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The Eastern woodchuck (Marmota monax) has been extensively used in research of chronic hepatitis B and liver cancer because its infection with the woodchuck hepatitis virus closely resembles a human hepatitis B virus infection. Development of novel immunotherapeutic approaches requires genetic information on immune pathway genes in this animal model. The woodchuck genome was assembled with a combination of high-coverage whole-genome shotgun sequencing of Illumina paired-end, mate-pair libraries and fosmid pool sequencing. The result is a 2.63 Gigabase (Gb) assembly with a contig N50 of 74.5 kilobases (kb), scaffold N50 of 892 kb, and genome completeness of 99.2%. RNA sequencing (RNA-seq) from seven different tissues aided in the annotation of 30,873 protein-coding genes, which in turn encode 41,826 unique protein products. More than 90% of the genes have been functionally annotated, with 82% of them containing open reading frames. This genome sequence and its annotation will enable further research in chronic hepatitis B and hepatocellular carcinoma and contribute to the understanding of immunological responses in the woodchuck.

Focused interviews were conducted with global pharmaceutical company representatives in order to derive a consistent view on drug target identification/validation challenges, collaborative strategies, and future developments in a precompetitive space. Analysis revealed translation into clinical utility as a major hurdle of novel drug target validation, originating from lack of biological understanding, irreproducibility of published results, and lack of valid animal models. Direct and close collaborations with academia are the preferred model to tackle basic research on novel drug targets in high-risk projects. Efforts to conduct target identification in large precompetitive consortia are acknowledged with some doubts about the pace of progress and data-sharing policies, while concept to extend the precompetitive space to target validation in phase II trials was curtailed to niche indications together with a revision of current intellectual property (IP) practice. Public-private partnerships in established areas are forecasted to increase. Novel emerging themes are toxicology data sharing, joint genetic patient data analysis, and reimbursement concepts.

Introduction Exon 19 deletions and the exon 21 L858R mutation of the epidermal growth factor receptor gene ( EGFR ) predict activity of EGFR tyrosine kinase inhibitors, including erlotinib; however, the ability of less common EGFR mutations to predict efficacy of erlotinib is unclear. Methods The efficacy of erlotinib in individual patients with rare EGFR mutations from the MERIT, SATURN, TITAN, TRUST, ATLAS, BeTa, and FASTACT-2 trials was analyzed and compared with data from the literature. Results In the patients tested for biomarkers, the frequency of rare mutations identified here ranged from 1.7% (eight of 467) in the SATURN study to 7.4% (27 of 364) in ATLAS. Some rare mutations were associated with greater clinical benefit from EGFR tyrosine kinase inhibitor therapy or improved prognosis independent of treatment, whereas others appeared to have a poorer prognosis. In particular, exon 18 G719 mutations, exon 19 K757R and E746G mutations, the exon 20 S768I mutation, and the exon 21 G836S mutation appeared to confer a good outcome with erlotinib treatment, whereas exon 18 S720I showed a particularly poor outcome. Owing to the small number of patients with each mutation, however, it is difficult to confirm whether these rare mutations do indeed confer sensitivity or resistance to erlotinib. Conclusions Erlotinib can have different efficacy depending on the specific EGFR mutation. More research is needed to create a central database such as the My Cancer Genome database of rare mutations to definitively confirm whether these mutations are activating, resistant, or neutral.

