F. Fina

Reliability and discriminant validity of HER2 gene quantification and chromosome 17 aneusomy analysis by real-time PCR in primary breast cancer

There is an increasing demand for the evaluation of HER2 status in breast cancer. In this study, sections from fixed tissues and triton extracts of tissue homogenates were obtained from 163 malignant breast tumors and analyzed in parallel using immunohistochemistry combined with fluorescence in situ hybridization, as gold standard tests, and an ELISA test (c-erbB2/c-neu Rapid Format ELISA, Oncogene Research Products, USA). Tumor DNA was employed to evaluate two quantitative PCR methods: the HER2/neu DNA Quantification Kit (Roche Diagnostics GmbH, Germany), which uses the gastrin chromosome 17 reference gene, and our recently developed Oncolab qPCR assay, where both a chromosome 17 gene (somatostatin receptor type II (SSTR2)) and a non-chromosome 17 reference gene (glyceraldehyde-3-phosphate deshydrogenase (GAPDH)) were used to detect an increase in HER2 gene copy number and to evaluate the aneusomy of chromosome 17, respectively. By IHC/FISH and ELISA, HER2 was overexpressed in 27 (16.6%) and 24 (14.7%) samples, respectively. With the Roche and Oncolab qPCR assays, 29 (17.8%) samples showed a ratio of HER2/gastrin > or = 2.0 and 26 (16.0%) showed a ratio of HER2/SSTR2 > or = 2.0, respectively. In samples presenting HER2/SSTR2 <2.0 and HER2/GAPDH > or = 2.0, which was indicative of a chromosome 17 polysomy, we observed a modest increase in HER2 protein expression. Complete agreement between the four methods for HER2 status determination was obtained for 154 (94.5%) samples. Overall, these results demonstrate that quantitative PCR is a reliable method for analyzing HER2 status and chromosome 17 polysomy.

McCune-Albright Syndrome: A Detailed Pathological And Genetic Analysis of Disease Effects in an Adult Patient.

CONTEXTnMcCune Albright syndrome (MAS) is a clinical association of endocrine and nonendocrine anomalies caused by postzygotic mutation of the GNAS1 gene, leading to somatic activation of the stimulatory α-subunit of G protein (Gsα). Important advances have been made recently in describing pathological characteristics of many MAS-affected tissues, particularly pituitary, testicular, and adrenal disease. Other rarer disease related features are emerging.nnnOBJECTIVEnThe objective of the investigation was to study the pathological and genetic findings of MAS on a tissue-by-tissue basis in classically and nonclassically affected tissues.nnnDESIGNnThis was a comprehensive autopsy and genetic analysis.nnnSETTINGnThe study was conducted at a tertiary referral university hospital.nnnPATIENTSnAn adult male patient with MAS and severe disease burden including gigantism was the subject of the study.nnnINTERVENTION(S)nInterventions included clinical, hormonal, and radiographic studies and gross and microscopic pathology analyses, conventional PCR, and droplet digital PCR analyses of affected and nonaffected tissues.nnnMAIN OUTCOME MEASUREnPathological findings and the presence of GNAS1 mutations were measured.nnnRESULTSnThe patient was diagnosed with MAS syndrome at 6 years of age based on the association of café-au-lait spots and radiological signs of polyostotic fibrous dysplasia. Gigantism developed and hyperprolactinemia, hypogonadotropic hypogonadism, and hyperparathyroidism were diagnosed throughout the adult period. The patient died at the age of 39 years from a pulmonary embolism. A detailed study revealed mosaiscism for the p.R201C GNAS1 mutation distributed across many endocrine and nonendocrine tissues. These genetically implicated tissues included rare or previously undescribed disease associations including primary hyperparathyroidism and hyperplasia of the thymus and endocrine pancreas.nnnCONCLUSIONSnThis comprehensive pathological study of a single patient highlights the complex clinical profile of MAS and illustrates important advances in understanding the characteristics of somatic GNAS1-related pathology across a wide range of affected organs.

