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Therapeutic guidance in non-small cell lung cancer (NSCLC) tumors that are positive for anaplastic lymphoma kinase (ALK) fluorescent in situ hybridization (FISH), but negative for ALK immunohistochemistry, is still challenging. Parallel routine screening of 4588 NSCLC cases identified 22 discordant cases. We rechecked these samples using ALK antibodies and selected reaction monitoring (SRM) quantitative multiplexed proteomics screening multiple protein targets, including ALK and MET for the ALK tyrosine kinase inhibitor (TKI), and FR-alpha, hENT1, RRM1, TUBB3, ERCC1, and XRCC1 for chemotherapy. The presence of ALK (31.8%), MET (36.4%), FR-alpha (72.7%), hENT1 (18.2%), RRM1 (31.8%), TUBB3 (72.9%), ERCC1 (4.5%), and a low level of XRCC1 (54.4%) correlated with clinical outcomes. SRM was more sensitive than the ALK D5F3 assay. Among the eight cases receiving ALK TKI, four cases with ALK or MET detected by SRM had complete or partial responses, whereas four cases without ALK or MET showed progression. Twenty-seven treatment outcomes from 20 cases were assessed and cases expressing more than half of the specific predictive proteins were sensitive to matching therapeutic agents and showed longer progression-free survival than the other cases (p &lt, 0.001). SRM showed a potential role in therapeutic decision making in NSCLC patients with ambiguous ALK test results.

Background A wide range of response rates have been reported in HER2-positive gastric cancer (GC) patients treated with trastuzumab. Other HER2-targeted therapies for GC have yet to show efficacy in clinical trials. These findings raise question about the ability of standard HER2 diagnostics to accurately distinguish between GC patients who would and would not benefit from anti-HER2 therapies. Patients and methods GC patients (n = 237), including a subset from the Trastuzumab in GC (ToGA) trial were divided into three groups based on HER2 status and history of treatment with standard chemotherapy or chemotherapy plus trastuzumab. We applied mass spectrometry-based proteomic analysis to quantify HER2 protein expression in formalin-fixed tumor samples. Using HER2 expression as a continuous variable, we defined a predictive protein level cutoff to identify which patients would benefit from trastuzumab. We compared quantitated protein level with clinical outcome and HER2 status as determined by conventional HER2 diagnostics. Results Quantitative proteomics detected a 115-fold range of HER2 protein expression among patients diagnosed as HER2 positive by standard methods. A protein level of 1825 amol/µg was predicted to determine benefit from the addition of trastuzumab to chemotherapy. Trastuzumab treated patients with HER2 protein levels above this cutoff had twice the median overall survival (OS) of their counterparts below the cutoff (35.0 versus 17.5 months, P = 0.011). Conversely, trastuzumab-treated patients with HER2 levels below the cutoff had outcomes similar to HER2-positive patients treated with chemotherapy. (Progression-free survival = 7.0 versus 6.5 months: P = 0.504; OS = 17.5 versus 12.6 months: P = 0.520). HER2 levels were not prognostic for response to chemotherapy. Conclusions Proteomic analysis of HER2 expression demonstrated a quantitative cutoff that improves selection of GC patients for trastuzumab as compared with current diagnostic methods.

BACKGROUNDCrizotinib has antitumor activity in ALK (anaplastic lymphoma receptor tyrosine kinase)-rearranged non–small cell lung cancer (NSCLC). The current diagnostic test for ALK rearrangement is breakapart fluorescence in situ hybridization (FISH), but FISH has low throughput and is not always reflective of protein concentrations. The emergence of multiple clinically relevant biomarkers in NSCLC necessitates efficient testing of scarce tissue samples. We developed an anaplastic lymphoma kinase (ALK) protein assay that uses multiplexed selected reaction monitoring (SRM) to quantify absolute amounts of ALK in formalin-fixed paraffin-embedded (FFPE) tumor tissue.METHODSAfter validation in formalin-fixed cell lines, the SRM assay was used to quantify concentrations of ALK in 18 FFPE NSCLC samples that had been tested for ALK by FISH and immunohistochemistry. Results were correlated with patient response to crizotinib.RESULTSWe detected ALK in 11 of 14 NSCLC samples with known ALK rearrangements by FISH. Absolute ALK concentrations correlated with clinical response in 5 of 8 patients treated with crizotinib. The SRM assay did not detect ALK in 3 FISH-positive patients who had not responded to crizotinib. In 1 of these cases, DNA sequencing revealed a point mutation that predicts a nonfunctional ALK fusion protein. The SRM assay did not detect ALK in any tumor tissue with a negative ALK status by FISH or immunohistochemistry.CONCLUSIONSALK concentrations measured by SRM correlate with crizotinib response in NSCLC patients. The ALK SRM proteomic assay, which may be multiplexed with other clinically relevant proteins, allows for rapid identification of patients potentially eligible for targeted therapies.

