Ludovic Lacroix

Characterization of a Large Panel of Patient-Derived Tumor Xenografts Representing the Clinical Heterogeneity of Human Colorectal Cancer

Purpose: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. Experimental Design: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. Results: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. Conclusions: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients. Clin Cancer Res; 18(19); 5314–28. ©2012 AACR.

Phase II Study of Cetuximab As First-Line Single-Drug Therapy in Patients With Unresectable Squamous Cell Carcinoma of the Skin

PURPOSE To evaluate the efficacy and safety of cetuximab, a monoclonal antibody that inhibits the epidermal growth factor receptor (EGFR), as a first-line monotherapy in patients with unresectable squamous cell carcinoma of the skin (SCCS). PATIENTS AND METHODS Thirty-six patients received cetuximab (initial dose of 400 mg/m(2) followed by subsequent weekly doses of 250 mg/m(2)) for at least 6 weeks with a 48-week follow-up. The primary end point was the disease control rate (DCR) at 6 weeks (according to Response Evaluation Criteria in Solid Tumors [RECIST] criteria). Secondary end points included best response rate, overall survival, progression-free survival (PFS), and toxicity assessment. Association of treatment efficacy with RAS mutations or FcγR genotypes was investigated. RESULTS Median age of the study population was 79 years. DCR at 6 weeks was obtained in 25 of 36 patients (69%; 95% CI, 52% to 84%) of the intention-to-treat population. The best responses were eight partial responses and two complete responses. There were no cetuximab-related deaths. There were three related serious adverse events: two grade 4 infusion reactions and one grade 3 interstitial pneumopathy. Grade 1 to 2 acne-like rash occurred in 78% of patients and was associated with prolonged PFS. One HRAS mutation was identified. Combined FcγRIIa-131H/H and/or FcγRIIIa-158V/V polymorphisms were not associated with the clinical outcomes. CONCLUSION As a first-line treatment in patients with unresectable SCCS, cetuximab achieved 69% DCR. A randomized phase III trial is warranted to confirm that cetuximab may be considered as a therapeutic option especially in elderly patients. The low frequency of RAS mutations in SCCS makes SCCS tumors attractive for EGFR inhibition.

Skin Tumors Induced by Sorafenib; Paradoxic RAS–RAF Pathway Activation and Oncogenic Mutations of HRAS, TP53, and TGFBR1

Purpose: The emergence of skin tumors in patients treated with sorafenib or with more recent BRAF inhibitors is an intriguing and potentially serious event. We carried out a clinical, pathologic, and molecular study of skin lesions occurring in patients receiving sorafenib. Experimental Design: Thirty-one skin lesions from patients receiving sorafenib were characterized clinically and pathologically. DNA extracted from the lesions was screened for mutation hot spots of HRAS, NRAS, KiRAS, TP53, EGFR, BRAF, AKT1, PI3KCA, TGFBR1, and PTEN. Biological effect of sorafenib was studied in vivo in normal skin specimen and in vitro on cultured keratinocytes. Results: We observed a continuous spectrum of lesions: from benign to more inflammatory and proliferative lesions, all seemingly initiated in the hair follicles. Eight oncogenic HRAS, TGFBR1, and TP53 mutations were found in 2 benign lesions, 3 keratoacanthomas (KA) and 3 KA-like squamous cell carcinoma (SCC). Six of them correspond to the typical UV signature. Treatment with sorafenib led to an increased keratinocyte proliferation and a tendency toward increased mitogen-activated protein kinase (MAPK) pathway activation in normal skin. Sorafenib induced BRAF–CRAF dimerization in cultured keratinocytes and activated CRAF with a dose-dependent effect on MAP-kinase pathway activation and on keratinocyte proliferation. Conclusion: Sorafenib induces keratinocyte proliferation in vivo and a time- and dose-dependent activation of the MAP kinase pathway in vitro. It is associated with a spectrum of lesions ranging from benign follicular cystic lesions to KA-like SCC. Additional and potentially preexisting somatic genetic events, like UV-induced mutations, might influence the evolution of benign lesions to more proliferative and malignant tumors. Clin Cancer Res; 18(1); 263–72. ©2011 AACR.

Defects in iodide metabolism in thyroid cancer and implications for the follow-up and treatment of patients

The two major steps of iodine metabolism—uptake and organification—are altered in thyroid cancer tissues. Organification defects result in a rapid discharge of radioiodine from thyroid cells, a short effective half-life of iodine, and a low rate of thyroid hormone synthesis. These defects are mainly due to decreased expression of functional genes encoding the sodium-iodide symporter and thyroid peroxidase and could result in a low radiation dose to thyroid cancer cells. TSH stimulation that is achieved with injections of recombinant human TSH, or long-term withdrawal of thyroid hormone treatment increases iodine-131 uptake in two-thirds of patients with metastatic disease and increases thyroglobulin production in all patients with metastases, even in the absence of detectable uptake. Serum thyroglobulin determination obtained following TSH stimulation and neck ultrasonography is the most sensitive combination for the detection of small tumor foci. Radioiodine treatment is effective when a high radiation dose can be delivered (in patients with high uptake and retention of radioiodine) and when tumor foci are sensitive to the effects of radiation therapy (younger patients, with a well-differentiated tumor and/or with small metastases). The other patients rarely respond to radioiodine treatment, and when progression occurs, other treatment modalities should be considered. Novel strategies are currently being explored to restore iodine uptake in cancer cells that are unable to concentrate radioiodine.

