b and miR-410 expression was significantly associated with disease-free survival of the non-MYCN-amplified favourable neuroblastoma: localised (stage 1, 2 and 3) and stage 4 of infant <18 months.Conclusion:Expression of miR-487b and miR-410 shows predictive value beyond the classical high-/low-risk stratification and is a biomarker of relapse in favourable neuroblastoma.

MYCN-non-amplified metastatic neuroblastoma with good prognosis and spontaneous regression: a molecular portrait of stage 4S.

Stage 4 neuroblastoma (NB) are heterogeneous regarding their clinical presentations and behavior. Indeed infants (stage 4S and non‐stage 4S of age <365days at diagnosis) show regression contrasting with progression in children (>365days). Our study aimed at: (i) identifying age‐based genomic and gene expression profiles of stage 4 NB supporting this clinical stratification; and (ii) finding a stage 4S NB signature. Differential genome and transcriptome analyses of a learning set of MYCN‐non amplified stage 4 NB tumors at diagnosis (n=29 tumors including 12 stage 4S) were performed using 1Mb BAC microarrays and Agilent 22K probes oligo‐microarrays. mRNA chips data following filtering yielded informative genes before supervised hierarchical clustering to identify relationship among tumor samples. After confirmation by quantitative RT‐PCR, a stage 4S NBs gene cluster was obtained and submitted to a validation set (n=22 tumors). Genomic abnormalities of infants tumors (whole chromosomes gains or loss) differ radically from that of children (intra‐chromosomal rearrangements) but could not discriminate infants with 4S from those without this presentation. In contrast, differential gene expression by looking at both individual genes and whole biological pathways leads to a molecular stage 4S NB portrait which provides new biological clues about this fascinating entity.

Integrated molecular portrait of non-small cell lung cancers

BackgroundNon-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.MethodsComparative 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.ResultsAt 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.ConclusionsIntegrated 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.

Array CGH and PIK3CA/AKT1 mutations to drive patients to specific targeted agents: A clinical experience in 108 patients with metastatic breast cancer

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%), investigators 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.

β-Catenin Inhibitor ICAT Modulates the Invasive Motility of Melanoma Cells

Inhibitor of β-catenin and TCF (ICAT) inhibits β-catenin transcriptional activity by competing with T-cell factor/lymphoid enhancer factor. We documented high ICAT levels in human melanoma cells, in which β-catenin signaling is frequently deregulated, finding a correlation with the capacity to form metastases in nude mice. Ectopic expression of ICAT in melanoma cells did not affect their proliferation but increased cell motility and Matrigel invasion of metastatic cells in a manner relying upon stable ICAT-β-catenin interaction. This effect was associated with conversion of an elongated/mesenchymal phenotype to a round/amoeboid phenotype in the absence of similar effects on elongated morphology of nonmetastatic melanoma cells. Transition from mesenchymal to amoeboid movement was associated with decreased levels of NEDD9 and activated Rac1, a positive regulator of mesenchymal movement. Ectopic ICAT promoted colonization of melanoma cells in the lungs of nude mice, suggesting an increase in metastatic potential. Together, our results showed that by downregulating Rac signaling in metastatic melanoma cells, ICAT increased their invasive motility by promoting a morphologic variation that facilitates a favorable adaptation to their microenvironment.

Gene copy number variations in breast cancer of Sub-Saharan African women

The goal of this study was CGH array profiling of breast cancer from Malian women in order to define differences with those from USA. CGH array was performed in 28 samples, 17 with a triple negative phenotype. The profiles were compared to those of 106 tumors from USA. 6 chromosomal regions (6p21, 9q34, 11q13, 12q24, 17q25 and 22q12.1-22q13.1) were identified with a significant higher rate of copy number alterations. These regions contain several genes of interest including BCR. FISH and IHC confirmed that BCR was amplified and overexpressed particularly in triple negative tumors. Finally, 5 regions presented a high level of amplification in two or more samples, including 2 regions located between 9p22.3-9p23 and 9p23-9p24.1. This study confirms that breast cancers from African women present biological differences with those from USA. Larger studies are needed to go further in the identification of therapeutic targets that would be specific to African women.