Trastuzumab has shown a survival benefit in cases of Her2-positive gastroesophageal cancer (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, these low-throughput assays often produce discordant or equivocal results. We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/μg) of Her2-SRM protein in cell lines (n = 27) and GEC tissues (n = 139). We compared Her2-SRM protein expression with IHC/FISH, seeking to determine optimal SRM protein expression cutoffs in order to identify HER2 gene amplification. After demonstrating assay development, precision, and stability, Her2-SRM protein measurement was observed to be highly concordant with the HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM expression of the covariates Met, Egfr, Her3, and HER2 heterogeneity, as well as their interactions (cell lines r 2 = 0.9842; FFPE r 2 = 0.7643). In GEC tissues, Her2-SRM protein was detected at any level in 71.2 % of cases. ROC curves demonstrated that Her2-SRM protein levels have a high specificity (100 %) at an upper-level cutoff of >750 amol/µg and sensitivity of 75 % at a lower-level cutoff of

Introduction Approximately 20% of breast cancer patients overexpress HER2 and are treated with anti-HER2 therapies. However, there is a great deal of disparity of HER2 levels in the patients that are classified as HER2 positive (IHC3+). Techniques like FISH or IHC do not allow for HER2 quantification and a significant proportion of patients are wrongly classified as HER2 positive. Liquid Tissue-Selected reaction monitoring (LT-SRM) is a multiplexed mass spectrometric technique that can objectively quantify levels of Her2 and other targets from formalin fixed paraffin embedded (FFPE) sections. Given the different available anti-HER2 therapies (trastuzumab, TDM1, lapatinib and pertuzumab) with different modes of action, it would be beneficial for a clinician to understand the levels EGFR and HER3 so as to personalize the therapy. In this work, we have used LT-SRM to quantitate HER2, EGFR and HER3 from FFPE samples (one slide) of patients treated with anti-HER2 agents and correlated the levels of these proteins to clinical outcome. Methods FFPE sections from 60 HER2 positive (IHC3+) primary breast cancer patients were microdissected and proteins were solubilized and digested by trypsin in Liquid Tissue® buffer. Of the 60 samples, 24 were from metastatic setting and 36 from adjuvant setting. After trypsin digestion, internal standards were added and absolute quantitation for multiple proteins was performed using selected reaction monitoring (SRM) mass spectrometry. In addition to LT-SRM, FISH for HER2 was also conducted. Results HER2 quantitation by LT-SRM revealed receptor level ranges from 283 to 14938 amol/µg. ROC analysis was conducted and a cut-off of 2758.75 amol/µg gave the optimal sensitivity and specificity. Survival analysis revealed statistically significant DFS (4.40 years vs 3.38 years; p=0.013) and OS (4.43 years vs 4.03 years; p= 0039) in patients expressing ≥ 2758.75 amol/µg in the adjuvant setting and also statistically significant OS (5.51 years vs 3.37 years; p=0.037) in the metastatic setting. Correlation of HER2 FISH and levels of HER2 is ongoing. Approximately 41% of samples expressed EGFR (range 45 to 2317 amol/µg) and similarly 51% of the samples expressed HER3 (range 84 to 360 amol/µg) with 18% of samples expressing all three targets. Correlation of EGFR/HER3 expression with clinical outcome is ongoing. Conclusion We used an objective multiplex non-antibody based method to quantify multiple targets from FFPE tissue. Clinical correlation analysis of HER2 revealed improved OS and DFS in samples with high HER2 protein levels. Currently, we are expanding these studies to a larger set of samples and taking into account also the expression of other markers such as EGFR and HER3. This approach can potentially identify those tumors that are more dependent on these receptors for survival and also those patients that are exquisitely sensitive to anti-HER2 therapy. Citation Format: Paolo Nuciforo, Sheeno Thyparambil, Claudia Aura, Ana Garrido-Castro, Vicente Peg, Jose Jimenez, William Hoos, Jon Burrows, Todd Hembrough, Jose Perez-Garcia, Javier Cortes, Maurizio Scaltriti. Quantitative measurement of HER2 levels by multiplexed mass spectrometry from FFPE tissue predicts survival in patients treated with anti-HER2 based therapy [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 P4-11-29.

