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2.  Validation of a preclinical model for assessment of drug efficacy in melanoma 
Oncotarget  2016;7(11):13069-13081.
The aim of personalized medicine is to improve our understanding of the disease at molecular level and to optimize therapeutic management. In this context, we have developed in vivo and ex vivo preclinical strategies evaluating the efficacy of innovative drugs in melanomas. Human melanomas (n = 17) of different genotypes (mutated BRAF, NRAS, amplified cKIT and wild type) were successfully engrafted in mice then amplified by successive transplantations. The exhaustive characterization of patient-derived xenografts (PDX) at genomic level (transcriptomic and CGH arrays) revealed a similar distribution pattern of genetic abnormalities throughout the successive transplantations compared to the initial patient tumor, enabling their use for mutation-specific therapy strategies. The reproducibility of their spontaneous metastatic potential in mice was assessed in 8 models. These PDXs were used for the development of histoculture drug response assays (ex vivo) for the evaluation of innovative drug efficacy (BRAF and MEK inhibitors). The pharmacological effects of BRAF and MEK inhibitors were similar between PDX-derived histocultures and their corresponding PDX, on 2 models of BRAF and NRAS-mutated melanomas. These models constitute a validated, effective tool for preclinical investigation of new therapeutic agents, and improve therapeutic strategies in the treatment of metastatic melanoma.
PMCID: PMC4914342  PMID: 26909610
histoculture drug response assay; xenograft; BRAF mutation; melanoma; model
3.  Diagnostic accuracy of procalcitonin in critically ill immunocompromised patients 
BMC Infectious Diseases  2011;11:224.
Recognizing infection is crucial in immunocompromised patients with organ dysfunction. Our objective was to assess the diagnostic accuracy of procalcitonin (PCT) in critically ill immunocompromised patients.
This prospective, observational study included patients with suspected sepsis. Patients were classified into one of three diagnostic groups: no infection, bacterial sepsis, and nonbacterial sepsis.
We included 119 patients with a median age of 54 years (interquartile range [IQR], 42-68 years). The general severity (SAPSII) and organ dysfunction (LOD) scores on day 1 were 45 (35-62.7) and 4 (2-6), respectively, and overall hospital mortality was 32.8%. Causes of immunodepression were hematological disorders (64 patients, 53.8%), HIV infection (31 patients, 26%), and solid cancers (26 patients, 21.8%). Bacterial sepsis was diagnosed in 58 patients and nonbacterial infections in nine patients (7.6%); 52 patients (43.7%) had no infection. PCT concentrations on the first ICU day were higher in the group with bacterial sepsis (4.42 [1.60-22.14] vs. 0.26 [0.09-1.26] ng/ml in patients without bacterial infection, P < 0.0001). PCT concentrations on day 1 that were > 0.5 ng/ml had 100% sensitivity but only 63% specificity for diagnosing bacterial sepsis. The area under the receiver operating characteristic (ROC) curve was 0.851 (0.78-0.92). In multivariate analyses, PCT concentrations > 0.5 ng/ml on day 1 independently predicted bacterial sepsis (odds ratio, 8.6; 95% confidence interval, 2.53-29.3; P = 0.0006). PCT concentrations were not significantly correlated with hospital mortality.
Despite limited specificity in critically ill immunocompromised patients, PCT concentrations may help to rule out bacterial infection.
PMCID: PMC3170614  PMID: 21864380
bacterial infection; neutropenia; HIV infection; immune deficiency; bone marrow transplantation; Sensitivity and Specificity.
4.  Dectin-1 Polymorphism: A Genetic Disease Specifier in Autism Spectrum Disorders? 
PLoS ONE  2015;10(9):e0137339.
In autism spectrum disorders (ASD), complex gene-environment interactions contribute to disease onset and progress. Given that gastro-intestinal dysfunctions are common in ASD, we postulated involvement of microbial dysbiosis in ASD and investigated, under a case-control design, the influence of DNA polymorphisms in the CLEC7A gene that encodes a pivotal fungal sensor, Dectin-1.