Background Preliminary reports suggested that circulating tumor DNA (ctDNA) can be used as a prognostic marker in a way akin to circulating tumor cells (CTC) in metastatic breast cancer patients. However ctDNA detection is often performed on multiple mutations, combining heterogeneous techniques. Here we used the high prevalence of TP53 mutations in triple-negative metastatic breast cancer (TNMBC) to compare CTC and ctDNA detection rates and prognostic value. Methods A cohort of 40 patients treated at the Institut Curie (Paris, France) was enrolled before starting a new line of treatment for TNMBC. CTC were detected by the CellSearch system (in 7.5 mL of blood). Using massively parallel sequencing (NGS), TP53 mutations were first characterized in tumor tissue, then in plasma DNA extracted from fresh frozen plasma samples (from 15-20 mL of blood). ctDNA detection was performed using high depth targeted sequencing using two platforms in parallel (Illumina HiSeq 2500 and Roche 454). Libraries for Illumina were prepared following the TAm-Seq procedure (Forshew et al, Sci Transl Med 2012), with preamplification of all coding TP53 exons and flanking untranslated regions followed by both paired-end 150bp Illumina and 454/Roche sequencing. CTC, ctDNA and usual patient characteristics were correlated with time to progression (TTP) and overall survival (OS). Results Archived tumor (FFPE or frozen) tissue was available for 36 patients, and 31 were successfully sequenced: TP53 mutations were found in 27 patients. As measured on the Illumina platform, ctDNA was detected in 21/27 patients (81%), ranging from 48 to 648,000 copies/mL of plasma (median 1620). Mutant allele fraction in circulating cell-free DNA ranged from 2 to 70% (median 5%). Comparison between ctDNA levels measured by Illumina and 454/Roche platforms in plasma displayed a good correlation (R2 = 0.903), with a single discordance. ≥1 CTC were detected in 19 of these 27 patients (70%). Strikingly, high ctDNA levels had prognostic impact neither on OS, nor on TTP, whatever the dataset used (Illumina or 454) whereas CTC≥5/7.5 mL were correlated with OS (p=0.04) and marginally with TTP (p=0.06). Other known usual factors, such as poor performance status, elevated LDH and number of previous treatment lines had also significant prognostic factors in this cohort. CTC and ctDNA early changes during treatment were available for 12 patients and changes (increase/decrease) of the two biomarkers were globally similar. Conclusion Demonstrating a good sensitivity (81%), ctDNA by the TAm-Seq is more frequently detected than CTCs in the 27 TNMBC with TP53 mutations. The observed correlation between the 2 massively parallel sequencing approaches suggested that ctDNA levels data were quantitative. In contrast to other usual prognostic factors, baseline ctDNA level did not demonstrate a prognostic impact,in this proof-of-principle study, suggesting that mechanisms of ctDNA release in TNMBC rely on biological features that do not dramatically impact patient’s outcome. Citation Format: Francois-Clement Bidard, Jordan Madic, Anna Kiialainen, Fabian Birzele, Guillemette Ramey, Quentin Leroy, Thomas Rio Frio, Virginie Raynal, Virginie Bernard, Alban Lermine, Inga Clausen, Nicolas Giroud, Roland Schmucki, Carsten Horn, Olivia Spleiss, Olivier Lantz, Marc-Henri Stern, Martin Weisser, Ronald Lebofsky, Jean-Yves Pierga. Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-8.

RO5263397 [(S)-4-(3-fluoro-2-methyl-phenyl)-4,5-dihydro-oxazol-2-ylamine], a new compound that showed promising results in animal models of schizophrenia, is mainly metabolized in humans by N-glucuronidation. Enzyme studies, using the (then) available commercial uridine 5'-diphosphate-glucuronosyltransferases (UGTs), suggested that UGT1A4 is responsible for its conjugation. In the first clinical trial, in which RO5263397 was administered orally to healthy human volunteers, a 136-fold above-average systemic exposure to the parent compound was found in one of the participants. Further administration in this trial identified two more such poor metabolizers, all three of African origin. Additional in vitro studies with recombinant UGTs showed that the contribution of UGT2B10 to RO5263397 glucuronidation is much higher than UGT1A4 at clinically relevant concentrations. DNA sequencing in all of these poor metabolizers identified a previously uncharacterized splice site mutation that prevents assembly of full-length UGT2B10 mRNA and thus functional UGT2B10 protein expression. Further DNA database analyses revealed the UGT2B10 splice site mutation to be highly frequent in individuals of African origin (45%), moderately frequent in Asians (8%) and almost unrepresented in Caucasians (1%). A prospective study using hepatocytes from 20 individual African donors demonstrated a100-fold lower intrinsic clearance of RO5263397 in cells homozygous for the splice site variant allele. Our results highlight the need to include UGT2B10 when screening the human UGTs for the enzymes involved in the glucuronidation of a new compound, particularly when there is a possibility of N-glucuronidation. Moreover, this study demonstrates the importance of considering different ethnicities during drug development.

Checkpoint inhibitor therapy has been a breakthrough in cancer research, but only some patients with cancer derive substantial benefit. Although mechanisms underlying sensitivity and resistance to checkpoint inhibitors are being elucidated, the importance of organ-specific regulation of immunity is currently underappreciated. Here, we call for a greater understanding of tissue-specific immunoregulation, namely, “tissue-specific immunostats,” to make advances in treatments for cancer. A better understanding of how individual organs at baseline regulate the immune system could enable an improved precision medicine approach to cancer immunotherapy. Cancer Discov; 8(4); 395–402. ©2018 AACR.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R2 = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

Purpose To assess pharmacodynamic effects and intrinsic and acquired resistance mechanisms of the BRAF inhibitor vemurafenib in BRAFV600-mutant melanoma, leading to an understanding of the mechanism of action of vemurafenib and ultimately to optimization of metastatic melanoma therapy. Methods In the phase II clinical study NP22657 (BRIM-2), patients received oral doses of vemurafenib (960 mg twice per day). Serial biopsies were collected to study changes in mitogen-activated protein kinase (MAPK) signaling, cell-cycle progression, and factors causing intrinsic or acquired resistance by immunohistochemistry, DNA sequencing, or somatic mutation profiling. Results Vemurafenib inhibited MAPK signaling and cell-cycle progression. An association between the decrease in extracellular signal-related kinase (ERK) phosphorylation and objective response was observed in paired biopsies (n = 22; P = .013). Low expression of phosphatase and tensin homolog showed a modest association with lower response. Baseline mutations in MEK1P124 coexisting with BRAFV600 were noted in seven of 92 samples; their presence did not preclude objective tumor responses. Acquired resistance to vemurafenib associated with reactivation of MAPK signaling as observed by elevated ERK1/2 phosphorylation levels in progressive lesions and the appearance of secondary NRASQ61 mutations or MEK1Q56P or MEK1E203K mutations. These two activating MEK1 mutations had not previously been observed in vivo in biopsies of progressive melanoma tumors. Conclusion Vemurafenib inhibits tumor proliferation and oncogenic BRAF signaling through the MAPK pathway. Acquired resistance results primarily from MAPK reactivation driven by the appearance of secondary mutations in NRAS and MEK1 in subsets of patients. The data suggest that inhibition downstream of BRAF should help to overcome acquired resistance.