This article examines the history of the British film industry's first trade union: the National Association of Cinematograph Operators (NACO), an organisation for projectionists established in 1907. The deterioration in pay and working conditions experienced by projectionists following the advent of permanent cinemas is outlined, but, contrary to prevailing wisdom, it is pointed out that NACO was not actually formed in response to these developments. NACO pre-dated the growth of fixed-site film exhibition venues and the reasons behind its inception are explained in relation to the politics of the music hall industry. It is shown that the union's executive steadfastly promoted a conception of projection work that was rapidly becoming anachronistic in several respects, and concentrated their campaigning efforts upon trying to police entry into the profession, primarily via a proposed parliamentary bill to make an annual examination of projectionists' fitness to ‘strike the arc’ compulsory. It is argued that this was an inadequate and blinkered response to the increasing exploitation of projectionists as sweated labour, and also that NACO's repeated denunciation of inexperienced projectionists as ‘handle-turners’ may have emboldened employers in their determination to drive wages down. Consequently, although NACO belatedly decided to relax its membership requirements, new subscriptions were in decline by 1912 and the union remained inactive throughout the First World War until it was wound up and replaced in 1919.

The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR and HER2), a third (HER3) currently under investigation for its possible role in the acquisition of multidrug resistance and a fourth (HER4), the role of which is still matter of debate. Drugs inhibiting EGFR or HER2 show significant antitumor activity in the clinic, however, acquisition of resistance is a hallmark of these and most targeted therapies. In the case of EGFR and HER2, one of the emerging resistance mechanisms is the co-expression of HER3. Indeed, recent reports show that inhibition of the PI3K pathway leads to upregulation of HER3, and subsequent resistance. Clinical analysis of protein levels in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative and requires significant amounts of tissue. Moreover, the vast majority of research groups consider specific HER3 staining by IHC particularly challenging. However, accurate measurement of these targets is critical both for properly defining treatment groups and predicting patterns of resistance. In order to address these issues, we used trypsin digestion mapping and stable isotope labeled peptides to develop a panel of quantitative mass spectrometric (MS) assays to measure the levels of EGFR, HER2, HER3 and other clinically relevant targets in FFPE breast cancer tissue. These quantitative MS assays were then multiplexed to analyze 1μg of tumor protein. In this study, we multiplexed HER family analysis on 31 HER2 positive breast cancers. Tumor tissue was microdissected from FFPE sections, and subjected to quantitative MS analysis of EGFR, HER2, HER3 as well as IGF1R and cMET. Quantitation of HER2 showed a broad range of HER2 expression in these tissues. The highest expresser measured 26 fmol/ug tumor tissue, representing amplification and massive protein over expression. In contrast, five tissues showed low levels of HER2 expression, below 1 fmol/ug, similar to HER2 non-amplified cell lines. This suggests that MS quantitation can identify patients with low expression of HER2 who are unlikely to respond to trastuzumab therapy. As a matter of fact, 3 of these 5 low expressing patients had outcome data and showed no response to trastuzumab treatment. In 28 of 31 patient tissue samples, HER3 showed low levels of expression (100–300 amol/ug tumor tissue) similar to HER3 expression in cell lines, and comparable to low expressing EGFR and HER2 cell lines. The remaining 3 patients had no detectable HER3. This study demonstrates the feasibility of measuring HER3 in multiplex in FFPE breast cancer tissue. Based on the low but widespread expression of HER3 in this cohort, it may be most useful to assess HER3 expression after initial treatment as a marker of potential resistance to targeted therapies. Taken together, these data demonstrate that a sensitive and quantitative assay to measure oncoproteins in FFPE clinical samples may help stratify patients with variable expression of these targets. Our quantitation of oncogene expression from clinical samples uses a small amount of tissue, is clinically applicable and alleviates the problem of scoring either positive or negative for the expression of a given protein. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-19.

