Ivan Bièche

The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer.

To investigate the clinical value of somatic TP53 mutations in breast cancer, we assembled clinical and molecular data on 1,794 women with primary breast cancer with long-term follow-up and whose tumor has been screened for mutation in exons 5 to 8 of TP53 by gene sequencing. TP53 mutations were more frequent in tumors of ductal and medullar types, aggressive phenotype (high grade, large size, node positive cases, and low hormone receptor content) and in women <60 years old. TP53 mutations within exons 5 to 8 conferred an elevated risk of breast cancer-specific death of 2.27 (relative risk >10 years; P < 0.0001) compared with patients with no such mutation. The prognostic value of TP53 mutation was independent of tumor size, node status, and hormone receptor content, confirming and reconciling previous findings in smaller series. Moreover, an interaction between TP53 mutation and progesterone receptor (PR) status was revealed, TP53 mutation combined with the absence of progesterone receptor being associated with the worst prognosis. Whereas previous studies have emphasized the fact that missense mutations in the DNA-binding motifs have a worse prognosis than missense mutations outside these motifs, we show that non-missense mutations have prognostic value similar to missense mutations in DNA-binding motifs. Nonetheless, specific missense mutants (codon 179 and R248W) seem to be associated with an even worse prognosis. These results, obtained on the largest series analyzed thus far, show that TP53 mutations identified by gene sequencing have an independent prognostic value in breast cancer and could have potential uses in clinical practice.

Molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer (SHIVA): a multicentre, open-label, proof-of-concept, randomised, controlled phase 2 trial.

BACKGROUND Molecularly targeted agents have been reported to have anti-tumour activity for patients whose tumours harbour the matching molecular alteration. These results have led to increased off-label use of molecularly targeted agents on the basis of identified molecular alterations. We assessed the efficacy of several molecularly targeted agents marketed in France, which were chosen on the basis of tumour molecular profiling but used outside their indications, in patients with advanced cancer for whom standard-of-care therapy had failed. METHODS The open-label, randomised, controlled phase 2 SHIVA trial was done at eight French academic centres. We included adult patients with any kind of metastatic solid tumour refractory to standard of care, provided they had an Eastern Cooperative Oncology Group performance status of 0 or 1, disease that was accessible for a biopsy or resection of a metastatic site, and at least one measurable lesion. The molecular profile of each patients tumour was established with a mandatory biopsy of a metastatic tumour and large-scale genomic testing. We only included patients for whom a molecular alteration was identified within one of three molecular pathways (hormone receptor, PI3K/AKT/mTOR, RAF/MEK), which could be matched to one of ten regimens including 11 available molecularly targeted agents (erlotinib, lapatinib plus trastuzumab, sorafenib, imatinib, dasatinib, vemurafenib, everolimus, abiraterone, letrozole, tamoxifen). We randomly assigned these patients (1:1) to receive a matched molecularly targeted agent (experimental group) or treatment at physicians choice (control group) by central block randomisation (blocks of size six). Randomisation was done centrally with a web-based response system and was stratified according to the Royal Marsden Hospital prognostic score (0 or 1 vs 2 or 3) and the altered molecular pathway. Clinicians and patients were not masked to treatment allocation. Treatments in both groups were given in accordance with the approved product information and standard practice protocols at each institution and were continued until evidence of disease progression. The primary endpoint was progression-free survival in the intention-to-treat population, which was not assessed by independent central review. We assessed safety in any patients who received at least one dose of their assigned treatment. This trial is registered with ClinicalTrials.gov, number NCT01771458. FINDINGS Between Oct 4, 2012, and July 11, 2014, we screened 741 patients with any tumour type. 293 (40%) patients had at least one molecular alteration matching one of the 10 available regimens. At the time of data cutoff, Jan 20, 2015, 195 (26%) patients had been randomly assigned, with 99 in the experimental group and 96 in the control group. All patients in the experimental group started treatment, as did 92 in the control group. Two patients in the control group received a molecularly targeted agent: both were included in their assigned group for efficacy analyses, the patient who received an agent that was allowed in the experimental group was included in the experimental group for the purposes of safety analyses, while the other patient, who received a molecularly targeted agent and chemotherapy, was kept in the control group for safety analyses. Median follow-up was 11·3 months (IQR 5·8-11·6) in the experimental group and 11·3 months (8·1-11·6) in the control group at the time of the primary analysis of progression-free survival. Median progression-free survival was 2·3 months (95% CI 1·7-3·8) in the experimental group versus 2·0 months (1·8-2·1) in the control group (hazard ratio 0·88, 95% CI 0·65-1·19, p=0·41). In the safety population, 43 (43%) of 100 patients treated with a molecularly targeted agent and 32 (35%) of 91 patients treated with cytotoxic chemotherapy had grade 3-4 adverse events (p=0·30). INTERPRETATION The use of molecularly targeted agents outside their indications does not improve progression-free survival compared with treatment at physicians choice in heavily pretreated patients with cancer. Off-label use of molecularly targeted agents should be discouraged, but enrolment in clinical trials should be encouraged to assess predictive biomarkers of efficacy.