Background. Vemurafenib has been approved for the treatment of patients with advanced BRAFV600E-mutant melanoma. This report by the Vemurafenib Dermatology Working Group presents the characteristics of dermatologic adverse events (AEs) that occur in vemurafenib-treated patients, including cutaneous squamous cell carcinoma (cuSCC). Methods. Dermatologic AEs were assessed from three ongoing trials of BRAFV600E mutation-positive advanced melanoma. Histologic central review and genetic characterization were completed for a subset of cuSCC lesions. Results. A total of 520 patients received vemurafenib. The most commonly reported AEs were dermatologic AEs, occurring in 92%–95% of patients. Rash was the most common AE (64%–75% of patients), and the most common types were rash not otherwise specified, erythema, maculopapular rash, and folliculitis. Rash development did not appear to correlate with tumor response. Photosensitivity occurred in 35%–63% of patients, and palmar-plantar erythrodysesthesia (PPE) occurred in 8%–10% of patients. The severity of rash, photosensitivity, and PPE were mainly grade 1 or 2. In all, 19%–26% of patients developed cuSCC, mostly keratoacanthomas (KAs). The majority of patients with cuSCC continued therapy without dose reduction after resection. Genetic analysis of 29 cuSCC/KA samples demonstrated HRAS mutations in 41%. Conclusions. Dermatologic AEs associated with vemurafenib treatment in patients with melanoma were generally manageable with supportive care measures. Dose interruptions and/or reductions were required in

Background Tuberous sclerosis complex (TSC) is a genetic disease characterized by benign tumor growths in multiple organs and neurological symptoms induced by mTOR hyperfunction. Because the molecular pathology is highly complex and the etiology poorly understood, we employed a defined human neuronal model with a single mTOR activating mutation to dissect the disease-relevant molecular responses driving the neuropathology and suggest new targets for treatment. Methods We investigate the disease phenotype of TSC by neural differentiation of a human stem cell model that had been deleted for TSC2 by genome editing. Comprehensive genomic analysis was performed by RNA sequencing and ribosome profiling to obtain a detailed genome-wide description of alterations on both the transcriptional and translational level. The molecular effect of mTOR inhibitors used in the clinic was monitored and comparison to published data from patient biopsies and mouse models highlights key pathogenic processes. Results TSC2-deficient neural stem cells showed severely reduced neuronal maturation and characteristics of astrogliosis instead. Transcriptome analysis indicated an active inflammatory response and increased metabolic activity, whereas at the level of translation ribosomal transcripts showed a 5’UTR motif-mediated increase in ribosome occupancy. Further, we observed enhanced protein synthesis rates of angiogenic growth factors. Treatment with mTOR inhibitors corrected translational alterations but transcriptional dysfunction persisted. Conclusions Our results extend the understanding of the molecular pathophysiology of TSC brain lesions, and suggest phenotype-tailored pharmacological treatment strategies. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0347-3) contains supplementary material, which is available to authorized users.