This article demonstrates that the film presentations of British public hall showmen in the 1900s were often more elaborate and oriented towards middle‐class audiences, but much less well‐travelled, than is typically realized. It attempts to explain how one such show, West’s Pictures at the Shaftesbury Hall, Bournemouth, established itself as a semi‐permanent attraction, and argues that T.J. West’s activities reveal the need for a revised morphology of the emergence and evolution of the permanent picture theatre that acknowledges the complexity of both the cinema’s journey towards ‘gentrification’ and the relationships between provincial and metropolitan screen entertainment practices.

This article revisits the various claims and counter-claims that have been made over the years as to whether the 1910s music hall comedian turned slapstick film star Billie Ritchie stole his costume and comic persona from Charlie Chaplin, or was himself the victim of plagiarism on Chaplin's part. It proposes that neither of these arguments can be definitively supported on the basis of evidence available in the public domain, but also draws attention to the fact that there are other factors in play that may plausibly explain the various similarities between the two comedians as the result of shared formative influences rather than outright imitation, and that some contemporaries deemed it eminently reasonable to take such a view. The article moves on to consider the significance of the fact that, although their costumes were practically identical, Ritchie typically played dissolute gentlemen of ambiguous class status rather than tramps. It concludes that there is a stronger argument to be made that, rather than seeking to represent vagrancy, both Chaplin and Ritchie originally used the iconic 'tramp' costume to poke fun at the culturally ubiquitous Edwardian figure of the 'masher' - a lower-middle- or working-class male pleasure-seeker trying (and in their incarnations, failing) to emulate the fashions of his social superiors.

During the first decade of mass cinema-going in Britain, the nascent film exhibition industry was beset by scandal. It continually received negative publicity and scrutiny from politicians, the pol...

Background: Trastuzumab showed survival benefit for Her2-positive gastroesophageal cancers (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, both assays are low throughput with various limitations. Methods: We developed a selected reaction monitoring mass spectrometric (SRM-MS) assay and quantified levels (amol/ug) of Her2-SRM in cell lines (n=27) and GEC tissues (n=139). We compared Her2-SRM expression with IHC/FISH, seeking to determine optimal SRM expression cut-offs to identify HER2 amplification. Results: After demonstrating assay development, precision, and stability, Her2-SRM measurement was observed to be highly concordant with HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM-expression of Met, Egfr, Her3, and HER2-heterogeneity covariates, and their interactions (cell lines r2=0.9842; FFPE r2=0.7643). In GEC tissues, Her2-SRM was detected in 71.2% of cases, and 12.3% were identified as ‘HER2+’. ROC curves demonstrated HER2-SRM levels to have high specificity (100%) at an upper-level cut-off of >750 amol/µg and sensitivity (75%) at lower-level cut-off of

This article explores the role played by British film companies in the International Congresses of Film Manufacturers held in Paris in 1909. It argues that British involvement in the attempt to create a European MPPC‐style film cartel was the result of both ongoing price wars with Pathe Freres and an increasing saturation of the domestic film market caused by the growth of the film rental sector. It concludes by suggesting that the failure to successfully implement the resolutions passed in Paris had severe repercussions for British film producers, opening up a bitter rift between domestic filmmakers and renters and exhibitors, and allowing American film companies to take the first significant step towards their future domination of the British film trade.

This paper pays close attention to the serial and 'cinematic' narratives produced in the British press of the 'teens in order to display film's widening reach as a narrative mode mingling action, sentiment, and gender and to historicize debates about genre in film studies. Many filmmakers, publicists and journalists in the 1910s fully understood that melodrama was a radically hybrid form, yet they did not have the same semantic anxieties about juxtaposing multiple, non-hierarchical definitions of melodrama as later genre historians.

Receptor tyrosine kinase (RTK) targeted agents such as trastuzumab, imatinib, bevacizumab, and gefitinib inhibitors have illustrated the utility of targeting this protein class for treatment of selected cancers. A unique member of the RTK family, c-Met, also represents an intriguing target for cancer therapy that is yet to be explored in a clinical setting. The proto-oncogene, c-Met, encodes the high-affinity receptor for hepatocyte growth factor (HGF) or scatter factor (SF). c-Met and HGF are each required for normal mammalian development and have been shown to be particularly important in cell migration, morphogenic differentiation, and organization of three-dimensional tubular structures (e.g. renal tubular cells, gland formation, etc.) as well as cell growth and angiogenesis. Both c-Met and HGF have been shown to be deregulated in and to correlate with poor prognosis in a number of major human cancers. New data describing the constitutive phosphorylation of c-Met in a number of human tumors is presented here along with a variety of mechanisms by which c-Met can become activated, including mutation and gene amplification. In support of the clinical data implicating c-Met activation in the pathogenesis of human cancers, introduction of c-Met and HGF (or mutant c-Met) into cells conferred the properties of motility, invasiveness, and tumorgenicity to the transformed cells. Conversely, the inhibition of c-Met with a variety of receptor antagonists inhibited the motility, invasiveness, and tumorgenicity of human tumor cell lines. Consistent with this observation, small-molecule inhibitors of c-Met were developed that antagonized c-Met/HGF-dependent phenotypes and tumor growth in mouse models. This review will address the potential for development of c-Met inhibitors for treatment of human cancers with particular emphasis on recent findings with small-molecule inhibitors.

