Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing

International audienceTargeted therapies and, more precisely, EGFR tyrosine kinase inhibitors (TKIs) have been a major improvement in the therapeutic management of EGFR-mutated non-small-cell lung cancers (NSCLCs). Earlier administration of these TKIs throughout tumor progression is imperative to improve patient outcomes. Consequently, studies have focused on refining the characterization of biomarkers, especially concerning the resistance mutation p.Thr790Met of EGFR. Herein, we developed peptide nucleic acid (PNA)-mediated PCR clamping followed by pyrosequencing, favoring enrichment of the mutated fraction. A preamplification step was first added to increase the amplifiable DNA fraction. Throughout the application of our method on DNA extracted from FFPE samples of 46 patients with NSCLC who had relapsed under first-generation EGFR TKI, we evaluated a sensitivity of 93.3% and a specificity of 100%. All 19 patients who were positive for the p.Thr790Met mutation with NGS were also found to be positive with our protocol. The only discordant case was a sample with no mutation detected with NGS, but which was positive with PNA. This protocol allows for the detection of the p.Thr790Met mutation with a sensitivity of 0.5% which will permit earlier detection and an improvement of therapeutic management

Tumor quantification of several fluoropyrimidines resistance gene expression with a unique quantitative RT-PCR method. Implications for pretherapeutic determination of tumor resistance phenotype.

International audiencePretherapeutic determination of tumor resistance to chemotherapy is a main challenge, hindered by the low number of mechanisms characterized at the same time, the small size of the clinical specimens and the heterogeneity of the techniques or the lack of true quantification. The aim of the present study was to determine in real time quantitative RT-PCR, tumor cell expression of several transcripts involved in cancer cell resistance with a unique cDNA sample from a tumor biopsy. The technique had to be suitable in clinical practice for determination of several factors involved in resistance to a given drug family, for example, fluoropyrimidines resistance factors: thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine kinase (TK), dihydrofolate reductase (DHFR), folylpolyglutamate synthetase (FPGS). A frame-shifted artificial construct was designed specifically to work within the same conditions. We validated our technique by quantifying expressions of these 5 genes starting from tissue samples of colorectal carcinoma and the surrounding normal mucosa of 33 different patients. That real time quantitative RT-PCR technique using the frame-shifted artificial construct as a standard provided a real comparison and quantification of different resistance factors. Tumor resistance phenotype determination based on that approach will be investigated in a control study

OLFM4 Expression in Ductal Carcinoma In Situ and in Invasive Breast Cancer Cohorts by a SWATH‐Based Proteomic Approach

International audienceHuman olfactomedin-4 (OLFM4) is a secreted protein involved in a variety of cellular functions including proliferation, differentiation, apoptosis, and cell adhesion. OLFM4 expression has been studied in several tumor types including gastric, colorectal, lung, and endometrioid cancers where it has been suggested to be an independent favorable or unfavorable prognostic marker. For breast cancer, the clinical significance of OLFM4 is still unclear. In the present study, SWATH-MS is used as a tool for the robust identification and quantification of breast tissue proteins. SWATH-MS data show that OLFM4 expression is higher in DCIS than in invasive breast cancer. In-depth analysis of the breast tumor proteome show that OLFM4 is a favorable pronostic marker. Serum OLFM4 levels in peripheral blood are also analyzed by ELISA in 825 cases, including 94 cases of healthy individuals, 61 cases of non-invasive breast tumor (DCIS) and 670 cases of breast cancer (BC). It is found that serum OLFM4 levels are significantly higher in the DCIS cohort and in the breast cancer cohort compared with the healthy controls. This result suggests that circulating OLFM4 could be an interesting biomarker of early breast cancer. Data are available via ProteomeXchange with identifier PXD014194

Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples

International audienceTo date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n = 42) and a test set (n = 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan–Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan–Meier method and immunohis-tochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments

Identification of three subtypes of triple-negative breast cancer with potential therapeutic implications

International audienceBACKGROUND:Heterogeneity and lack of targeted therapies represent the two main impediments to precision treatment of triple-negative breast cancer (TNBC), and therefore, molecular subtyping and identification of therapeutic pathways are required to optimize medical care. The aim of the present study was to define robust TNBC subtypes with clinical relevance.METHODS:Gene expression profiling by means of DNA chips was conducted in an internal TNBC cohort composed of 238 patients. In addition, external data (n = 257), obtained by using the same DNA chip, were used for validation. Fuzzy clustering was followed by functional annotation of the clusters. Immunohistochemistry was used to confirm transcriptomics results: CD138 and CD20 were used to test for plasma cell and B lymphocyte infiltrations, respectively; MECA79 and CD31 for tertiary lymphoid structures; and UCHL1/PGP9.5 and S100 for neurogenesis.RESULTS:We identified three molecular clusters within TNBC: one molecular apocrine (C1) and two basal-like-enriched (C2 and C3). C2 presented pro-tumorigenic immune response (immune suppressive), high neurogenesis (nerve infiltration), and high biological aggressiveness. In contrast, C3 exhibited adaptive immune response associated with complete B cell differentiation that occurs in tertiary lymphoid structures, and immune checkpoint upregulation. External cohort subtyping by means of the same approach proved the robustness of these results. Furthermore, plasma cell and B lymphocyte infiltrates, tertiary lymphoid structures, and neurogenesis were validated at the protein levels by means of histological evaluation and immunohistochemistry.CONCLUSION:Our work showed that TNBC can be subcategorized in three different subtypes characterized by marked biological features, some of which could be targeted by specific therapies