Dopazo, Joaquín et al., Recent results from large-scale genomic projects suggest that allele frequencies, which are highly relevant for medical purposes, differ considerably across different populations. The need for a detailed catalog of local variability motivated the whole-exome sequencing of 267 unrelated individuals, representative of the healthy Spanish population. Like in other studies, a considerable number of rare variants were found (almost one-third of the described variants). There were also relevant differences in allelic frequencies in polymorphic variants, including ~10,000 polymorphisms private to the Spanish population. The allelic frequencies of variants conferring susceptibility to complex diseases (including cancer, schizophrenia, Alzheimer disease, type 2 diabetes, and other pathologies) were overall similar to those of other populations. However, the trend is the opposite for variants linked to Mendelian and rare diseases (including several retinal degenerative dystrophies and cardiomyopathies) that show marked frequency differences between populations. Interestingly, a correspondence between differences in allelic frequencies and disease prevalence was found, highlighting the relevance of frequency differences in disease risk. These differences are also observed in variants that disrupt known drug binding sites, suggesting an important role for local variability in population-specific drug resistances or adverse effects. We have made the Spanish population variant server web page that contains population frequency information for the complete list of 170,888 variant positions we found publicly available (http://spv.babelomics.org/), We show that it if fundamental to determine population-specific variant frequencies to distinguish real disease associations from population-specific polymorphisms. © 2016 The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution., The MGP is a joint initiative between the Consejería de Salud de la Junta de Andalucía and Roche, supported by the “Programa Nacional de Proyectos de investigación Aplicada,” I+D+i 2008, “Subprograma de actuaciones Científicas y Tecnológicas en Parques Científicos y Tecnológicos” (ACTEPARQ 2009), and European Regional Development Funds (ERDF). This work is also supported by grants BIO2014-57291-R and BFU2012-38236 from the Spanish Ministry of Economy and Competitiveness and “Plataforma de Recursos Biomoleculares y Bioinformáticos” PT 13/0001/0030 from the ISCIII, both cofunded with ERDF; grants PI1102923 and PI1001290 from the Fondo de Investigación Sanitaria, PROMETEOII/2014/025 from the Generalitat Valenciana (GVA-FEDER), FP7-PEOPLE-2012-ITN MLPM2012 318861 from the EU FP7, Fundació la Marató TV3 [20133134], and by Direcció General de Recerca, Generalitat de Catalunya (2014SGR1311). The CIBER de Enfermedades Raras is an Instituto de Salud Carlos III initiative.

The accuracy of common markers for PI3K/AKT and MAPK pathway activation in preclinical and clinical cancer biomarker studies depends on phosphoepitope stability and changes of phosphorylation under ischemia. Herein, we define conditions under which phosphoepitope-specific duplex immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tumor tissues reflects pathway activation in situ as accurately as possible, and identify activation patterns linked to mutational status, pathway dependency and tumor microenvironment in clinical tumor samples, cell culture and xenograft tissues. Systematically assessing robustness of pAKT, pERK1/2, pMEK1/2 and pmTOR detection and related markers in xenograft tissues exposed to ischemia, we show that control of preprocessing and ischemia times allows accurate interpretation of staining results. Phosphorylation patterns were then analyzed in 33 xenograft models and in 58 cases with breast cancer, including 21 paired samples of core-needle biopsies with corresponding mastectomy specimens, and 37 mastectomy samples obtained under rigorously controlled conditions minimizing ischemia time. Patterns of pAKT and pERK1/2 staining (predominant PI3K/AKT, predominant MAPK and concomitant activation) were associated with sensitivity to pathway inhibition and partially with the mutational status in cell lines and corresponding xenograft tumors. In contrast, no clear correlation between mutational status and staining patterns was observed in clinical breast cancer samples, suggesting that interaction with the human tumor microenvironment may interfere with the use of phosphoepitope-specific IHC as potential markers for pathway dependency. In contrast to core needle biopsies, surgically resected breast cancer samples showed evidence of severe signal changes comparable to those effects observed in xenograft tumors exposed to controlled ischemia.

Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors.We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed.Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor.Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

Tocilizumab, a monoclonal antibody to interleukin-6 receptor, was recently approved for the treatment of moderate-to-severe rheumatoid arthritis. Two patients during clinical development met laboratory, but not clinical, criteria for Hy's law with bilirubin elevations suspected as a result of genetic variation in uridine diphosphoglucose glucuronosyltransferase (UGT1A1) typical of Gilbert syndrome.Genotyping of the two cases potentially meeting with Hy's law was performed using commercially available procedures. UGT1A1 single nucleotide polymorphism data were extracted from a genome-wide array database for 1187 patients from tocilizumab trials, and associations of UGT1A1 genotypes with bilirubin elevations were analyzed using logistic regression for associations with baseline and change from baseline in bilirubin levels as continuous variables.Bilirubin elevations were not associated with clinical adverse events. Both patients potentially meeting Hy's law carry homozygous UGT1A1*28 alleles and UGT1A1*60 alleles. UGT1A1*28 and three additional single nucleotide polymorphisms showed odds ratios greater than 25 for associations with elevated bilirubin. The presence of rs6742078 accounted for 32% of the total variance in bilirubin (P=2.2×10).Bilirubin increases occurring with tocilizumab appear to be related to anti-inflammatory effects extending to the liver. Thus, in the absence of other signs of hepatic dysfunction, bilirubin elevations after treatment with tocilizumab have a high probability of association with UGT1A1 polymorphism, which should alleviate concerns of serious hepatotoxicity. Our results underscore the value of genotyping in the clinical trial setting to avoid misinterpretations that could lead to terminating development of a promising new agent.