Paleogenetic study of ancient DNA suggestive of X-Linked acrogigantism

Pituitary gigantism is caused by chronic growth hormone (GH) hypersecretion by a pituitary lesion before epiphyseal fusion. Genetic causes have been identified in nearly 50% of patients with pituitary gigantism, with germline mutations in the AIP gene being the most frequent cause (Rostomyan et al. 2015). Recently, a new form of pituitary gigantism, X-linked acrogigantism (X-LAG), was described (Trivellin et al. 2014). X-LAG is due to chromosome Xq26.3 duplication, and GPR101 is the disease-associated gene (Trivellin et al. 2014, Iacovazzo et al. 2016). X-LAG is characterized by mixed GH/prolactin-secreting pituitary macroadenomas and/or hyperplasia in early childhood (Beckers et al. 2015). X-LAG typically occurs sporadically in females, but somatic mosaicism also occurs in males; familial mother-to-son transmission of the Xq26.3 duplication has been reported in three familial isolated pituitary adenoma families (Trivellin et al. 2014, Daly et al. 2016, Gordon et al. 2016, Iacovazzo et al. 2016). The clinical presentation of X-LAG syndrome differs from other genetic forms of pituitary gigantism (Rostomyan et al. 2015), and many well-known historical cases of gigantism share the clinical characteristics of X-LAG syndrome (Beckers et al. 2015, Rostomyan et al. 2015). If untreated during childhood, X-LAG leads to established extreme gigantism (>1.9 m) before puberty (Daly et al. 2016). We studied a historical case of severe acrogigantism. The subject, J.K., was born in 1872 in Reutlingen, in what is now Baden-Württemberg, Germany. His parents and brother were of normal size. It was reported by his doctor that J.K. had always been ‘very large’ and he was reputed to have a huge appetite; he measured 1.94 m at the age of 14 and never stopped growing thereafter (Launois & Roy 1904). In contemporary Württemberg, the average adult male height was only 164 cm. By the 1890s he was exhibiting himself as Giant Constantine/Le Geant Constantin (Fig. 1A). In 1898, he was 259 cm in height (8 feet 6 inches) and weighted 168 kg (370 pounds). He fell ill while in the Walloon region of Belgium and was hospitalized on November 15, 1901, at the Hôpital Civil in Mons, Belgium with a fever (39.3°C) due to severe lower limb gangrene. Hospital records show his height as 256 cm and weight as 180 kg. He improved initially after an amputation of the right leg, but the following year, he fell and the other leg was amputated below the knee. He developed post-operative septicemia and died on March 30, 1902. At autopsy, the pituitary was grossly enlarged to the size of ‘a large walnut’ (Launois & Roy 1904). The sella turcica was also greatly enlarged, so much so that it was remarked that ‘after removing the cerebral hemispheres and the cerebellum, the sella was so broad and deep that it brought to mind two juxtaposed spinal canals’ (Launois & Roy 1904) (Fig. 1B and C). The long bones and extremities were elongated, and the proximal humeral epiphyses remained unfused (Launois & Roy 1904). Concomitant hypogonadism (testicular atrophy) was present on examination and post-mortem. Current forensic analysis of the skeleton demonstrates bleaching of the bones consistent with reported preservation by prolonged boiling. Given the clinical history of early-onset acrogigantism, an underlying genetic cause was thought to be likely. DNA extraction from teeth was unreliable as the skull was edentulous when originally photographed in 1904 (Launois & Roy 1904); subsequently, teeth were added to the skull but they could not be confirmed as having been obtained from the subject himself. The skeleton was fragile after preservation by prolonged boiling, and DNA extractions from a metatarsal and the femur were unsuccessful. Based on results obtained from ancient skeletal remains, tissue from the cochlea was obtained via the petrous temporal bone (Pinhasi et al. 2015) as reported in Supplemental materials (see section on supplementary data given at the end of this article). His history of earlyonset, severe pituitary gigantism led us to suspect X-LAG (Trivellin et al. 2014, Daly et al. 2016). The DNA sample was assayed using a ddPCR technique as previously described (Daly et al. 2016). Briefly, the ddPCR compared copy number variations (CNV) at the GPR101 gene as compared with ZIC3, a gene that is not duplicated in X-LAG syndrome. Daly and coworkers recently showed this method was 24:2 Research Letter A Beckers et al. Historical XLAG syndrome

BIABooster: Online DNA Concentration and Size Profiling with a Limit of Detection of 10 fg/μL and Application to High-Sensitivity Characterization of Circulating Cell-Free DNA

We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/μL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces. Using a set of DNA ladders in the range of 100-1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of ∼20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA.

Bénéfice à l’évaluation moléculaire en routine pour les cancers bronchiques métastatiques

BACKGROUNDnEGFR tyrosine kinase inhibitors and crizotinib are nowadays the optimal treatment for metastatic lung cancer with activation of EGFR mutations and ALK rearrangement. In addition, several targeted agents are in development for lung cancer with other oncodrivers. In France, since 2011, six oncodrivers are routinely tested in patients with stage IV. The aim of this study was to assess whether systematic detection of oncodrivers and matched targeted therapy improve overall survival in patients with advanced lung adenocarcinoma.nnnMETHODSnThis study included all consecutive patients treated in our department for advanced lung adenocarcinoma from January 2012 to December 2013. We studied the impact in survival according to the presence of the driver and the targeted therapy.nnnRESULTSnAmong the 261 patients included, oncodrivers alterations were found in 43.5% of patients: EML4-ALK fusion genes (2.1%), EGFR (10.3%), KRAS (27.7%), BRAF (2.5%), HER2 (0.8%), and PI3KCA (0.8%) mutations. Twenty-nine percent of patients (n=32) with oncodrivers received matched targeted therapy. Patient treated by targeted agent appropriate to an oncogenic driver had a median survival of 21.1 months (95% CI: 14.7-27.5). The patients (n=79) who did not receive targeted therapy had a median survival of 6.6 months (95% CI: 4.3-8.9). The patients (n=150) without identified driver had a median survival of 9.7 months (95% CI: 6.7-11.7); P<0.001.nnnCONCLUSIONnAn actionable oncodriver was routinely detected in nearly half of patients with advanced lung adenocarcinoma. This systematic detection may influence treatment outcomes, notably with matched targeted therapy.