801 ORAL High Throughput Molecular Analyses to Select Patients for Targeted Agents

Background: Rapid advancements in genomics paired with significant growth in the availability of targeted therapies offers clinicians expanding opportunities to provide increasingly effective cancer treatment. Currently, individual gene sequencing (e.g. EGFR) from formalin-fixed paraffinembedded (FFPE) tissue is widely used in cancer diagnosis. Shifting this paradigm towards NGS-based, comprehensive mutation testing in routinely collected FFPE cancer specimens will enable more complete and accurate characterization of patients’ cancers for individualized targeted therapy selection. Materials and Methods: DNA was extracted from 2×20micron sections of 83 specimens consisting of colorectal cancer, non-small cell lung cancer and melanoma. Hybridization-capture of 2574 exons across 176 oncogenes, tumour suppressor genes and ADME-related genes was performed to produce libraries appropriate for paired-end sequence analysis on the Illumina HiSeq2000 platform. Results: In-depth sequence analysis of 176 genes in 50 CRC, 29 NSCLC, and 4 melanoma specimens with median coverage averaging 213-fold (range 8 to 461) detected a per-sample average of 2 previously-described mutations, 7 novel mutations and 2 CNAs in the colon specimens, including frequent alterations in TP53 (33), APC (27), KRAS (12) and BRAF (6). The lung specimens averaged 1 previously described mutation, 8 novel mutations and 1 CNA per sample, most frequently KRAS (10), TP53 (7), JAK2 (3), EGFR (2) and BRAF (2). The melanoma cases exhibited on average 1 previously described mutation, 7 novel mutations and 3 CNAs including TP53 (4) and BRAF (2). In addition to validated clinically actionable mutations in EGFR, KRAS, and BRAF, and multiple alterations in well-known cancer genes such as TP53, STK11, APC,MLH1, BRCA2, and SMAD4, we detected many other mutations that are plausibly clinically actionable. These included activating mutations in the PI3 kinase subunit gene PIK3CA, as well as mutations in MET , KIT , ERBB2 and CDKN2A. Conclusions: It is feasible to perform highly sensitive and specific sequence analysis of hundreds of genes from routinely collected FFPE tissues. This approach detects not only the “hot spot” mutations commonly tested for in CRC, NSCLC and melanoma but also many additional mutations that could plausibly inform therapeutic decision-making. We suggest that clinical-grade next-generation sequencing should become a routine part of all clinical trials, and increasingly, of clinical care.

High Resolution Oligonucleotide Array-CGH and Hot Spot Mutations To Select Patients for Targeted Therapies.

Background. A high number of genomic alterations (gene amplification, mutations) have been reported in breast cancers, and could potentially be targeted in early clinical trials. In the present molecular screening program, we aim at performing CGH array and hot spot mutations for 200 patients with metastatic breast cancer, in order to drive these patients in specific early clinical trials.Patients and Methods. Patients were selected to present a metastatic breast cancer under treatment in four French Centers. DNA was extracted by Qiagen method from either primary breast cancer or metastatic lesions. The hybridizations were done on 4*44K Agilent Array CGH. The analysis was done with Analytics v3.4.40 software. The file reported amplified zones (log2ratio>0.84 for biopsy, 1.58 for FNA) and focused on predefined genes that encode a candidate target. Hot spot mutations cover PI3K, AKT, PTEN genes. The primary endpoint was to determine how many patients were driven to a specific clinical trial because of molecular analyses. The secondary endpoints included PFS and response rates.Results. As june 12 th , 109 patients were included in the molecular screening program. CGH array was feasible and could be interpreted within one month in 85 (78%) patients. Array-CGH identified high level amplification in 57 patients (67%). As expected, the two most frequent amplicons included ERBB2 (17q11-12, 24%) and FGFR1 (8p11, 16%).The accuracy to detect ERBB2 amplification was 90%. A significant number of patients presented rare gene amplifications including ERBB4 amplification (n=1), CCND1/FGF4 amplification (n=6), FGFR2 (n=2), EGFR (n=1), VEGFB (n=4), BCR (n=1)Interestingly, only 4 patients presented TOP2A amplification. Hot spot mutations for PI3KCA, AKT, PTEN is being performedConclusion. This preliminary analysis shows that array CGH allows to identify patients who are candidate for targeted agents in phase I/II trials. Accrual will continue until 200 patients. Selected patients will be included in clinical trials in second semester of 2009 after a target validation by IHC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5067.

