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Determining the status of HER2-neu amplification and overexpression in breast cancer is crucial for prognosis but mostly for treatment purposes. Standard techniques include the determination of IHC in combination with in situ hybridization techniques to confirm a HER2-neu amplification in case of IHC2+ using either a core-needle biopsy or a surgical specimen. qPCR has been also demonstrated to be able to determine HER2 status, mostly in core biopsies or in surgical specimens. Fine-needle aspiration is a reliable, quicker and less invasive technique that is widely used for diagnosis of invasive breast cancer. In this study, we assessed the performance of qPCR in invasive breast carcinomas to determine HER2-neu status by using fine-needle aspiration samples and comparing to standard IHC and FISH. From a total of 154 samples from patients who had nodular breast lesions and attended the 1-day-stop clinic at the Gustave Roussy from March 2013 to October 2014, qPCR was able to determine the HER2 status in a mean of 3.7 days (SD 3.1). The overall concordance with standard HER2-testing was very high: 97% (95% CI 0.94 to 0.99); sensitivity was 96% (0.87–1), specificity 98% (0.95–1) and positive and negative predictive values 88% (0.75–1) and 99% (0.98–1), respectively. In conclusion, our study demonstrates that qPCR performed using fine-needle aspiration samples from a primary tumour is a reliable and fast method to determine HER2/neu status in patients with early breast cancer.

Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs.RETmutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somaticRETmutations (RETmut group) and 34 with wild-typeRET(RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared withRETwt tumors,RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels inRETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis inRETmut MTCs may be more intense and complete than that found inRETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density,RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors.

Medullary thyroid carcinoma (MTC) is characterized by proto-oncogene RET mutations in almost all hereditary cases as well as in more than 40% of sporadic cases. Recently, a high prevalence of RAS mutations was reported in sporadic MTC, suggesting an alternative genetic event in sporadic MTC tumorigenesis.This study aimed to extend this observation by screening somatic mutational status of RET, BRAF, and the three RAS proto-oncogenes in a large series of patients with MTC.Direct sequencing of RET (exons 8, 10, 11, 13, 14, 15, 16), BRAF (exons 11 and 15), and KRAS, HRAS, and NRAS genes (exons 2, 3, and 4) was performed on DNA prepared from 50 MTC samples, including 30 sporadic cases.Activating RET mutations were detected in the 20 hereditary cases (germline mutations) and in 14 sporadic cases (somatic mutations). Among the 16 sporadic MTC without any RET mutation, eight H-RAS mutations and five K-RAS mutations were found. Interestingly, nine RAS mutations correspond to mutation hot spots in exons 2 and 3, but the other four mutations were detected in exon 4. The RET and RAS mutations were mutually exclusive. No RAS gene mutation was found in hereditary MTC, and no BRAF or NRAS mutation was observed in any of the 50 samples.Our study confirms that RAS mutations are frequent events in sporadic MTC. Moreover, we showed that RAS mutation analysis should not be limited to the classical mutational hot spots of RAS genes and should include analysis of exon 4.

Purpose: Liquid biopsies based on circulating cell-free DNA (cfDNA) analysis are described as surrogate samples for molecular analysis. We evaluated the concordance between tumor DNA (tDNA) and cfDNA analysis on a large cohort of patients with advanced or metastatic solid tumor, eligible for phase I trial and with good performance status, enrolled in MOSCATO 01 trial (clinical trial NCT01566019). Experimental Design: Blood samples were collected at inclusion and cfDNA was extracted from plasma for 334 patients. Hotspot mutations were screened using next-generation sequencing for 50 cancer genes. Results: Among the 283 patients with tDNA–cfDNA pairs, 121 had mutation in both, 99 in tumor only, 5 in cfDNA only, and for 58 patients no mutation was detected, leading to a 55.0% estimated sensitivity [95% confidence interval (CI), 48.4%–61.6%] at the patient level. Among the 220 patients with mutations in tDNA, the sensitivity of cfDNA analysis was significantly linked to the number of metastatic sites, albumin level, tumor type, and number of lines of treatment. A sensitivity prediction score could be derived from clinical parameters. Sensitivity is 83% in patients with a high score (≥8). In addition, we analyzed cfDNA for 51 patients without available tissue sample. Mutations were detected for 22 patients, including 19 oncogenic variants and 8 actionable mutations. Conclusions: Detection of somatic mutations in cfDNA is feasible for prescreening phase I candidates with a satisfactory specificity; overall sensitivity can be improved by a sensitivity score allowing to select patients for whom cfDNA constitutes a reliable noninvasive surrogate to screen mutations. Clin Cancer Res; 22(12); 2960–8. ©2016 AACR.

Abnormal expression of non-coding micro RNA (miRNA) has been described in medullary thyroid carcinoma (MTC). Expression of genes encoding factors involved in miRNA biogenesis results often deregulated in human cancer and correlates with aggressive clinical behavior. In this study, expression of four genes involved in miRNA biogenesis (DICER, DROSHA, DCGR8, and XPO5) was investigated in 54 specimens of MTC. Among them, 33 and 13 harbored RET and RAS mutations, respectively. DICER, DGCR8, and XPO5 mRNA levels were significantly overexpressed in MTC harboring RET mutations, in particular, in the presence of RET634 mutation. When MTCs with RET and RAS mutations were compared, only DGCR8 displayed a significant difference, while MTCs with RAS mutations did not show significant differences with respect to non-mutated tumors. We then attempted to correlate expression of miRNA biogenesis genes with tumor aggressiveness. According to the TNM status, MTCs were divided in two groups and compared (N0 M0 vs. N1 and/or M1): for all four genes no significant difference was detected. Cell line experiments, in which expression of a RET mutation is silenced by siRNA, suggest the existence of a causal relationship between RET mutation and overexpression of DICER, DGCR8, and XPO5 genes. These findings demonstrate that RET- but not RAS-driven tumorigenic alterations include abnormalities in the expression of some important genes involved in miRNA biogenesis that could represent new potential markers for targeted therapies in the treatment of RET-mutated MTCs aimed to restore the normal miRNA expression profile.