Heterogeneity in the underlying mechanisms of disease processes and inter-patient variability in drug responses are major challenges in drug development. To address these challenges, biomarker strategies based on a range of platforms, such as microarray gene-expression technologies, are increasingly being applied to elucidate these sources of variability and thereby potentially increase drug development success rates. With the aim of enhancing understanding of the regulatory significance of such biomarker data by regulators and sponsors, the US Food and Drug Administration initiated a programme in 2004 to allow sponsors to submit exploratory genomic data voluntarily, without immediate regulatory impact. In this article, a selection of case studies from the first 5 years of this programme - which is now known as the voluntary exploratory data submission programme, and also involves collaboration with the European Medicines Agency - are discussed, and general lessons are highlighted.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R(2) = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

Glucocorticoid-attenuated response genes (GARG) belong to a recently described family of genes responsive to the action of dexamethasone. Full-length cDNA of one member of this family, GARG16, has been cloned from rat microglia and regulation of its mRNA expression has been studied. Moreover, regulation of retinoid/retinoic acid activated transcription factor (RXR/RAR) mRNAs in mixed astrocyte and in purified microglia cultures has been investigated. RARbeta mRNA was undetectable in microglia by RT-PCR, whereas clearly present in the mixed cultures. RXRalpha, RARgamma, and GARG16 mRNAs were found in both culture systems. RXRalpha mRNA was strongly expressed in control microglia but rapidly declined upon treatment with LPS. Conversely, GARG16 mRNA was almost untraceable in control microglia but rapidly increased by LPS. Time-course studies revealed an oscillating behavior of expression of both mRNAs during the first 6 hr, which receded to control levels (RXRalpha high, GARG16 low) at 72 hr of LPS-treatment. Additionally, p38 MAPK and SEK phosphorylations peaked at 1 hr followed by steady declines, whereas MEK and c-Jun showed double peaks at 1+4 hr and 1+6 hr, respectively, before subsiding to control levels. This behavior was not observed in comparative studies with TNF-alpha, interleukin-10 (IL-10), or interferon-gamma inducible protein 10 (IP-10). Finally, inhibitors of p38 MAPK, p42/p44 ERK, and PKCalpha as well as the use of dexamethasone revealed major influences of the p38 MAPK-c-Jun-AP-1 signaling pathway on RXRalpha and GARG16 mRNA expressions. The counter regulatory control of GARG16 and RXRalpha mRNA expression is believed to be an example of a fine-tuned cellular mechanism to react to inflammatory stimuli.

We have investigated the functional expression of the beta-chemokine receptors CCR1 to 5 in cultured rat microglia. RT-PCR analysis revealed constitutive expression of CCR1, CCR2 and CCR5 mRNA. The beta-chemokines MCP-1 (1-30 nM) as well as RANTES and MIP-1alpha (100-1000 nM) evoked calcium transients in control and LPS-treated microglia. Whereas, the response to MCP-1 was dependent on extracellular calcium the response to RANTES was not. The effect of MCP-1 but not that of RANTES was inhibited by the calcium-induced calcium release inhibitor ryanodine. Calcium responses to MCP-1- and RANTES were observed in distinct populations of microglia.

Disturbances of events associated with intracellular signaling pathways have been suspected of involvement in the development or progression of affective disorders. Often, heterotrimeric G proteins are located at the beginning of these pathways as modulators of extracellular messages. For this reason, messenger RNA expression of three G protein alpha-subunits and of phosphatidylinositol-3 kinase (PI-3 K) regulatory subunit p85 was examined in granulocytes from patients with bipolar or unipolar affective disorder and compared to healthy controls. Messenger RNA expression of the G protein subunit alpha(q) and of p85 was identical in unipolar and bipolar patients and in controls. Furthermore, mRNAs of G protein subunits alpha(s) and alpha(i2) were not different in unipolar patients as compared to healthy controls. Alpha(s) mRNA, however, was markedly increased in bipolar patients. This increase was observed in lithium-treated (more than 12 months) and in unmedicated patients. Elevated levels of alpha(i2) mRNA in unmedicated bipolar patients did not reach statistical significance, whereas mRNA in bipolar patients receiving lithium was significantly above controls. Finally, long-term medication of unipolar patients with lithium had no influence on alpha(i2) mRNA levels. The data reveal elevated mRNA levels of G alpha(s) as a robust feature of bipolar affective disorder. Moreover, despite responsiveness of alpha(i2) gene expression to cAMP-related events, no substantial upregulation of alpha(i2) mRNA was observed in bipolar patients. The lack of alpha(i2) mRNA upregulation, hence, could be an additional abnormality in these patients. Even though lithium was able to reinstate this upregulation, there was no feedback downregulation of alpha(s). This strongly supports the notion of major disturbances of the cAMP signaling system in bipolar illness.