Abstract 1217: Chromosomal imbalances detected by microarray CGH in malignant pleural mesothelioma.

Background CDKN2A (9p21 locus) and NF2 (22q locus) genes are inactivated in most Malignant Pleural Mesotheliomas (MPM). Few other recurrent molecular abnormalities have been described as well. A better understanding of the molecular pathways is needed to identify potential additional driver genes. We did an integrated analysis of chromosomal copy number variations from MPM specimens and cells lines. Patients and Methods In order to describe genomic profiles, we performed microarray Comparative Genomic Hybridization (CGH) (Agilent 400K). Analyses were carried out using genomic DNA samples extracted from 26 MPM primary tumours (frozen sections) and 5 MPM cell lines (Four ATCC cell lines and one cell line established in our laboratory from a pleural effusion). Subsequently the results were correlated with the baseline characteristics of the patients. Results The mean age of the patients was 60 years (30-81). The sex ratio (M/F) and the histologic epithelioid type were 1.6 and 69% respectively. Our results showed that MPM are characterized by a complex pattern of genetic changes where losses of genomic regions are more frequent than gains. Common losses (30-70%) were clustered at chromosomal regions: 1p36.13, 1p13.3, 1q21.3-1q24.2, 1p22.3-1p12 (41%) (NRAS gene), 1p36.33-1q21.2 (30%) (JUN gene) 3p22.1-3p14.2 (59%) (BAP1, RASSF1 genes), 4q13.2, 6p21.32, 6q14.1-6q27 (63%) (ROS1 gene), 7q36.1, 8p23.1, 9p24.1, 9p22.1-9p21 (52%) (CDKN2A gene), 10p11.1-10q26.3 (30%) (RET, PTEN genes), 11q25, 14q11.2, 14q32.33, 15q11.2, 15q14, 22q11.22-22q13.33 (63%) (NF2 gene). Common gains (30-50%) were located at chromosomal regions 1q44, 8p11.22, 11q11, 12p13.31, 15q11.2, 16p11, 20p13, 22q11.23, Xq22.2. The most common deletions (D) and amplifications (A) (>20%) defined as having a log2(ratio) ≤-1.0 and ≥1.0 respectively were located at chromosomal regions: 1p31.1 (A, D), 1q21, 1q24.2 (D), 6p21.32, 6q16.3, 6q27 (D), 7q22.1 (D), 8p11.22 (A), 9p21.3, 9q34.2 (D), 12p13.2 (D), 14q32.33 (D), 16q23.1, 16q22.1 (WWOX gene)(D), 20p13 (A, D), 22q11.23 (D). Higher frequency of genetic alterations was noted in cell lines than tumour specimens. Different patterns of genetic changes were observed according to gender, stage, histology, smoking history, asbestos exposure and performance status. Conclusion Our study confirmed the complexity pattern of genetic changes in MPM through high-resolution microarray CGH analysis. We found high number of chromosomal imbalances not yet well identified. Some correspond to known genes and others could be the areas of unknown genes involved in its development that we will try to identify thereafter. Moreover, identifying patient subgroups seems essential for having a more individualized therapy and personalized medicine in MPM (e.g. MOSCATO trial, ongoing in our cancer center). Citation Format: Jacques Raphael, Nicolas Dorvault, Cedric Orear, Bastien Job, Vincent Thomas de Montpreville, Francois Leroy-Ladurie, Philippe Girard, Pierre Validire, Gwenael Le Teuff, David Planchard. Chromosomal imbalances detected by microarray CGH in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1217. doi:10.1158/1538-7445.AM2013-1217