Targeted therapies are increasingly being evaluated for patients with Ewing sarcoma (EWS). Optimal strategies for quantifying key signaling proteins in EWS remain unclear. We sought to quantify tumor expression of signaling pathway proteins in EWS using 3 methodologies. A total of 46 blocks of formalin-fixed paraffin-embedded tissue were obtained from 40 patients with EWS. Tumor was evaluated for the expression of proteins in the insulin-like growth factor type 1 receptor (IGF-1R), epithelial growth factor receptor (EGFR), and mTOR pathways using standard immunohistochemical analysis (IHC), automated quantitative analysis (AQUA) immunohistochemical analysis, and mass spectrometry quantification. The mean age at diagnosis was 14 years (range, 1 to 49 y). About 67.5% were male and 57.5% had localized disease. Samples displayed a wide range of expression by AQUA: mean (range) IGF-1R=10,702 (393 to 14,424); EGFR=2750 (672 to 9798); and phosphatase and tensin homolog (PTEN)=2250 (251 to 6557). Mean IGF-1R expression by AQUA did not differ between standard IHC expression categories (low IHC=11,255; medium IHC=11,070; high IHC=11,023; P=0.98). Mean PTEN expression by AQUA was higher in the medium and high IHC categories (low IHC=1229; medium IHC=2715; high IHC=2940; P=0.064). Only 2 samples expressed EGFR by standard IHC. Mass spectrometry trended toward correlation with standard IHC but did not yield interpretable results in the majority of samples. This study demonstrates that the relative quantification of signaling protein expression in EWS is dependent on the methodology used. Optimization and validation of these tools are necessary before clinical application for risk stratification of patients or measurement of biomarker expression.

Background Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. Methods After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). Results Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (

Application of mass spectrometry to proteomic analysis of tissue is a highly desirable approach to discovery of disease biomarkers due to a direct correlation of findings to tissue/disease histology and in many respects obviating the need for model systems of disease. Both frozen and formalin-fixed, paraffin-embedded (FFPE) tissue can be interrogated; however, worldwide access to vastly larger numbers of highly characterized FFPE tissue collections derived from both human and model organisms makes this form of tissue more advantageous. Here, an approach to large-scale, global proteomic analysis of FFPE tissue is described that can be employed to discover differentially expressed proteins between different histological tissue types and thus discover novel protein biomarkers of disease.

Application of mass spectrometry to proteomic analysis of tissue is a highly desirable approach to discovery of disease biomarkers due to a direct correlation of findings to tissue/disease histology and in many respects obviating the need for model systems of disease. Both frozen and formalin-fixed, paraffin-embedded (FFPE) tissue can be interrogated; however, worldwide access to vastly larger numbers of highly characterized FFPE tissue collections derived from both human and model organisms makes this form of tissue more advantageous. Here, an approach to large-scale, global proteomic analysis of FFPE tissue is described that can be employed to discover differentially expressed proteins between different histological tissue types and thus discover novel protein biomarkers of disease.

It is traditionally thought that during the first ten years in which permanent cinemas became established in Britain, the accompaniment to silent films was typically supplied by lone pianists who improvised musical themes that subserviently complemented narrative developments as they unfolded on screen. This chapter acknowledges that, following a period in which mechanical musical devices predominated, this model was widely espoused around 1909–10. But it argues that such practices quickly fell out of fashion. Using the evidence of cinema licensing records, as well as debates published in the film-industry trade press, the chapter demonstrates that miniature orchestras had been installed in the majority of cinemas in London by 1913 and that such orchestras rarely attempted to tightly synchronize their music with the films they accompanied.

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R2 > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.

