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Background: IDE196 is a potent and selective PKC inhibitor targeting the novel (δ, ϵ, η, θ) and classical (α, β) PKC isoforms that has demonstrated preliminary clinical activity in patients with metastatic uveal melanoma (MUM). IDE196 is being further evaluated for safety and efficacy as a monotherapy in a Phase1/2 basket trial in non-MUM patients whose tumors harbor GNAQ/GNA11 hotspot mutations (Clinicialtrials.gov: NCT03947385). Patients with solid tumors harboring PRKC fusions or GNAQ/11 non-hotspot mutations may be eligible after preclinical validation of the activity of these fusions and mutations in non-MUM indications. PRKC gene fusions exist at very low frequencies with various gene partners across multiple indications. Hotspot mutations in GNAQ/GNA11 (codons Q209 and R183) constitutively activate the PKC pathway in 90% of MUM patients and may also occur in non-MUM indications such as colorectal cancer (CRC) and cutaneous melanoma. Furthermore, there are additional non-hotspot mutations found in these GTPases across various non-MUM indications. To help identify patients that may benefit from IDE196 in this basket trial, we assessed the sensitivity of non-MUM cell lines that have PRKC gene fusions or GNAQ/11 hotspot/non-hotspot mutations to IDE196. Methods The most prevalent fusions in patient samples listed in TCGA are the HIF1A-PRKCH (n=20) and RP11-47I22.3-PRKCH (n=3) fusions, therefore cell lines containing PRKCH fusions listed in CCLE were prioritized for cell viability screening using short (3-day) and long-term (10-day) assays after IDE196 treatment. For PD analyses, cells were treated for 3hrs with IDE196. Expression of the fusions and pharmacodynamic modulation of phospho-MARCKS was assessed using western blotting (WB). RNASeq data was used to determine fusion structure, size and whether the fusion was in-frame. Results 8/10 PRKCH fusion lines were highly resistant to IDE196 (IC50>10μM, including HIF1A-PRKCH and RP11-47I22.3-PRKCH fusion lines), whereas 2/10 showed sensitivity at high doses in the long-term viability assay (IC50≥1μM, lines with PRKCH-SPTB or VWA2-PRKCH fusions). PD assessment showed that the PKC substrate, pMARCKS, was decreased in all cell lines by IDE196 at doses similar to those seen in the MUM cell line, 92.1 (≥0.3μM). Fusion protein expression was not detectable by WB consistent with literature suggesting that PKC fusions are unstable (1). In summary, cell lines with clinically prevalent PRKCH fusions were insensitive to IDE196 despite robust PD modulation. Assessment of cell lines harboring GNAQ/GNA11 hotspot/non-hotspot mutations for IDE196 sensitivity is ongoing. (1) An-Angela N. Van, Timothy R. Baffi, Maya T. Kunkel, Corina E. Antal and Alexandra C. Newton, FASEB J. (2018); vol 32, suppl.no. 1, abstract 687.6. Citation Format: Marie Wagle, Nandini Ravindran, Carol O'Brien, Christian Frey, Julie Hambleton, Mark R. Lackner, Zineb Mounir. Preclinical evaluation of PRKC fusions and GNA11/GNAQ mutations as genetic drivers of PKC activation in non-MUM indications to support a phase 1/2 basket trial of IDE196 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4152.