ANRIL, a long, noncoding RNA, is an unexpected major hotspot in GWAS

A large noncoding RNA called ANRIL (for antisense noncoding RNA in the INK4 locus) has been identified within the p15/CDKN2B‐p16/CDKN2A‐p14/ARF gene cluster. While the exact role of ANRIL awaited further elucidation, common disease genome‐wide association studies (GWAS) have surprisingly identified the ANRIL gene as a genetic susceptibility locus shared associated by coronary disease, intracranial aneurysm and also type 2 diabetes. Expression studies have confirmed the coregulation of p15/CDKN2B, p16/CDKN2A’, p14/ARF, and ANRIL. Among the cluster, ANRIL expression showed the strongest association with the multiple phenotypes linked to the 9p21.3 region. More recent GWAS also identified ANRIL as a risk locus for gliomas and basal cell carcinomas in accordance with the princeps observation. Moreover’ a mouse model has confirmed the pivotal role of ANRIL in regulation of CDKN2A/B expression through a cis‐acting mechanism and its implication in proliferation and senescence. The implication of ANRIL in cellular aging has provided an attractive unifying hypothesis to explain its association with various susceptibility risk factors. ANRIL identification emphasizes the underestimated role of long noncoding RNAs. Many GWAS have identified trait‐associated SNPs that felt in non‐coding genomic regions. It is conceivable to anticipate that long’ noncoding RNAs will map to many of these “gene deserts.”—Pasmant, E., Sabbagh, A., Vidaud, M., Bieche, I. ANRIL, a long, noncoding RNA, is an unexpected major hotspot in GWAS. FASEB J. 25, 444–448 (2011). www.fasebj.org

Reverse transcriptase-PCR quantification of mRNA levels from cytochrome (CYP)1, CYP2 and CYP3 families in 22 different human tissues.

Objective The aim of this work was to study simultaneously the expression profile of the 23 CYP mRNAs of CYP1, CTP2 and CYP3 families in 22 different human tissues namely adrenal gland, bladder, bone marrow, colon, fetal liver, heart, kidney, liver, lung, mammary gland, ovary, placenta, prostate, salivary gland, skeletal muscle, small intestine, spleen, testis, thymus, thyroid, trachea and uterus. Methods Analysis of the mRNA levels of each of these CYP isoforms was performed on total RNA from pooled specimens of human organs using reverse transcriptase-PCR-based CYP mRNA assays previously validated for their sensitivity and their specificity. Results Our results confirmed previously reported data in the literature concerning isoforms expression in the most currently studied tissues. Moreover, they provided a great deal of new information, mainly about the expression of mRNA of little-known CYP isoforms. Among the 23 CYP isoforms studied, 12 were mainly hepatic (CYP1A2, 2A6, 2A7, 2A13, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4 and 3A43). Two CYP mRNAs were predominantly expressed in several extrahepatic tissues: CYP1B1 mRNA was the predominant CYP in seven extrahepatic tissues (bone marrow, kidney, mammary gland, prostate, spleen, thyroid and uterus) and CYP2J2 in four extrahepatic tissues (heart, placenta, salivary gland and skeletal muscle). Finally, some CYPs were nearly exclusively expressed in only one extrahepatic tissue. CYP2R1 was found in testis, CYP2U1 in the thymus and CYP2F1 in the respiratory tract (lung and trachea). Conclusion This description will broaden the understanding of the physiological functions of these CYPs.