L’article discute l’impact des films d’art français sur la cinématographie britannique et examine deux tentatives d’imitation de cette forme de production, Henry VIII (1911) et Richard III (1910/1911). Il développe l’idée que les films d’art continentaux furent regardés comme des exemples d’une forme d’interprétation expressive que les acteurs britanniques devaient suivre. Il montre également que les films d’art britanniques représentèrent un moment d’expérimentation de nouveaux types de discours adaptés aux publics émergeant avec le développement considérable de l’exploitation en Grande-Bretagne dans les années 1908-1911. This essay discusses the impact of the French films d’art within British film culture and examines two native attempts to imitate this form of filmmaking: Henry VIII (1911) and Richard III (1910/1911). It argues that European films d’art were taken very seriously as salutary exemplars of a gesturally expressive mode of film acting which British actors were encouraged to imitate. It also aims to demonstrate that British films d’art represented important experiments in formulating new modes of address suitable for the emerging constituencies of spectators created by the enormous expansion of film exhibition in Britain between 1908 and 1911.

Targeted Mass Spectrometry Todd Hembrough1, Wei-Li Liao1, Christopher P. Hartley2, Patrick C. Ma3, Vamsidhar Velcheti3, Manish Monga4, Sheeno Thyparambil1, Eunkyung An1, Jon Burrows1, and Laura J. Tafe2 1OncoPlex Diagnostics, Rockville, MD, 2Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 3Cleveland Clinic Taussig Cancer Institute, Cleveland, OH, 4West Virginia University, Morgantown, WV

Introduction: Selection of drugs to treat patients with cancer is typically based on the anatomical site in which the tumor is located. Here we report a treatment decision for a patient with relapsed, advanced cervical cancer that was based on a comprehensive omics analysis using whole genome sequencing (WGS) combined with quantitative proteomics. Methods: The patient was a 44-year-old female whose disease had progressed following surgery and more than 4 lines of chemotherapy. WGS was performed on the patient's formalin-fixed, paraffin-embedded (FFPE) metastatic tumor sample and a matched-normal reference sample. Quantitative proteomics was performed on the FFPE tumor sample by Selected Reaction Monitoring Mass Spectrometry and was quantitated at the atomolar level. Results: WGS found somatic mutations and rearrangements and reads mapping to human papillomavirus type 18 (HPV 18). Mutations more commonly found in breast cancer (ERBB2, CDH1, and CLTCL1) were noted. The HPV 18 genome was integrated into chromosome 17 in close proximity to a 7-fold amplification of the ERBB2 gene. Proteomic analysis of the FFPE tumor validated and quantitated overexpression of HER2 protein resulting from ERBB2 gene amplification, with 11,322 amol/μg of tissue protein. Clinically observed ranges for breast or gastric cancer are 150-500 amol/μg, with levels above 750 amol/μg correlating with FISH-positive amplification and clinical efficacy of trastuzumab (unpublished observation). Based on these comprehensive omic findings, trastuzumab, a therapy approved for breast and gastric cancer, was administered. The patient experienced a reduction in the size of her tumor (by CT/PET) and stabilization of her disease for 5 months. Conclusion: WGS and proteomic profiling of this patient's disease identified, confirmed, and quantitated an appropriate target for pharmaceutical intervention. The patient presented with cervical cancer; however, the WGS analysis pointed towards a potentially causative integration of the HPV 18 genome resulting in ERBB2 amplification along with genomic mutations more commonly found in breast cancer. Proteomic analysis further validated and quantitated the HER2 expression resulting from ERBB2 gene amplification, leading to the patient's treatment with trastuzumab. Our findings argue for the use of comprehensive omics analysis to guide decision support for personalized management of cancer care with therapies determined based on a quantitative proteomic signature, independent of anatomical tumor type. Citation Format: Stephen Benz, J Zackary Sanborn, Nicole S. Hensley, Todd Hembrough, Charles J. Vaske, Jon Burrows, Shahrooz Rabizadeh, Ivor Royston, Patrick Soon-Shiong. Whole genome sequencing and quantitative proteomics reveal HPV integration and HER2 overexpression in a patient with cervical cancer: Comprehensive omics analysis driving clinical treatment decisions. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 25.