Deletions in Methylthioadenosine phosphorylase (MTAP) are observed in 15% of all cancers including pancreatic, lung, bladder and GBM cancers (1). MTAP loss results in an accumulation of MTA which partially inhibits Protein Arginine Methyltransferase 5 (PRMT5). MTAP deletions confer enhanced sensitivity to inhibition of Methionine Adenosyltransferase 2A (Mat2A), which is involved in the synthesis of the universal methyl donor and PRMT5 substrate S-Adenosyl Methionine (SAM). In combination with MTA accumulation, Mat2A inhibition depletes SAM levels resulting in further inhibition of PRMT5 activity and a synthetic lethal (SL) relationship in MTAP null cancers. Inhibition of PRMT5 activity as a result increases RNA splicing defects and chromatin dysregulation which impacts tumor cell viability in different models. We developed MAT2A inhibitors for treatment of MTAP-deleted tumors, and in this study sought to determine the mechanism of action of these inhibitors in MTAP null models. These inhibitors were assessed for activity against both MTAP-wt and MTAP-null tumor models using cellular proliferation assays. MTAP-null (KP4, BxPC3, RT112/84, MiaPaCa-2, H647) models were more sensitive to Mat2A inhibition than MTAP-wt (HCT116, NCI-H460) models. Inhibition of Mat2A increased the expression of markers associated with cellular senescence and cell cycle arrest. Cellular SAM levels were diminished in both MTAP-wt and MTAP-null cells and xenografts while selective reduction of Symmetric Dimethyl Arginine (SDMA) levels were observed in MTAP-null models. Cellular response to Mat2A inhibitors is unaltered by RNAi-mediated knockdown of Mat1A. Signaling changes that were observed due to decreased PRMT5 activity included increased stability of p53, which may be due to the inefficient splicing of MDM4 mRNA, and was seen across multiple MTAP-null models but not in MTAP WT. Furthermore, increased protein and mRNA expression of the p53 target gene p21 was also observed in MTAP-null models. In conclusion, Mat2A inhibition effectively decreases SAM and SDMA levels, leading to increased stability of p53 and its targets and resulting in decreased cellular proliferation. Ongoing exploratory studies will further delineate the molecular mechanisms mediated by Mat2A inhibition in MTAP-null cell lines. The genes and metabolites identified from these studies will be validated in Mat2A-dependent and non-dependent MTAP-null cell lines identified from DepMap studies to inform further preclinical and clinical studies. Citation Format: Neil Bhola, Atieh Givmanesh, Marie-Claire Wagle, Candy Garcia, Kedar Vaidya, Leah Cleary, Zhonghua Pei, Mark Lackner, Zineb Mounir. Mat2A Inhibitors decrease growth, increase senescence and p53 stability in MTAP-deleted cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 631.

ER-targeted therapeutics provide valuable treatment options for patients with ER+ breast cancer, however, current relapse and mortality rates emphasize the need for improved therapeutic strategies. The recent discovery of prevalent ESR1 mutations in relapsed tumors underscores a sustained reliance of advanced tumors on ERα signaling, and provides a strong rationale for continued targeting of ERα. Here we describe GDC-0810, a novel, non-steroidal, orally bioavailable selective ER downregulator (SERD), which was identified by prospectively optimizing ERα degradation, antagonism and pharmacokinetic properties. GDC-0810 induces a distinct ERα conformation, relative to that induced by currently approved therapeutics, suggesting a unique mechanism of action. GDC-0810 has robust in vitro and in vivo activity against a variety of human breast cancer cell lines and patient derived xenografts, including a tamoxifen-resistant model and those that harbor ERα mutations. GDC-0810 is currently being evaluated in Phase II clinical studies in women with ER+ breast cancer.

To identify important druggable targets and immunological determinants associated with the overall survival (OS) of hepatocellular carcinoma (HCC) patients, we interrogated 279 Chinese HCC samples, mostly in early stage, with the NanoString platform assessing the expression of 1,173 genes involved in cell-growth signaling pathways, cellular machineries and tumor immunity. A prognostic model was constructed using 17 genes with bootstrap-corrected C-index of 0.81 for the OS. Among the genes associated with worse OS, cell-growth related druggable targets CHK1 and EZH2 were selected for in vitro and in vivo tumor growth studies. HCC cell line clonogenic assays and mice xenograft models demonstrated that the combinatorial application of CHK1 inhibitor and EZH2 inhibitor resulted in significant suppression of HCC tumor growth. On the tumor immunity front, we discovered that genes enriched in regulating CD8+ T cells and activating NK cells contributed to prolonged OS. Our findings provide potential rationale for investigating combinatorial targeted therapies and immunotherapies in specific subsets of early-stage HCC with distinct gene expression characteristics. Funding: This work was supported by National Natural Science Fund of China (Grant numbers: No.81672330; No. 81871916; No. 81472218), Talent Development Program of Zhongshan Hospital (2015ZSYXGG), and by Genentech Inc., a member of the Roche Group. Declaration of Interest: Chun Sun, Bonnie Liu, Kwame Okrah, Xu Cao, Jian-Jun Guo, Shi-Jing Fu, Su-Chun Li, Hang-jun Dai, Mark R. Lackner, Astrid Kiermaier, Shih-Min A. Huang and Gang Cheng are employees of F. Hoffmann-La Roche/Genentech Inc. All other authors declare no conflict of interest. Ethical Approval: This study was approved, and the collection of written informed consent forms was waived, by the institutional review board of SZH under the condition that all samples were anonymized in this study.

