Jérôme Solassol

Establishment and Characterization of a Cell Line from Human Circulating Colon Cancer Cells

Circulating tumor cells (CTC) in blood are promising new biomarkers potentially useful for prognostic prediction and monitoring of therapies in patients with solid tumors including colon cancer. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in patients with cancer. However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of patients with colorectal cancer. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. Here, we describe, for the first time, the establishment of cell cultures and a permanent cell line from CTCs of one patient with colon cancer. The cell line designated CTC-MCC-41 has been cultured for more than one year, and the cells have been characterized at the genome, transcriptome, proteome, and secretome levels. This thorough analysis showed that CTC-MCC-41 cells resemble characteristics of the original tumor cells in the patient with colon cancer and display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem cell-like properties, and an osteomimetic signature, indicating a bone marrow origin. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. The establishment of this first colon cancer CTC line allows now a wealth of functional studies on the biology of CTCs as well as in vitro and in vivo drug testing.

Anti‐PrP antibodies block PrPSc replication in prion‐infected cell cultures by accelerating PrPC degradation

The use of anti‐PrP antibodies represents one of the most promising strategies for the treatment of prion diseases. In the present study, we screened various anti‐PrP antibodies with the aim of identifying those that would block PrPSc replication in prion‐infected cell culture. Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein, and SAF61 directed against PrP amino acid residues (144–152), not only inhibited PrPSc formation in prion‐infected neuroblastoma cells but also decreased the PrPC levels in non‐infected N2a cells. In addition, treatment with both SAF34 and SAF61 antibodies decreased PrPC and PrPSc levels in the cells synergistically. In the presence of both antibodies, our results showed that the mode of action which leads to the disappearance of PrPSc in cells is directly coupled to PrPC degradation by reducing the half‐life of the PrPC protein.

Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network

BACKGROUND There is scarce data available about epidermal growth factor receptor (EGFR) mutations other than common exon 19 deletions and exon 21 (L858R) mutations. PATIENTS AND METHODS EGFR exon 18 and/or exon 20 mutations were collected from 10 117 non-small-cell lung cancer (NSCLC) samples analysed at 15 French National Cancer Institute (INCa)-platforms of the ERMETIC-IFCT network. RESULTS Between 2008 and 2011, 1047 (10%) samples were EGFR-mutated, 102 (10%) with rare mutations: 41 (4%) in exon 18, 49 (5%) in exon 20, and 12 (1%) with other EGFR mutations. Exon 20 mutations were related to never-smoker status, when compared with exon 18 mutations (P < 0.001). Median overall survival (OS) of metastatic disease was 21 months [95% confidence interval (CI) 12-24], worse in smokers than in non-smoker patients with exon 20 mutations (12 versus 21 months; hazard ratio [HR] for death 0.27, 95% CI 0.08-0.87, P = 0.03). Under EGFR-tyrosine kinase inhibitors (TKIs), median OS was 14 months (95% CI 6-21); disease control rate was better for complex mutations (6 of 7, 86%) than for single mutations (16 of 40, 40%) (P = 0.03). CONCLUSIONS Rare EGFR-mutated NSCLCs are heterogeneous, with resistance of distal exon 20 insertions and better sensitivity of exon 18 or complex mutations to EGFR-TKIs, probably requiring individual assessment.

Proteomics-Based Identification of HSP60 as a Tumor-Associated Antigen in Early Stage Breast Cancer and Ductal Carcinoma in situ

The detection of autoantibodies in cancer patients has been shown to constitute an excellent tool for early diagnosis. Because breast cancer still lacks early diagnostic markers, we investigated novel tumor-associated antigens and related autoantibodies in sera from patients with early stage breast cancer compared to autoimmune disease, other cancers, and healthy volunteers, using a proteomics-based approach. Among the 26 protein antigens specifically recognized by early stage breast cancer sera, we focused on Heat Shock Protein 60 (HSP60). Using ELISA, we investigated the frequency of autoantibodies directed against this protein in the sera of 240 individuals, comprising patients with either ductal carcinoma in situ (DCIS) ( n = 49) or early stage breast cancer ( n = 58), other cancers ( n = 20), autoimmune disease ( n = 20), and healthy subjects ( n = 93). Autoantibodies directed against HSP60 were present in 16/49 (31%) early stage breast cancer and 18/58 (32.6%) DCIS patients, compared to 4/93 (4.3%) healthy subjects. In particular, autoantibodies were present in 11/23 patients (47.8%) with high-grade DCIS, compared to 5/26 (19.2%) with low-grade DCIS. HSP60 mRNA levels were significantly higher in primary breast cancer compared to healthy breast tissues. Using immunohistochemistry, we found that HSP60 expression gradually increases from normal through DCIS to invasive tissues. Our results indicate that HSP60 autoantibodies may be of interest in terms of clinical utility for the early diagnosis of breast cancer and more particularly in DCIS. Moreover, HSP60 overexpression during the first steps of breast carcinogenesis may be functionally correlated to tumor growth and/or progression.