Background: Antibody-Drug Conjugates (ADCs) are poised to become an extremely important class of therapeutics in oncology. By conjugating cytotoxic payloads to antibodies that target proteins found primarily on cancer cells, ADCs represent a novel mechanism for directing extremely toxic small molecules specifically to tumor cells. Due to the unique mechanism of ADCs, patient selection should involve screening not only for the presence of the antibody target, but also screening for the presence of any markers of resistance or response to the payload. Several proteins, such as multi-drug effluxers and tubulin-beta 3, have been implicated in resistance to small molecule cytotoxins and microtubule inhibitor drugs. OncoPlex Diagnostics has built a multiplexed ADC biomarker panel that simultaneously quantifies the levels of the antibody target and putative resistance markers for several known payloads, such as maytansinoids, auristatins and taxanes, as well as response markers for the topoisomerase inhibitor payloads SN-38 and doxorubicin. Methods: Liquid Tissue-Selected Reaction Monitoring (LT-SRM) is a multiplexed, quantitative method that uses mass spectrometry to quantify proteins based on a unique sequence of amino acids, and thus does not have the same limitations as traditional antibody-based, semi-quantitative protein detection methods, such as immunohistochemistry. We developed a LT-SRM assay to quantify protein levels of EGFR, FRalpha, Her2, CD30 and Mesothelin (antibody targets) and MCL1, MDR, MRP1, tubulin-beta3, Topo1 and Topo2a (payload response and resistance markers) simultaneously from FFPE biopsies. Calibration curves for all the proteins in the ADC panel are linear over 5-orders of magnitude, with limits of detection for each analyte between 25 and 400 amol/ug of tissue. Results: Analysis of FFPE tumor tissues show a broad range of expression for the ADC proteins, with some tissues showing no detectable levels of some payload markers. Clinical analysis of FRalpha showed a range of expression from Conclusions: The OncoPlexDx ADC panel can determine of a cutoff for expression levels of the antibody-target protein necessary for ADC response as well as identify markers of payload response or resistance to further understand how these markers affect therapeutic efficacy. This panel can be used to predict which patients will derive the most benefit from ADC therapy based on the specific biology of their tumor. Citation Format: Adele Blackler, Wei-Li Liao, Sheeno Thyparambil, Eunkyung An, Fabiola Cecchi, Marlene Darfler, Todd Hembrough, Jon Burrows. Development of a mass spectrometry based antibody-drug conjugate biomarker panel. [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 3398. doi:10.1158/1538-7445.AM2015-3398

Many available oncology therapies are targeted to specific proteins, the most notable examples being therapies targeted to EGFR and Her2. For targeted therapies to have maximal efficacy, it is necessary to identify patients whose tumors express the target protein. As more pathways and proteins are identified as tumor drivers and therapies are developed that target those proteins, and more patient screening tools are needed to efficiently direct patients to correct therapeutic regimens. While chemotherapy regimens are not often considered targeted therapies, protein biomarkers for chemotherapy efficacy have been identified; for example amplification of TOPO2A is known to improve response to anthracycline-based therapy combinations. Though many chemotherapy biomarkers have been identified, screening is not routine, and chemotherapy regimens are not being adjusted for individual tumor biology. To address the growing need for efficient patient screening using minimal tissue, OncoPlex Diagnostics has built a comprehensive protein quantification panel that allows for the simultaneous quantitation of multiple actionable proteins from formalin-fixed, paraffin-embedded patient biopsies using multiplex mass spectrometry. This panel currently quantifies nine proteins that are markers of targeted therapy (including ALK, AR, EGFR, HER2, HER3, MET, MSLN, and PD-L1) and includes the ChemoPlex panel, which quantifies chemotherapy biomarkers (ERCC1, FRalpha, hENT1, RRM1, SPARC, TOPO1, TOPO2A). Since 2013, over 270 biopsies from multiple indications have been analyzed for protein expression in the OncoPlex Diagnostics CAP-qualified, CLIA-certified laboratory, revealing large ranges of expression for many drug targets that are not routinely assayed. Because of the importance of TOPO2A in anthracycline-based therapies, which are commonly prescribed in breast cancer patients, sixty-two breast cancer biopsies were retrospectively reviewed. Of the 62 samples, 41 were positive for TOPO2A expression; ranging from 233-1750amol/ug. Of the primary biopsies, 80% of them expressed TOPO2A; however only two of seven liver metastases were positive for TOPO2A. These data suggest that anthracycline-based therapy might not be as efficacious in metastatic sites due to the lack of TOPO2A. Also of interest, quantification of FRalpha, a biomarker for folate-targeted therapies, showed a 50-fold range of expression in NSCLC cases, and TOPO1, the target of irinotecan and topotecan, showed a 10-fold range of expression in various indications, with 4% of biopsies having no detectable TOPO1. These wide expression ranges of known biomarkers suggest that certain therapies might have vastly different response rates based on biomarker expression. To derive the best therapeutic response to both targeted and chemotherapy regimens, it is necessary to understand each patient tumor biology individually. Citation Format: Fabiola Cecchi, Adele Blackler, Heather Jordan, Marlene Darfler, Todd Hembrough, Michael Stocum, Jon Burrows. Clinical Survey of 19 actionable proteins in multiple indications using multiplex mass spectrometry. [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 3395. doi:10.1158/1538-7445.AM2015-3395