Chemokine receptors play a crucial role in the recruitment of immune cells to sites of inflammation. Although chronic diseases of the brain are often accompanied by inflammatory events, there is presently no information about the occurrence and regulation of these receptors in the central nervous system (CNS), Moreover, one CC-chemokine receptor, CKR5, has recently been identified as coreceptor for HIV-1 entry into macrophages, HIV-1 target cells in brain are macrophage-related microglia, which suggests that they are infected by the same mechanism (He et al,,: Nature 385:645-649, 1997), Although rats are not susceptible to HIV-1 infection, they can be used to study chemokine receptor regulation in a variety of brain pathologies. After cloning CC-CKR5 and establishing reverse transcriptase polymerase chain reaction (RT-PCR) for its ligands macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and regulated on activation, normal T cell-expressed and secreted (RANTES), we studied expression of these four mRNAs in purified microglia and compared it with their expression in rat brain, Lipopolysaccharide (LPS)-treated microglia showed transiently increased mRNA levels of both CKR5 and its ligands, Similar data were obtained from brains of LPS-injected rats, In middle cerebral artery occluded (MCAO)-animals, RANTES mRNA was unaffected, whereas CKR5 mRNA showed a sustained rise until 96 hr after surgery, MIPs exogenously added to microglial cultures markedly reduced CKR5 mRNA expression, whereas RANTES did not, MIP mRNAs, in contrast to RANTES and CKR5 mRNAs, were undetectable in normal brain, RANTES appears to play a role distinct from MIPs in brain, In summary, upregulation of CC-chemokines and CKR5 in the CNS upon bacterial infection or in ischemia may impact on microglial activation stage and result in increased risk of HIV-1 infection, J, Neurosci, Res. 53:16-28, 1998 (C) 1998 Wiley Liss, Inc.

Currently there are multiple approaches for measuring gene expression from human tissue samples on the market. These methods range from measuring the expression of a few genes using quantitative RT-PCR to measuring every gene in a sample by whole transcriptome RNA sequencing (RNA-Seq). Although many techniques and products for measuring gene expression are available, the highly degraded RNA isolated from formalin-fixed, paraffin embedded (FFPE) tumor tissues still poses a challenge for many of them. FFPE tumor samples are an abundant source of biomarker information and gene expression analysis in these samples has the potential to identify new signatures, biomarkers, or diagnostics that could predict patient response to treatment. Accurately measuring gene expression in a high throughput manner from this sample type has long been a struggle in the field. To determine the ability of RNA-Seq to accurately measure gene expression from FFPE-derived RNA, we utilized a large collection of matched fresh frozen (FF) and FFPE samples from 12 different tumor indications. By comparing the results to the matching FF sample, we were able to determine the accuracy and sensitivity of each platform when using degraded FFPE-derived RNA. The FFPE samples in the collection had a wide range of RNA quality scores (RIN score: 0-6.8, DV200:0-78) representing what is typically isolated from clinical trial samples. We further compared whole transcriptome RNA-Seq with a hybrid capture method, RNA Access, to determine the accuracy and feasibility of using this technology on degraded RNA. RNA Access selects for protein coding transcripts by hybridization, whereas whole transcriptome RNA-seq removes ribosomal RNA, but analyzes everything else i.e. the global transcriptome. All samples were sequenced to an average of 50 or 25 million paired-end reads for whole transcriptome RNA-Seq and RNA Access, respectively and 100 ng of RNA was used as input in both assays. We conclude that both methods can be used for analyzing FFPE tumor samples. A similar dynamic range was observed for both methods and both show similar correlation between FF and FFPE samples within the method (0.76 vs 0.74). The overall concordance between methods was 0.69 and 0.63 for FF and FFPE, respectively. When looking specifically at the detection of 90 low expressing immune related genes, both assays were able to detect >94% with an inter-platform correlation of 0.66 and 0.56 in FF and FFPE, respectively. Whole transcriptome RNA-seq provides the maximum amount of information from clinical samples including anti-sense and non-coding RNA detection while RNA Access can be applied more cost-efficiently when one is mostly interested in the protein coding transcriptome. Citation Format: Erica B. Schleifman, Anna Kiialainen, Andreas Roller, Sabine Bader, Maipelo Motlhabi, Priti Hegde, Ian McCaffery, Garret Hampton, Michael Cannarile, Craig Cummings, Olivia Spleiss, Eric Peters. Whole transcriptome and exome targeted RNA sequencing for FFPE tumor samples from clinical trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3631.

Polymorphisms of activating Fc-γ receptors (FCGRs) on natural killer cells and macrophages result in variable affinity for immunoglobulin G1 monoclonal antibodies and subsequently modulate antibody-dependent cellular cytotoxicity (ADCC) activity. Whether single-nucleotide polymorphisms of FCGRs correlate with survival of chronic lymphocytic leukemia (CLL) patients treated with a monoclonal antibody containing regimen is unclear. We assessed the FCGR3A and FCGR2A genotype of patients enrolled in the REACH trial, where patients received fludarabine and cyclophosphamide (FC) or rituximab plus FC (R-FC). FCGR3A and FCGR2A polymorphisms did not demonstrate prognostic significance in the FC arm (P = .42 and P = .64, respectively) or R-FC arm (P = .41 and P = .88, respectively) with respect to progression free survival. Patients with intermediate affinity genotypes (FV and HR) benefited significantly from addition of rituximab (hazard ratio = 0.55 [0.37-0.8 CI]; P = .0017 and hazard ratio = 0.63 [0.44-0.9 CI]; P = .011, respectively). Similar benefit was suggested for patients with high- affinity VV and HH (hazard ratio = 0.86 [0.4-1.84 CI]; P = .7 and hazard ratio = 0.7 [0.41-1.18 CI]; P = .18, respectively) and low-affinity FF and RR (hazard ratio = 0.85 [0.56-1.29 CI]; P = .44 and hazard ratio = 0.82 [0.47-1.42 CI]; P = .48, respectively). Overall, our results suggest that FCGR2A and FCGR3A polymorphisms do not significantly influence the outcomes of relapsed or refractory CLL patients treated with FC or the monoclonal antibody regimen R-FC.