Autoantibody signatures: progress and perspectives for early cancer detection.

•  Introduction •  Unknown origins of autoantibody production in cancer •  The use of proteomics to identify autoantibodies •  Clinical utility of autoantibody signatures for early detection of cancer ‐  Autoantibodies as potential cancer biomarkers ‐  Defining autoantibody signatures in cancer: still many challenges •  Autoantibody detection to build screening tests in high‐risk populations •  Conclusion and perspectives

Identification of a new panel of serum autoantibodies associated with the presence of in situ carcinoma of the breast in younger women.

Purpose: We examined the feasibility of using a panel of autoantibodies to multiple tumor-associated proteins as a method for early detection of breast cancer and, more particularly, carcinoma in situ (CIS). Experimental Design: PPIA, PRDX2, and FKBP52 were identified as early-stage breast cancer autoantigens by proteomic approaches. The seroreactivity of a panel of antibodies consisting of these three antigens and two previously described autoantigens, HSP60 and MUC1, was tested on 235 samples (60 from primary breast cancer patients, 82 from CIS patients, and 93 from healthy controls) with the use of specific ELISAs. FKBP52, PPIA, and PRDX2 mRNA and protein expression levels were evaluated by reverse transcription-PCR and immunohistochemistry in early-stage breast tumors. Results: Three of five autoantibodies, FKBP52, PPIA, and PRDX2, showed significantly increased reactivity in primary breast cancer and CIS compared with healthy controls. When combined, the five markers significantly discriminated primary breast cancer [receiver operating characteristic area under the curve, 0.73; 95% confidence interval (95% CI), 0.60-0.79] and CIS (receiver operating characteristic area under the curve, 0.80; 95% CI, 0.71-0.85) from healthy individuals. Importantly, the receiver operating characteristic–area under the curve value of the autoantibody panel was able to distinguish CIS, including high grades, from healthy controls in women under the age of 50 years (receiver operating characteristic area under the curve, 0.85; 95% CI, 0.61-0.92). Finally, only FKBP52 mRNA and protein levels were found to be increased in CIS and primary breast cancer compared with healthy breast tissue. Conclusions: This autoantibody assay against a panel of five antigens allows for an accurate discrimination between early-stage breast cancer, especially CIS, and healthy individuals. These results could be of interest in detecting early breast cancer as an aid to mammography, especially in women under the age of 50 years with aggressive cancers.

Impact of Systematic EGFR and KRAS Mutation Evaluation on Progression-Free Survival and Overall Survival in Patients with Advanced Non–Small-Cell Lung Cancer Treated by Erlotinib in a French Prospective Cohort (ERMETIC Project—Part 2)

Background: Epidermal growth factor and v-Ki-ras2 Kirsten ras sarcoma (KRAS) mutation status, although associated with EGFR- tyrosine kinase inhibitor (TKI) efficacy, has not been used in clinical practice until recently. The prospective Evaluation of the EGFR Mutation status for the administration of EGFR-TKIs in non small cell lung Carcinoma (ERMETIC) study aimed to implement these biomarkers in France. Methods: Between March 2007 and April 2008, EGFR and KRAS were studied by sequencing DNA tumor specimens from 522 consecutive advanced non–small-cell lung cancer patients treated with EGFR-TKI, mostly in second- or third-line settings. Cox models were used to investigate the impact of patient characteristics and mutations on progression-free survival (PFS) and overall survival (OS). Added value from mutation status was evaluated using likelihood ratio (LR) tests. Classification and regression tree analysis aimed to identify homogeneous groups in terms of survival. Results: Among the 522 patients, 87% were white, 32% were women, and 18% were never-smokers, with 65% presenting with adenocarcinoma. Biological data were available for 307 patients, showing 44 EGFR mutations (14%) and 42 KRAS (14%) mutations. Median PFS was 2.4 months (interquartile range, 1.4–4.6) and median OS 5.6 months (interquartile range, 2.2–14.0). Factors independently associated with PFS were performance status 1 or 2 to 3 (hazards ratio [HR] = 1.5, 95% confidence interval [CI] 1.1–1.9; and HR = 2.3, CI 1.7–3.1, respectively; p < 0.001); former or current smoker status (HR = 1.8, CI 1.4–2.4 and 2.0,CI 1.4–2.8, respectively; p < 0.001); nonadenocarcinoma histology (squamous cell: HR = 0.9 CI 0.7–1.2]; others: HR = 1.6, 1.3–2.1; p < 0.001); at least two metastatic sites (HR = 1.3, CI 1.1–1.6 and 1.6, CI 1.3–2.1, respectively; p < 0.001); prior taxane-based chemotherapy (HR = 1.3, CI 1.0–1.3, p = 0.01); non-white (HR = 0.7, CI 0.5–0.9, p = 0.009). Similar results were found for OS. In addition, EGFR and KRAS mutations were significantly associated with PFS (HR = 0.5, CI 0.3–0.7 and HR = 1.2, CI 0.8–1.8, respectively, versus no mutation; LR p = 0.001). In the OS model, adjusted HR was 0.7 (0.4–1.0) for EGFR mutation and 1.7 (1.1–2.4) for KRAS (LR p = 0.004). Classification and regression tree analysis revealed EGFR mutation to be the primary factor for identifying homogeneous patient subgroups in terms of PFS. Conclusions: EGFR and KRAS status independently impacts outcomes in advanced non–small-cell lung cancer patients treated with EGFR-TKI. However, EGFR status impacts both PFS and OS whereas KRAS only impacts OS. These findings support the nationwide use of EGFR status for patient selection before EGFR-TKI therapy. The role of KRAS mutations remains to be elucidated.