Optimisation du délai d’analyse pour l’utilisation en routine de l’ADN tumoral circulant dans le cancer bronchique

Introduction Le traitement des CBNPC de stade IV est base sur les analyses genomiques de prelevements tissulaires qui peuvent etre difficiles a obtenir chez les sujets âges ou fragiles. L’ADN tumoral circulant semble etre une bonne alternative a la biopsie et les plateformes de biologie moleculaire commencent a l’utiliser en routine. La faible sensibilite de cette technique et la necessite de realiser les analyses dans les 3xa0h apres le prelevement en limitent son utilisation. Nous avons etudie la possibilite d’allonger ce delai a 24xa0h pour permettre aux patients un meilleur acces a cette technique. Methodes Les patients atteints de CBNPC de stade IV hospitalises dans notre unite et connus pour avoir une mutation de KRAS ont ete selectionnes et deux echantillons sanguins ont ete preleves et stockes a 4xa0°C. L’ADN tumoral circulant a ete extrait 3xa0h apres le prelevement pour le 1er echantillon et 24xa0h apres le prelevement pour le 2e echantillon. La mutation du gene KRAS a ete analysee par deux techniquesxa0: PCR-HRM et sequencage ou droplet digital PCR (ddPCR, technique plus sensible et permettant une quantification du nombre de copies d’ADN). Resultats Vingt et un patients ont ete preleves. Il n’y avait pas de difference significative du nombre de copies d’ADN extraites a 3xa0h et 24xa0h du prelevement (pxa0=xa00.91). Avec la technique conventionnelle, la mutation de KRAS a ete retrouvee chez 7 patients (33xa0%) a 3xa0h et 24xa0hxa0; avec la ddPCR, la mutation de KRAS a ete retrouvee chez 12 patients (57xa0%) a 3xa0h et 24xa0h. Les resultats etaient donc identiques a 3xa0h et 24xa0h du prelevement. Conclusion Cette etude montre qu’il est possible d’analyser l’ADN tumoral circulant 24xa0h apres le prelevement sans perte de sensibilite, permettant aux patients un meilleur acces a cette technique innovante, y compris lorsque la prise en charge n’est pas faite dans en CHU a proximite d’une plateforme de biologie moleculaire. De plus, l’utilisation de ddPCR augmente la sensibilite de cette technique par rapport aux techniques conventionnelles.

New oncogenes drivers in lung cancer—new therapeutic targets

Many advances have been achieved during the last decade in the field of lung cancer molecular biology, leading to the identification of potential new oncogene drivers and new therapeutic targets. However, only two targetable biomarkers are currently approved for lung cancer treatment: EGFR activating mutations and EML4-ALK rearrangements. Adenocarcinoma is the most common type of non-small cell lung cancer and was the first to be studied. Indeed, most of lung adenocarcinoma biomarkers were identified several years ago. Nevertheless, new therapies targeting these biomarkers are still investigated. More recently, new oncogene drivers have been identified in the field of lung squamous cell carcinoma and small cell lung cancer, and new agents are being developed to target these biomarkers.

Abstract 5549: Recipient-donor contradictory genotype with impact on anticancer drug pharmacogenetics after liver transplant: A deadly gift

A 5-year old girl with liver transplant was scheduled for a therapy including high-dose cytarabine for her Burkitt lymphoma. Cytarabine pharmacogenetics focus on cytidine deaminase (CDA) , the polymorphic liver enzyme responsible for its detoxification. Preliminary double testing for her CDA status on DNA lymphocytes showed none of the germline polymorphisms usually associated with CDA deficiency (ie, 79A>C (K27Q), 208G>A), but surprisingly with a functional mild deficiency syndrom. Despite 30% reduction in cytarabine dosing, life-threatening toxicities showed quickly and treatment was discontinued. Genetic HRM-based investigations performed in the patient on liver biopsy failed in generating a clear genotype. Further HRM+sequencing retrospective investigations on liver biopsy collected from the donor showed that he was actually bearing the homozygous CDA*2 allelic variant (ie, 79AA), a genotype usually associated with severe CDA deficiency. Based upon the donor liver genotype, and not the recipient genotype, treatement was resumed with further dose reduction, close monitoring and better tolerance eventually. The patient is now in complete remission more than a year after completion of the treatment. This case report illustrates the limits and risks of searching germline polymorphisms in patients with liver transplant when the story plays in the liver. Citation Format: Cindy Serdjebi, Raphaelle Fanciullino, Frederic Fina, Arnaud Verschuur, Bertrand Roquelaure, Bruno Lacarelle, L9Houcine Ouafik, Joseph Ciccolini, Nicolas Andre. Recipient-donor contradictory genotype with impact on anticancer drug pharmacogenetics after liver transplant: A deadly gift. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5549. doi:10.1158/1538-7445.AM2014-5549