BackgroundImmune check point proteins play a pivotal role in immune evasion by the tumor. Recent trials involving inhibitors of the immune checkpoint protein pairs, PD-1 and PD-L1 have demonstrated anti-tumor activity. Measuring the levels of immune check point proteins and other members of the immunological synapse will help clinicians personalize therapy. Currently, immunohistochemistry (IHC) is the preferred diagnostic to assess PD-L1 status; however, PD-L1 positivity varies based on the antibody that is used. Additionally, PD-L1 negative patients by IHC have responded to anti-PD-L1 therapy implicating disconnect between PD-L1 diagnostics and response. We have developed and clinically validated a quantitative mass spectrometric technique that not only quantitates PD-L1 in formalin fixed paraffin embedded (FFPE) tissue but can concurrently quantitate other members (B7H3, B7.1, B7.2, OX40L) of the immunological synapse using the same tissue section. MethodRecombinant PD-L1 protein was used to identify optimal quantitative peptides for PD-L1 assay. Standard curves were generated using labeled and unlabeled peptides. The PD-L1 assay was pre-clinically validated on 14 cell lines with known expression levels of PD-L1. The assay was then run on archived FFPE sections from in 9 normal tissues, 21 early staged (stage 1 and 2) and 4 advanced staged (stage 3) NSCLC patients. In addition PD-L1 was also assayed in bladder, breast and gastric cancer. Results PD-L1 protein expression was detected in 7 out of 14 cell lines The regression analysis between SRM and mRNA analysis demonstrated moderate correlation (R2 = 0.8894). Normal lung tissue did not express PD-L1; ∼24% of early stage (5/21) and 50% of advanced stage NSCLC (2/4) expressed measurable PD-L1 protein. PD-L1 was detected more frequently in squamous cell carcinoma than adenocarcinoma. We are currently assessing the levels of PD-L1 and other targets of the immunological synapse using multiplex mass spectrometry and comparing it with IHC in 100 cholangiocarcinoma and possible inclusion of PD-L1diagnostics in clinical trials. DiscussionThe need to characterize expression levels of druggable targets in small biopsies is becoming ever more critical as new drug targets and biomarkers are identified. Initial PD-L1 screening using clinical NSCLC samples suggests that more advanced NSCLC patients are more likely to be PD-L1 positive compared to early stage NSCLC patients. Laser microdissection (LMD) can be used to specifically microdissect the immunological synapse. Additional quantitative assays for both lymphocyte (CD8, CD68) and immunotargets (B7-H3,B.1, B7.2 etc) have been developed for assessing the ‘immune profile’ in tumor associated stroma via LMD. This immuno-proteomic assay of the key immunological synapse members within tumor and/or stroma may lead to improved personalized immunotherapy. Citation Format: Sheeno P. Thyparambil, Fabiola Cecchi, Eunkyung An, Wei-Li Liao, Jon Burrows, Todd Hembrough, Daniel Catenacci. Development and clinical validation of a quantitative mass spectrometric assay for immuno-oncology targets in FFPE samples. [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 4255. doi:10.1158/1538-7445.AM2015-4255

BACKGROUND: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. METHODS: After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). RESULTS: Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/μg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (1500 amol/μg was 100% sensitive (95% CI 0.69-1) and 100% specific (95% CI 0.92-1) for MET amplification. CONCLUSIONS: The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC. Citation Format: Fabiola Cecchi, Wei-Li Liao, Sheeno Thyparambil, Marlene Darfler, David Krizman, Todd Hembrough, Jon Burrows, Don Bottaro, Daniel V.T. Catenacci. A novel clinical tool that provides quantitative and accurate measurement of Met protein. [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 3397. doi:10.1158/1538-7445.AM2015-3397