Heterogeneity in the underlying mechanisms of disease processes and inter-patient variability in drug responses are major challenges in drug development. To address these challenges, biomarker strategies based on a range of platforms, such as microarray gene-expression technologies, are increasingly being applied to elucidate these sources of variability and thereby potentially increase drug development success rates. With the aim of enhancing understanding of the regulatory significance of such biomarker data by regulators and sponsors, the US Food and Drug Administration initiated a programme in 2004 to allow sponsors to submit exploratory genomic data voluntarily, without immediate regulatory impact. In this article, a selection of case studies from the first 5 years of this programme - which is now known as the voluntary exploratory data submission programme, and also involves collaboration with the European Medicines Agency - are discussed, and general lessons are highlighted.

Renal transplant outcomes exhibit large inter-individual variability, possibly on account of genetic variation in immune-response mediators and genes influencing the pharmacodynamics/pharmacokinetics of immunosuppressants. We examined 21 polymorphisms from 10 genes in 237 de novo renal transplant recipients participating in an open-label, multicenter study [Cyclosporine Avoidance Eliminates Serious Adverse Renal-toxicity (CAESAR)] investigating renal function and biopsy-proven acute rejection (BPAR) with different cyclosporine A regimens and mycophenolate mofetil. Genes were selected for their immune response and pharmacodynamic/pharmacokinetic relevance and were tested for association with BPAR. Four polymorphisms were significantly associated with BPAR. The ABCB1 2677T allele tripled the odds of developing BPAR (OR: 3.16, 95% CI [1.50-6.67]; P = 0.003), as did the presence of at least one IMPDH2 3757C allele (OR: 3.39, 95% CI [1.42-8.09]; P = 0.006). BPAR was almost fivefold more likely in patients homozygous for IL-10 -592A (OR: 4.71, 95% CI [1.52-14.55]; P = 0.007) and twice as likely in patients with at least one A allele of TNF-alpha G-308A (OR: 2.18, 95% CI [1.08-4.41]; P = 0.029). There were no statistically significant interactions between polymorphisms, or the different treatment regimens. Variation in genes of immune response and pharmacodynamic/pharmacokinetic relevance may be important in understanding acute rejection after renal transplant. (Less)

The high-affinity sodium/myo-inositol cotransporter (SMIT) is involved in osmoregulation in several cells and tissues. In the CNS the activity of SMIT also determines the individual susceptibility of neural cells to the inositol depleting effect of lithium, which is considered to be important in lithium's therapeutic effects in manic-depressive illness. Among neural cells SMIT is particularly active in astrocytes. In the present work we have cloned the cDNA of SMIT of the rat and assessed its activity, expression and regulation in primary astroglia cultures derived from five different rat brain regions: cerebellum, cortex, diencephalon, hippocampus and tegmentum. After an incubation period of 24 h in medium containing 3[H]labeled myo-inositol different steady-state concentrations were detected which were dependent on the brain region from which the astrocytes were cultured. In addition, myo-inositol uptake in astrocytes from different areas was characterized by two different Km values (27 microM for cerebellum and diencephalon, 50 microM for cortex, hippocampus and tegmentum) and by three different v(max) values (approx. 200 pmol/mg protein/min for astrocytes from cerebellum and tegmentum, 298 for hippocampus and 465 for cortex), indicating that the active myo-inositol uptake into astroglial cells is distinct in the various brain regions. The efficacy of uptake as determined by v(max) values of 3[H]myo-inositol uptake correlated with the level of mRNA of SMIT in the astrocyte cultures from the various brain regions as determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Both 3[H]myo-inositol uptake and SMIT mRNA content was upregulated by incubation of astrocytes in medium of increased osmolarity. In astrocytes from cerebellum, cortex, hippocampus and tegmentum 3[H]myo-inositol uptake was downregulated by chronic incubation with 400 microM inositol. This effect was not observed in astrocytes from diencephalon. Furthermore, in astrocytes from cortex and hippocampus but not from cerebellum, diencephalon and tegmentum incubation with corticosterone for three days upregulated 3[H]myo-inositol uptake. It is concluded that SMIT is differentially expressed and regulated in astrocytes from distinct brain regions. These regional differences suggest particular consideration of localized effects in investigations of the role of myo-inositol in the mechanism of action of antibipolar drugs.