HDL Proteome in Hemodialysis Patients: A Quantitative Nanoflow Liquid Chromatography-Tandem Mass Spectrometry Approach

Aside from a decrease in the high-density lipoprotein (HDL) cholesterol levels, qualitative abnormalities of HDL can contribute to an increase in cardiovascular (CV) risk in end-stage renal disease (ESRD) patients undergoing chronic hemodialysis (HD). Dysfunctional HDL leads to an alteration of reverse cholesterol transport and the antioxidant and anti-inflammatory properties of HDL. In this study, a quantitative proteomics approach, based on iTRAQ labeling and nanoflow liquid chromatography mass spectrometry analysis, was used to generate detailed data on HDL-associated proteins. The HDL composition was compared between seven chronic HD patients and a pool of seven healthy controls. To confirm the proteomics results, specific biochemical assays were then performed in triplicate in the 14 samples as well as 46 sex-matched independent chronic HD patients and healthy volunteers. Of the 122 proteins identified in the HDL fraction, 40 were differentially expressed between the healthy volunteers and the HD patients. These proteins are involved in many HDL functions, including lipid metabolism, the acute inflammatory response, complement activation, the regulation of lipoprotein oxidation, and metal cation homeostasis. Among the identified proteins, apolipoprotein C-II and apolipoprotein C-III were significantly increased in the HDL fraction of HD patients whereas serotransferrin was decreased. In this study, we identified new markers of potential relevance to the pathways linked to HDL dysfunction in HD. Proteomic analysis of the HDL fraction provides an efficient method to identify new and uncharacterized candidate biomarkers of CV risk in HD patients.

Proteomic profile determination of autosomal aneuploidies by mass spectrometry on amniotic fluids

BackgroundPrenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time-consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance of women with normal results can be performed by molecular analysis of uncultured foetal cells. In the present study, we developed a proteomic fingerprinting approach coupled with a statistical classification method to improve diagnosis of aneuploidies, including trisomies 13, 18, and 21, in amniotic fluid samples.ResultsThe proteomic spectra obtained from 52 pregnant women were compiled, normalized, and mass peaks with mass-to-charge ratios between 2.5 and 50 kDa identified. Peak information was combined together and analysed using univariate statistics. Among the 208 expressed protein peaks, 40 differed significantly between aneuploid and non aneuploid samples, with AUC diagnostic values ranging from 0.71 to 0.91. Hierarchical clustering, principal component analysis and support vector machine (SVM) analysis were performed. Two class predictor models were defined from the training set, which resulted in a prediction accuracy of 92.3% and 96.43%, respectively. Using an external and independent validation set, diagnostic accuracies were maintained at 87.5% and 91.67%, respectively.ConclusionThis pilot study demonstrates the potential interest of protein expression signature in the identification of new potential biological markers that might be helpful for the rapid clinical management of high-risk pregnancies.

Proteomic analysis of RCL2 paraffin‐embedded tissues

Histopathological diagnosis in most of the worlds hospitals is based upon formalin‐fixed and paraffin‐embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non‐toxic fixative, for sparing proteins preserved in paraffin‐embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi‐dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western‐blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser‐captured microdissection were found to be identical for frozen and RCL2‐fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin‐embedded tissue sample, and can be a new method for investigating protein biomarkers.