Epigenetic control of gene expression plays a major influence in the development and progression of many cancer types. Aim of the present study was to investigate the expression of epigenetic regulators in a large cohort of medullary thyroid carcinomas (MTC), correlating the data with the clinical outcome and mutational status of the patients. Taqman Low Density Arrays (TLDAs) were used to analyze expression levels of several genes involved in the epigenetic control of transcription in a series of 54 MTCs. The patients cohort included 13 familial MTCs and 41 sporadic forms; 33 hosted a RET mutation and 13 a RAS somatic mutation. The expression profiling revealed in the more aggressive diseases (i.e. occurrence of metastases; persistent disease; disease-related death) a significant increase of EZH2 and SMYD3 gene expression. The increased levels of EZH2 and SMYD3 did not correlate significantly with mutational status of RET or RAS genes. Thus, the histone methyltransferases EZH2 and SMYD3 mRNA expression may represent useful prognostic biomarkers tailoring the most appropriate follow-up and timing of therapeutic approaches.

The extensive molecular characterization of tumors with high throughput technologies has led to the segmentation of different tumors into very small molecularly defined subgroups. Many ongoing clinical trials are conducted only when specific molecular alterations are identified in tumor samples. In this review, we will describe the implementation of genome analysis in the clinical setting as it has expanded over the last four years in our Precision Medicine Program. This manuscript will also highlight the main limitations and challenges related to the development of broader and deeper genome analysis.

Background Precision-medicine initiatives are driven by the molecular analysis of tumor samples (fresh or FFPE material). Such an approach is limited by the availability of the tumor material and the challenges related to on-purpose tumor biopsies. A very appealing alternative to advance precision-medicine initiatives is the development of liquid biopsies using cfDNA. Methods We investigated the use of NGS on tumor biopsy and plasma, and evaluated the consistency between the tissue biopsy (tDNA) and cfDNA analysis on a prospective cohort of patients with metastatic or locally advanced solid tumors enrolled in the MOSCATO 01 trial (NCT01566019). Blood samples were collected at inclusion before tumor biopsy and cfDNA was extracted from 500μl plasma. Hot-spot mutations from 50 genes were screened with Ampliseq CHP2 panel (IonTorrent, Life Technologies, Dramstadt). Only variants reported by the Torrent Suite Variant Caller v4.2 were retained for the analysis. Paired results in tumor and plasma were described using Cohen's Kappa agreement coefficient (κ). Results From November 2011 to May 2014, among the 516 patients enrolled in the MOSCATO 01 trial, 190 patients (37%) were analyzed for tDNA and cfDNA. In addition, cfDNA was evaluated in 43 patients for whom no tumor analysis was performed because of low cellularity (< 10% of tumor cells). Patient characteristics were as follows: median age at biopsy: 57 years (range, 18-78); main tumor types: lung (19%), ENT (14%), colorectal (10%), breast (10%); median of 3 previous lines of treatment. Overall, 325 mutations were identified in the tDNA of 184 patients: 146 mutations were identified both in tumor and plasma and 179 in tumor, but not in plasma. 15 mutations were only found in cfDNA, thereby providing additional information. The κ.was 59% (95%CI, 0.54-0.64). The sensibility of using NGS for 50 targeted hot-spot genes analysis in cfDNA compared to tDNA was 44.9% and the specificity was 99.8%, with a positive predictive value of 90.7% and negative predictive value of 98.1%. The ten most frequent pathogenic mutations found in the tDNA or cfDNA (>5 cases each) included KRAS (33 cases), PIK3CA (22 cases) and TP53 genes (22 cases). When considering only these ten most frequent mutations, 77 mutations were identified in 71 patients: 39 in tumor and plasma, 36 in tumor, but not in plasma and 2 mutations only in plasma, with a κ of 66% (95%CI, 56-76%). The p.H1047R PIK3CA mutation, was found only in tumor (5 cases); for the other nine mutations, the κ coefficient varied from 56% to 89%, with a median of 75%. The cfDNA analysis of the 43 patients without tDNA analysis revealed at least one mutation in 24 patients (56%), including 11 pathogenic variants of therapeutic interest. Conclusion The analysis of cfDNA using NGS represents an attractive and noninvasive alternative to tumor biopsies, and can be used as a surrogate method to screen for mutations. Further prospective validation is warranted. Citation Format: Ecaterina Ileana, Cécile Jovelet, Marie-Cécile Le Deley, Christophe Massard, Nelly Motté, Antoine Hollebecque, Amélie Boichard, Charles Ferté, Sophie Postel-Vinay, Silvia Rosellini, Maud Ngo-Camus, Thierry De Baere, Philippe Vielh, Catherine Richon, Mélanie Laporte, Siham Gouissem, Yohann Loriot, Rastilav Bahleda, Anas Gazzah, Andrea Varga, Gilles Vassal, Alexander Eggermont, Fabrice André, Jean-Charles Soria, Ludovic Lacroix. Circulating cell-free tumor DNA (cfDNA) analysis of 50-genes by next-generation sequencing (NGS) in the prospective MOSCATO trial. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2401. doi:10.1158/1538-7445.AM2015-2401