Activation of brain microglial cells can be subdivided into a number of stages. Early stages likely are proliferation and migration to sites of cell damage. These two stages have been studied exemplarily on the IL-3 receptor beta-subunit and on the CC-chemokine receptor 5 using molecular biological methods. First, IL-3 receptor beta-subunit cDNA has been cloned in full length from rat microglia. Since cultured microglia are already activated to some extent, mRNA of this subunit has been detected in the isolated cells, but was absent in normal rat brain. Lipopolysaccharide (LPS) increased this mRNA in the cultured cells and LPS injected into the circulation of rats induced the mRNA specifically in brain microglia as revealed by in situ hybridizations. Next, we obtained partial cDNAs of receptor-coupled protein tyrosine kinases JAK 1 and JAK 2. These mRNAs were present both in cultured microglia and in rat brain, but were not influenced by LPS. Finally, a full-length cDNA of the rat chemokine receptor 5 has been obtained by PCR methodology. Its mRNA was increased by administration of LPS both in cultured microglia and in vivo. It is expected, that further investigations on these receptors could help to develop improved strategies to combat chronic inflammatory events in the brain.

8503^ Background: Vem induces frequent clinical responses (RR >50%) and improved survival in patients (pts) with BRAF-mutated metastatic melanoma. Multiple mechanisms of escape from vem have been proposed. We performed a centralized analysis of pretreatment, cycle 1, day 15 (D15), and progression (DP) tumor samples collected during the phase II BRIM-2 trial (Sosman et al, NEJM 2012). Methods: Of 132 pts enrolled, archival tumor samples, biopsies taken at baseline (BL), D15, and DP were obtained from 84, 38, 26, and 22 pts, respectively, and analyzed by immunohistochemistry (IHC) for signaling molecules in the MAPK, PI3K/AKT, and cell cycle pathways. Genetic analyses of signaling genes were performed using Sequenom MASSarray and direct DNA sequencing. Results: High levels of ERK phosphorylation were seen at BL indicating constitutive MAPK signaling due to the BRAF mutation. In 19/22 paired samples, pERK levels were reduced following vem (BL vs D15). Mean absolute pERK reduction in pts with clinical response (RECIST criteria) to vem (n=14) was significantly greater than in non-responders (n=8; p=0.013). Baseline cytoplasmic PTEN H-score was higher in responders vs non-responders (H-score=88 vs 68; p=0.042). At progression, upregulation of pERK (H-score=181) was frequently, but not uniformly found vs D15 tumors (H-score=48.5). At progression, increases in Cyclin D1 and Ki67 were seen, without obvious changes in PTEN or pAKT. NRAS mutations occurred in 3/13 DP; 2 had paired BL samples without NRAS mutations. Only 1/82 pts had a concomitant NRAS and BRAF mutation at BL, and did not respond to vem. MAP2K1 (MEK1) codon 124 mutations occurred in 7/92 BL and 1/20 DP samples. 2 pts with BL MEK1 mutations had partial responses. Conclusions: MAPK signaling was effectively inhibited by vem early in treatment, and in the subset of patients with matched tumor samples the degree of pathway inhibition correlated with clinical response. MAPK signaling is upregulated in many lesions at progression. NRAS mutations appear to be a mechanism of acquired resistance in a few tumors (3/13), while the role of MEK1 mutations in resistance is less clear.

8520 Background: Vemurafenib (V; RG7204, PLX4032), an oral inhibitor of oncogenic V600E mutant BRAF, was evaluated for safety, efficacy, and pharmacokinetics in dose escalation and melanoma extensi...

8502 Background: Vemurafenib (PLX4032), an oral, selective inhibitor of oncogenic V600E mutant BRAF, was evaluated for safety and pharmacokinetics in the Phase I dose escalation (DE) study PLX06-02 (Flaherty, et al. NEJM, 2010). Exploratory analyses included the investigation of MOA, pathway inhibition, and primary or acquired drug resistance from pts participating in the DE and melanoma extension phase. Methods: Biopsies were taken at baseline (BL), on vemurafenib therapy Day 15 (D15), and at progressive disease (PD). Protein expression (PTEN, Cyclin D1, p27, and Ki67), phosphorylation (pERK, pMEK and pAKT), apoptosis (TUNEL), gene sequence (MEK1), and mutations in oncogenes and tumour suppressor genes (404 mutations incl. BRAF, H-, K-, N-RAS, and PIK3CA using Sequenom MassArray) were analyzed. Results: Tumor tissue of 5 pts from the DE and 17 pts from the extension cohort was obtained (total samples: 18 BL, 12 D15, 20 PD). In 7/7 pts with paired BL/D15 samples, high levels of pERK were observed at BL fo...

e22056 Background: Triple negative breast cancer (TNBC) is associated with aggressive histological features, high relapse rates, and poor overall survival. To improve the management of TNBC patient...