Material and methods
DNAs from 478 ASD patients and 351 healthy controls (HC) were analyzed for the CLEC7A rs16910631G/A and rs2078178 A/G single nucleotide polymorphisms (SNPs). Differences in the distribution of allele, genotype and haplotype by Chi-square testing and nonparametric analysis by Kruskal-Wallis/Mann–Whitney tests, where appropriate, were performed. The free statistical package R.2.13 software was used for the statistical analysis.
We found that the CLEC7A rs2078178 G allele and GG genotype were more prevalent in HC as compared to ASD but failed to reach statistical significance for the latter (pc = 0.01, 0.06 respectively). However, after phenotype-based stratification, the CLEC7A rs2078178 G allele and GG genotype were found to be significantly more frequent in the Asperger group as compared to other ASD subsets (pc = 0.02, 0.01), a finding reinforced by haplotype analysis (rs2078178/rs16910631 G-G/G-G) (pc = 0.002). Further, intellectual quotient (IQ)-based stratification of ASD patients revealed that IQ values increase linearly along the CLEC7A rs2078178 AA, AG and GG genotypes (p = 0.05) and in a recessive manner (GG vs. AA+AG p = 0.02), further confirmed by haplotype distribution (CLEC7A rs2078178-16910631; A-G/A-G, A-G/G-G and G-G/G-G, p = 0.02, G-G/G-G vs. others, p = 0.01).
Our data suggest that the genetic diversity of CLEC7A gene influences the ASD phenotype by behaving as a disease specifier and imply that the genetic control of innate immune response could determine the ASD phenotype.
PMCID: PMC4564239  PMID: 26352598
5.  Tracking sub-clonal TP53 mutated tumor cells in human metastatic renal cell carcinoma 
Oncotarget  2015;6(22):19279-19289.
Renal Cell Carcinomas (RCCs) are heterogeneous tumors with late acquisition of TP53 abnormalities during their evolution. They harbor TP53 abnormalities in their metastases. We aimed to study TP53 gene alterations in tissue samples from primary and metastatic RCCs in 36 patients followed up over a median of 4.2 years, and in xenografted issued from primary RCCs.
In 36 primary RCCs systematically xenografted in mice, and in biopsies of metastases performed whenever possible during patient follow-up, we studied p53-expressing tumor cells and TP53 gene abnormalities.
We identified TP53 gene alterations in primary tumors, metastases and xenografts.
Quantification of tumors cells with TP53 gene alterations showed a significant increase in the metastases compared to the primary RCCs, and, strikingly, the xenografts were similar to the metastases and not to the primary RCCs from which they were derived.
Using laser-microdissection of p53-expressing tumor cells, we identified TP53-mutated tumor cells in the xenografts derived from the primary RCC, and in a lung metastasis later developed in one patient. The mutation enabled us to track back their origin to a minority sub-clone in the primary heterogeneous RCC.
Combining in situ and molecular analyses, we demonstrated a clonal expansion in a living patient with metastatic RCC.
PMCID: PMC4662490  PMID: 26002555
metastases; human RCC; tumor heterogeneity; sub-clonal tumor cells; TP53 mutation
6.  Stability of preclinical models of aggressive renal cell carcinomas 
Renal-cell carcinomas (RCC) are often resistant to conventional cytotoxic agents. Xenograft models are used for in vivo preclinical studies and drug development. The validity of these studies is highly dependent on the phenotypic and genotypic stability of the models. Here we assessed the stability of six aggressive human RCC xenografted in nude/NMRI mice. We compared the initial samples (P0), first (P1) and fifth (P5) passages for the following criteria: histopathology, immunohistochemistry for CK7, CD10, vimentin and p53, DNA allelic profiles using 10 microsatellites and CGH-array. Next we evaluated the response to sunitinib in primary RCC and corresponding xenografted RCC. We observed a good overall stability between primary RCC and corresponding xenografted RCC at P1 and P5 regarding histopathology and immunohistochemistry except for cytokeratin 7 (one case) and p53 (one case) expression. Out of 44 groups with fully available microsatellite data (at P0, P1 and P5), 66% (29 groups) showed no difference from P0 to P5 while 34% (15 groups) showed new or lost alleles. Using CGH-array, overall genomic alterations at P5 were not different from those of initial RCC. The xenografted RCC had identical response to sunitinib therapy compared to the initial human RCC from which they derive. These xenograft models of aggressive human RCC are clinically relevant, showing a good histological and molecular stability and are suitable for studies of basic biology and response to therapy.