Novel approach to quantitative polymerase chain reaction using real‐time detection: Application to the detection of gene amplification in breast cancer

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase‐chain‐reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real‐time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real‐time PCR does not require post‐PCR sample handling, thereby preventing potential PCR‐product carry‐over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real‐time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccnd1 and erbB2) in breast tumors. Extra copies of myc, ccnd1 and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast‐tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi‐automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings. Int. J. Cancer 78:661–666, 1998.

Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C

Background and Aims: The gold standard treatment of chronic hepatitis C (CHC) is combined pegylated interferon and ribavirin. Considering side effects and treatment cost, prediction of treatment response before therapy is important. The aim of this study was to identify a liver gene signature to predict sustained virological response in patients with CHC. Methods: Group A (training set) comprised 40 patients with CHC including 14 non-responders (NRs) and 26 sustained virological responders (SVRs). Group B (validation set) comprised 29 patients including 9 NRs and 20 SVRs. Eleven responder–relapsers were also included. A total of 58 genes associated with liver gene expression dysregulation during CHC were selected from the literature. Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of these 58 selected genes in liver biopsy specimens taken from the patients before treatment. Results: From the Group A data, three genes whose expression was significantly increased in NRs compared with SVRs were identified: IFI-6-16/G1P3, IFI27 and ISG15/G1P2. These three genes also showed significant differences in their expression profiles between NRs and SVRs in the independent sample (Group B). Supervised class prediction analysis identified a two-gene (IFI27 and CXCL9) signature, which accurately predicted treatment response in 79.3% (23/29) of patients from the validation set (Group B), with a predictive accuracy of 100% (9/9) and of 70% (14/20) in NRs and SVRs, respectively. The expression profiles of responder–relapsers did not differ significantly from those of NRs and SVRs, and 73% (8/11) of them were predicted as SVRs with the two-gene classifier. Conclusion: NRs and SVRs have different liver gene expression profiles before treatment. The most notable changes occurred mainly in interferon-stimulated genes. Treatment response could be predicted with a two-gene signature (IFI27 and CXCL9).

Molecular Profiling of Inflammatory Breast Cancer Identification of a Poor-Prognosis Gene Expression Signature

Purpose: Inflammatory breast cancer (IBC) is a rare but particularly aggressive form of primary breast cancer. The molecular mechanisms responsible for IBC are largely unknown. Experimental Design: To obtain further insight into the molecular pathogenesis of IBC, we used real-time quantitative reverse transcription (RT)-PCR to quantify the mRNA expression of 538 selected genes in IBC relative to non-IBC. Results: Twenty-seven (5.0%) of the 538 genes were significantly up-regulated in IBC compared with non-IBC. None were down-regulated. The 27 up-regulated genes mainly encoded transcription factors (JUN, EGR1, JUNB, FOS, FOSB, MYCN, and SNAIL1), growth factors (VEGF, DTR/HB-EGF, IGFBP7, IL6, ANGPT2, EREG, CCL3/MIP1A, and CCL5/RANTES) and growth factor receptors (TBXA2R, TNFRSF10A/TRAILR1, and ROBO2). We also identified a gene expression profile, based on MYCN, EREG, and SHH, which discriminated subgroups of IBC patients with good, intermediate, and poor outcome. Conclusion: Our study has identified a limited number of signaling pathways that require inappropriate activation for IBC development. Some of the up-regulated genes identified here could offer useful diagnostic or prognostic markers and could form the basis of novel therapeutic strategies.

VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms

Studies comparing tumor neovascularity with pathological findings suggest that angiogenesis contributes to the pathogenesis of prostate cancer. We have examined 42 primary sporadic prostate tumors at different clinical stages, together with 3 prostate cancer cell lines (DU145, PC3 and LNCaP), for expression of VEGF and the gene encoding the recently identified VEGF165 isoform‐specific receptor neuropilin‐1, by using a quantitative reverse transcription (RT)‐PCR method. We also evaluated the VEGF transcription pattern. Upregulation of VEGF and neuropilin‐1 was observed in 12 and 14 tumors, respectively. The VEGF165 isoform was slighly overrepresented in tumors that overexpressed VEGF. VEGF overexpression correlated with stage II disease (p < 0.05); neuropilin‐1 overexpression correlated with advanced disease (p < 0.01) and a high Gleason grade (p < 0.02). Our observations suggest that VEGF expression could be used as a prognostic marker in early‐stage prostate tumors, whereas neuropilin‐1 overexpression might be a marker of aggressiveness. Int. J. Cancer (Pred. Oncol.) 89:167–171, 2000.

A refined molecular taxonomy of breast cancer

The current histoclinical breast cancer classification is simple but imprecise. Several molecular classifications of breast cancers based on expression profiling have been proposed as alternatives. However, their reliability and clinical utility have been repeatedly questioned, notably because most of them were derived from relatively small initial patient populations. We analyzed the transcriptomes of 537 breast tumors using three unsupervised classification methods. A core subset of 355 tumors was assigned to six clusters by all three methods. These six subgroups overlapped with previously defined molecular classes of breast cancer, but also showed important differences, notably the absence of an ERBB2 subgroup and the division of the large luminal ER+ group into four subgroups, two of them being highly proliferative. Of the six subgroups, four were ER+/PR+/AR+, one was ER−/PR−/AR+ and one was triple negative (AR−/ER−/PR−). ERBB2-amplified tumors were split between the ER−/PR−/AR+ subgroup and the highly proliferative ER+ LumC subgroup. Importantly, each of these six molecular subgroups showed specific copy-number alterations. Gene expression changes were correlated to specific signaling pathways. Each of these six subgroups showed very significant differences in tumor grade, metastatic sites, relapse-free survival or response to chemotherapy. All these findings were validated on large external datasets including more than 3000 tumors. Our data thus indicate that these six molecular subgroups represent well-defined clinico-biological entities of breast cancer. Their identification should facilitate the detection of novel prognostic factors or therapeutical targets in breast cancer.

Prognostic value of ERBB family mRNA expression in breast carcinomas

The ErbB‐driven autocrine growth pathway has been implicated in the development and progression of most common human epithelial malignancies; its blockade is therefore a promising therapeutic strategy, and several candidate drugs are currently undergoing clinical trials. Paradoxically, little is known of the expression pattern of these 4 genes in human tumors, and the clinical significance of the 2 most recently discovered ERBB genes, ERBB3 and ERBB4, is unclear. We used a real‐time quantitative RT‐PCR assay to quantify ERBB family mRNA copy numbers in a large series of breast tumors from patients with known long‐term outcome. ERBB gene expression varied widely, by more than 2 orders of magnitude for ERBB1 and ERBB3, more than 3 orders for ERBB2 and more than 4 orders for ERBB4. We found a positive correlation between ERBB3 and ERBB4 mRNA levels, and a negative correlation between the expression of these 2 latter genes and that of ERBB1. Compared to normal breast tissue, ERBB1 was underexpressed (82.3% of tumors), ERBB2 (16.9%) and ERBB3 (46.2%) were overexpressed and ERBB4 was both underexpressed (24.6%) and overexpressed (29.2%). Links were also found between ERBB status on the one hand and Scarff‐Bloom‐Richardson (SBR) histopathological grade and estrogen receptor alpha (ERa) status on the other hand. Relapse‐free survival (RFS) was shorter among patients with ERBB3‐overexpressing tumors (p=0.0092) and longer among those with ERBB4‐underexpressing tumors (p=0.0085) relative to patients with normal expression of the respective genes; in contrast, RFS was not significantly influenced by ERBB1 or ERBB2 mRNA status. Only ERBB4 status retained prognostic significance in Cox multivariate regression analysis (p=0.015). Our results point to the involvement of several ErbB‐specific ligands (amphiregulin and neuregulin 1) and enzymes or adaptor molecules (PI3K, Src, Shc and Grb7) in the ErbB pathway dysregulation associated with breast cancer. These findings reveal a complex expression pattern of ERBB gene family members in breast tumors and suggest that it is this pattern of expression, rather than the expression of individual family members, that should be taken into account when evaluating antitumoral drugs designed to target these receptors.