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.

Somatic RAS Mutations Occur in a Large Proportion of Sporadic RET-Negative Medullary Thyroid Carcinomas and Extend to a Previously Unidentified Exon

CONTEXT 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. OBJECTIVE 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

Vemurafenib in pediatric patients with BRAFV600E mutated high-grade gliomas

We present three pediatric patients with BRAFV600E mutant high‐grade gliomas treated by vemurafenib on a nominative authorization level at our institution. One patient with anaplastic ganglioglioma experienced confirmed partial tumor response and significant clinical improvement and she is alive 20 months after start of treatment. A second patient with ganglioglioma responded transiently to re‐introduction of vemurafenib after immunotherapy. Pharmacokinetic studies suggest that maximum concentration and exposure of vemurafenib at steady‐state is dose‐dependent and similar in children to that reported in adults. These cases suggest that BRAFV600 is an oncogenic driver in pediatric gliomas. Further exploration in clinical studies is ongoing. Pediatr Blood Cancer 2014;61:1101–1103.

Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

NADPH oxidase 4 (NOX4) belongs to the NOX family that generates reactive oxygen species (ROS). Function and tissue distribution of NOX4 have not yet been entirely clarified. To date, in the thyroid gland, only DUOX1/2 NOX systems have been described. NOX4 mRNA expression, as shown by real-time PCR, was present in normal thyroid tissue, regulated by TSH and significantly increased in differentiated cancer tissues. TSH increased the protein level of NOX4 in human thyroid primary culture and NOX4-dependent ROS generation. NOX4 immunostaining was detected in normal and pathologic thyroid tissues. In normal thyroid tissue, staining was heterogeneous and mostly found in activated columnar thyrocytes but absent in quiescent flat cells. Papillary and follicular thyroid carcinomas displayed more homogeneous staining. The p22(phox) protein that forms a heterodimeric enzyme complex with NOX4 displayed an identical cellular expression pattern and was also positively regulated by TSH. ROS may have various biological effects, depending on the site of production. Intracellular NOX4-p22(phox) localization suggests a role in cytoplasmic redox signaling, in contrast to the DUOX localization at the apical membrane that corresponds to an extracellular H(2)O(2) production. Increased NOX4-p22(phox) in cancer might be related to a higher proliferation rate and tumor progression but a role in the development of tumors has to be further studied and established in the future.

Follicular Thyroid Tumors with the PAX8-PPARγ1 Rearrangement Display Characteristic Genetic Alterations

Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) gamma1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARgamma1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARgamma target genes were up-regulated in PAX8-PPARgamma-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARgamma expression profile presented a putative PPARgamma-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARgamma and as members of novel molecular pathways involved in the development of thyroid carcinomas.

High-Throughput Genomics and Clinical Outcome in Hard-to-Treat Advanced Cancers: Results of the MOSCATO 01 Trial

High-throughput genomic analyses may improve outcomes in patients with advanced cancers. MOSCATO 01 is a prospective clinical trial evaluating the clinical benefit of this approach. Nucleic acids were extracted from fresh-frozen tumor biopsies and analyzed by array comparative genomic hybridization, next-generation sequencing, and RNA sequencing. The primary objective was to evaluate clinical benefit as measured by the percentage of patients presenting progression-free survival (PFS) on matched therapy (PFS2) 1.3-fold longer than the PFS on prior therapy (PFS1). A total of 1,035 adult patients were included, and a biopsy was performed in 948. An actionable molecular alteration was identified in 411 of 843 patients with a molecular portrait. A total of 199 patients were treated with a targeted therapy matched to a genomic alteration. The PFS2/PFS1 ratio was >1.3 in 33% of the patients (63/193). Objective responses were observed in 22 of 194 patients (11%; 95% CI, 7%-17%), and median overall survival was 11.9 months (95% CI, 9.5-14.3 months).Significance: This study suggests that high-throughput genomics could improve outcomes in a subset of patients with hard-to-treat cancers. Although these results are encouraging, only 7% of the successfully screened patients benefited from this approach. Randomized trials are needed to validate this hypothesis and to quantify the magnitude of benefit. Expanding drug access could increase the percentage of patients who benefit. Cancer Discov; 7(6); 586-95. ©2017 AACR.See related commentary by Schram and Hyman, p. 552This article is highlighted in the In This Issue feature, p. 539.