PMCID: PMC4097235  PMID: 25031714
Xenograft; preclinical model; renal cell carcinoma; tumor phenotype; tumor genotype
7.  Determination of Angptl4 mRNA as a Diagnostic Marker of Primary and Metastatic Clear Cell Renal-Cell Carcinoma 
PLoS ONE  2010;5(4):e10421.
We have previously shown that angiopoietin-like 4 (angptl4) mRNA, a hypoxia-inducible gene, is highly expressed in clear cell renal-cell carcinoma (ccRCC), the most common subtype of RCC for which no specific marker is available. We here investigated whether angptl4 mRNA 1) could be a useful diagnostic and/or prognostic marker of ccRCC in a large and comprehensive retrospective series, 2) induction is dependent on the VHL status of tumors.
Methodology/Principal Findings
Using in situ hybridization, we report that angptl4 mRNA is expressed in 100% of both sporadic (n = 102) and inherited (n = 6) primary ccRCCs, without any statistical association with nuclear grade (p = 0.39), tumor size (p = 0.09), stage grouping (p = 0.17), progression-free survival (p = 0.94), and overall survival (p = 0.80). Angptl4 mRNA was also expressed in 26 (87%) of 30 secondary ccRCCs but neither in any other secondary RCCs (n = 7). In contrast, angptl4 mRNA was neither expressed in 94% non-ccRCC renal tumors (papillary RCCs (n = 46), chromophobe RCCs (n = 28), and oncocytomas (n = 9)), nor in non-renal clear cell carcinomas (n = 39). Angptl4 expression was also examined in tumors associated (n = 23) or not associated (n = 66) with VHL disease. 40 (98%) hemangioblastomas expressed angptl4 whereas all pheochromocytomas (n = 23) and pancreatic tumors (n = 25) were angptl4-negative, whatever their VHL status.
Angptl4 mRNA expression was highly associated with ccRCC (p = 1.5 10−49, Chi square test) allowing to define its expression as a diagnosis marker for primary ccRCC. Moreover, angptl4 mRNA allows to discriminate the renal origin of metastases of clear-cell carcinomas arising from various organs. Finally, inactivation of VHL gene is neither necessary nor sufficient for angptl4 mRNA induction.
PMCID: PMC2861680  PMID: 20454689
8.  Exquisite Sensitivity of TP53 Mutant and Basal Breast Cancers to a Dose-Dense Epirubicin−Cyclophosphamide Regimen 
PLoS Medicine  2007;4(3):e90.
In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown.
Methods and Findings
In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m2 epirubicin and 1,200 mg/m2 cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status.
This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin–cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features.
Hugues de The and colleagues report thatTP53 status is a predictive factor for responsiveness in breast cancers to a dose-dense epirubicin-cyclophosphamide chemotherapy regimen, and suggests that this regimen might be well suited for patientsTP53 mutant tumors.
