Personalized biomarker-based treatment strategy for patients with squamous cell carcinoma of the head and neck: EORTC position and approach.

The molecular landscape of squamous cell carcinoma of the head and the neck (SCCHN) has been characterized and actionable or targetable genomic alterations have been identified. However, targeted therapies have very limited activity in unselected SCCHN, and the current treatment strategy is still based on tumor location and disease stage and not on tumor biology. Trying to select upfront the patients who will benefit from a specific treatment might be a way to improve patients' outcome. With the objective of optimizing the activity of targeted therapies and immunotherapy, we have designed an umbrella biomarker-driven study dedicated to recurrent and/or metastatic SCCHN patients (EORTC-1559-HNCG, NCT03088059). In this article, we review not only the different trial designs for biomarker-driven studies with their respective advantages and opportunities but also the potential pitfalls that led to the design of the EORTC-1559-HNCG protocol. We also discuss the scientific and logistic challenges of biomarker-driven trials.

Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer.

BACKGROUND: Molecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies. PATIENTS AND METHODS: The Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations. RESULTS: Evaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44-90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information. CONCLUSION: Plasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs.

Evolution of microRNA expression during human bronchial squamous carcinogenesis.

MicroRNAs, negative post-transcriptional regulators of gene expression, are involved in cancer. Their role in early bronchial carcinogenesis was analysed in 60 biopsies obtained by fluorescence bronchoscopy (six per stage: normal tissue of nonsmokers, normal normofluorescent and hypofluorescent bronchial tissue of smokers, hyperplasia, metaplasia, mild, moderate and severe dysplasia, in situ carcinoma and invasive squamous cell carcinoma (SQCC)). In total, 69 microRNAs were found to be differentially expressed in the course of bronchial carcinogenesis. Among them, some microRNAs showed a linear evolution of their expression level, such as miR-32 and miR-34c, whose expression progressively decreased from normal bronchial tissues of nonsmokers to SQCC. Others behaved differently at successive stages, such as miR-142-3p or miR-9, or are only altered from a specific stage, such as miR-199a or miR-139. MicroRNAs globally followed a two-step evolution, first decreasing (a reverse of their increase during embryogenesis) during the earliest morphological modifications of bronchial epithelium, and thereafter increasing at later stages of lung carcinogenesis. Moreover, microRNA expression was very efficient for the prediction of the histological classification between low- and high-grade lesions and between in situ and invasive carcinoma. The present data show, for the first time, that microRNAs are involved in bronchial carcinogenesis from the very early steps of this process and, thus, could provide tools for early detection of lung cancer.

Gene regulation by phorbol 12-myristate 13-acetate in MCF-7 and MDA-MB-231, two breast cancer cell lines exhibiting highly different phenotypes.

We have examined the effects of the protein kinase C (PKC)-activator phorbol 12-myristate 13-acetate (PMA) on gene expression in two breast cancer cell (BCC) lines exhibiting highly different phenotypes. These are the estrogen receptor alpha (ERalpha)-positive, weakly invasive, luminal epithelial-like MCF-7 and the ERalpha-negative, highly invasive, fibroblast-like MDA-MB-231. They express constitutively low and high PKC activities, respectively. After a 24-h exposition to 100 nM PMA, the number of genes showing an altered expression at the 2-fold change level was much higher in MCF-7 (n=435) than in MDA-MB-231 (n=18) BCC. Four of these genes, namely CDC2, CENPA, NR4A1 and MMP10, were altered in the same way in both cell lines. Two genes were regulated in an opposite way: ID1 and EVA1. Many of the genes down-regulated in MCF-7 BCC appeared to be preferentially expressed in the G1, S, and/or G2 phases of the cell cycle. The ERalpha gene, ESR1, and other genes associated to the ERalpha-positive, luminal epithelial-like BCC phenotype were down-regulated, while a series of genes related to a more aggressive, fibroblast-like BCC phenotype were up-regulated. Other altered genes were notably linked to cell architecture, supporting profound effects of PMA on cell morphology and motility, as well as on the interactions between BCC and their neighboring proteins. Of note, all the modulated genes involved in proteolysis and its control were up-regulated. In summary, PMA effects suggest that PKC activation may induce, to some extent, a more fibroblast-like phenotype in the ERalpha-positive, luminal epithelial-like MCF-7 BCC, and significantly modulate the interactions of these cells with their environment.

Improved radiation hybrid map of rat chromosome 2: colocalization of the genes encoding corticotropin-releasing hormone and IL6-receptor with quantitative trait loci regulating the inflammatory response.

We established a radiation hybrid (RH) map of several genes and anonymous markers in the lower half of rat chromosome 2, a chromosome region that contains quantitative trait loci (QTLs) for blood pressure, diabetes and inflammatory response. Two of the newly localized genes (Crh and Il6r) encode proteins involved in the regulation of inflammatory and immune events. Our data show that they reside within regions that were genetically defined as QTLs controlling the inflammatory response. These genes are thus both functional and positional candidates.

Analysis of candidate genes included in the mammary cancer susceptibility 1 (Mcs1) region.

The rat strain COP is resistant to spontaneous and carcinogen-induced mammary cancer, whereas the strain WF is susceptible. Using genetic linkage analysis of (WF x COP) F1 x WF backcrosses, LC Hsu, LA Shepel and co-workers showed that a region at the centromeric end of Chromosome (Chr) 2 (2q1) segregates with the sensitivity to mammary cancer development. The responsible locus was named Mcs1 (for mammary cancer susceptibility 1). We have developed the chromosome map of the 2q1 region by localizing 18 genes, 4 ESTs, and several anonymous markers, using radiation hybrids and fluorescence in situ hybridization. The region containing Mcs1 was delineated to 2q12-q14. Five of the genes (Mef2c, Map1b, Ccnh, Rasa, Rasgrf2) were assigned to this region and were shown to be expressed in the rat mammary glands, while three possible functional candidate genes, Pi3kr1, Rad17, and Naip, were excluded from the critical region. Since cyclin H, encoded by Ccnh, plays an important role in the control of the cell cycle and since the proteins encoded by Rasa and Rasgrf2 control the activity of the RAS oncoprotein, the corresponding genes appeared as both functional and positional Mcs1 candidates. RT-PCR experiments on RNA extracted from mammary glands of the two rat strains (COP, WF) was done. No amino acid sequence difference was found between the two strains. These results argue against the hypothesis that any of these three genes is Mcs1.

Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1.

The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development. To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1). To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically. The radiation map generated in region 2q1 extends its centromeric end of about 150 cR.