Editors' Summary
One woman in eight will develop breast cancer during her life. As with other cancers, breast cancer arises when cells accumulate genetic changes (mutations) that allow them to grow uncontrollably and to move around the body. These altered cells are called malignant cells. The normal human breast contains several types of cell, any of which can become malignant. In addition, there is more than one route to malignancy—different sets of genes can be mutated. As a result, breast cancer is a heterogeneous disease that cannot be cured with a single type of treatment. Ideally, oncologists would like to know before they start treating a patient which therapeutic approach is going to be successful for that individual. Recently, researchers have begun to identify molecular changes that might eventually allow oncologists to make such rational treatment decisions. For example, laboratory studies in cell lines or animals indicate that the status of a gene called TP53 determines the chemotherapy agents (drugs that preferentially kill rapidly dividing cancer cells) to which cells respond. p53, the protein encoded by TP53, is a tumor suppressor. That is, in normal cells it prevents unregulated growth by controlling the expression of proteins involved in cell division and cell death. Consequently, p53 is often inactivated during cancer development.
Why Was This Study Done?
Although laboratory studies have linked TP53 status to chemotherapy responses, little is known about this relationship in human breast cancers. The clinical studies that have investigated whether TP53 status affects chemotherapy responses have generally found that patients whose tumors contain mutant TP53 have a poorer response to therapy and/or a shorter survival time than those whose tumors contain normal TP53. In this study, the researchers have asked whether TP53 status affects tumor responses to a dose-intense chemotherapy regimen (frequent, high doses of drugs) given to women with advanced noninflammatory breast cancer before surgery. This type of treatment is called neoadjuvant chemotherapy and is used to shrink tumors before surgery.
What Did the Researchers Do and Find?
The researchers collected breast tumor samples from 80 women before starting six fortnightly cycles of chemotherapy with epirubicin and cyclophosphamide. After this, each woman had her affected breast removed and examined to see whether the chemotherapy had killed the tumor cells. The researchers determined which original tumor samples contained mutated TP53 and used a technique called microarray expression profiling to document gene expression patterns in them. Overall, 28 tumors contained mutated TP53. Strikingly, all 15 tumors that responded completely to neoadjuvant chemotherapy (no tumor cells detectable in the breast tissue after chemotherapy) contained mutated TP53. Nine of these responsive tumors were basal-cell–like breast tumors, a particularly aggressive type of breast cancer; only one basal-cell–like, TP53-mutated tumor did not respond to chemotherapy. Patients whose tumors were unresponsive to the neoadjuvant chemotherapy but contained mutated TP53 tended to die sooner than those whose tumors contained normal TP53 or those with chemotherapy-responsive TP53-mutated tumors. Finally, expression profiling identified changes in the expression of many p53-regulated genes, but did not identify an expression profile in the TP53-mutated tumors unique to those that responded to chemotherapy.
What Do These Findings Mean?
These findings indicate that noninflammatory breast tumors containing mutant TP53—in particular, basal-cell–like tumors—are very sensitive to dose-dense epirubicin and cyclophosphamide chemotherapy. Intensive regimens of this type have rarely been used in previous studies, which might explain the apparent contradiction between these results and the generally poor response to chemotherapy of TP53-mutated breast tumors. More tumors now need to be examined to confirm the association between complete response, TP53 status and basal-cell–like tumors. In addition, although complete tumor responses generally predict good overall survival, longer survival studies than those reported here are needed to show that the tumor response to this particular neoadjuvant chemotherapy regimen translates into improved overall survival. If the present results can be confirmed and extended, dose-dense neoadjuvant chemotherapy with epirubicin and cyclophosphamide could considerably improve the outlook for patients with aggressive TP53-mutant, basal-cell–like breast tumors.
Additional Information.
Please access these Web sites via the online version of this summary at
The US National Cancer Institute provides patient and physician information on breast cancer and general information on understanding cancer
Cancer Research UK offers patient information on cancer and breast cancer
The MedlinePlus encyclopedia has pages on breast cancer
Emory University's CancerQuest discusses the biology of cancer, including the role of tumor suppressor proteins
Wikipedia has pages on p53 (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1831731  PMID: 17388661

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