Purpose: Four consensus molecular subtypes (CMS1–4) of colorectal cancer were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for colorectal cancer–related mortality, we examined the molecular and clinical attributes of CMS in metastatic colorectal cancer (mCRC). Experimental Design: We developed a colorectal cancer–focused NanoString-based CMS classifier that is ideally suited to interrogate archival tissues. We successfully used this panel in the CMS classification of formalin-fixed paraffin-embedded (FFPE) tissues from mCRC cohorts, one of which is composed of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modeling of colorectal cancer in vivo at relevant sites. Results: Using our classifier, we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in nonmetastatic early-stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared with other CMS. Assigning CMS to primary tumors and their matched metastases reveals mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs reveals differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic colorectal cancer. Conclusions: We describe the utility of a NanoString panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on colorectal cancer heterogeneity during disease progression.

Introduction The advent of autonomous automobiles raises new challenges for maintaining passenger safety and comfort. The challenge addressed here is how to predict and mitigate motion sickness when passengers read in a moving vehicle. Methods We utilized a car equipped with a commercial active suspension system developed for attenuating the transmission of road surface fluctuations to passengers. The system was used to reproduce, in a parked car, either the vibrations that would be experienced in a moving car equipped with a conventional suspension system (unmitigated ride) or the attenuated vibrations that would occur on the road with the active cancellation system engaged (mitigated ride). We evaluated the consequences of these two simulated ride conditions for reading performance, comfort, and evocation of motion sickness. Results Both ride conditions reduced the 0 to 0.8 Hz vibrations to below threshold for evoking motion sickness during passive exposure [corrected]. Only the mitigated ride condition attenuated frequencies in the 0.8 to 8 Hz band where visual suppression of the vestibulo-ocular reflex is known to break down, and this condition also reduced the motion sickness induced by reading and increased reading comprehension and comfort relative to the unmitigated ride. Discussion The palliative effects of 0.8 to 8 Hz attenuation are discussed in terms of the different mechanisms underlying motion sickness evoked by reading in a vehicle versus mere exposure to vehicle motion without reading. Implications for ISO-2631 standards for human exposure to vibration are also discussed.DiZio P, Ekchian J, Kaplan J, Ventura J, Graves W, Giovanardi M, Anderson Z, Lackner JR. An active suspension system for mitigating motion sickness and enabling reading in a car. Aerosp Med Hum Perform. 2018; 89(9):822-829.

Bei der Stimme reagiert der Korper auf Storungen durch Kompensation, diese reduzieren das Mas der Leistungsfahigkeit. Wenn die Kompensation nicht mehr moglich ist, kommt es zum erkennbaren Vollbild der Storung. Der interdisziplinare Zugang der manuellen Medizin beschaftigt sich mit der Moglichkeit einer Akuthilfe einerseits und der Vorsorge andererseits. Im vorliegenden Beitrag werden anatomische und fur den Manualmediziner „fassbare“ Grundlagen der Stimmbildung mit einem Uberblick uber die Funktion, die faszialen- und muskularen Verhaltnisse des Halses und die neuronalen Zusammenhange dargestellt.

Our objective was to examine how the control of orientation is learned in a task involving dynamically balancing about an unstable equilibrium point, the gravitational vertical, in the absence of leg reflexes and muscle stiffness. Subjects (n = 10) used a joystick to set themselves to the gravitational vertical while seated in a multi-axis rotation system (MARS) device programmed with inverted pendulum dynamics. The MARS is driven by powerful servomotors and can faithfully follow joystick commands up to 2.5 Hz with a 30-ms latency. To make the task extremely difficult, the pendulum constant was set to 600°/s(2). Each subject participated in five blocks of four trials, with a trial ending after a cumulative 100 s of balancing, excluding reset times when a subject lost control. To characterize performance and learning, we used metrics derived from joystick movements, phase portraits (joystick deflections vs MARS position and MARS velocity vs angular position), and stabilogram diffusion functions. We found that as subjects improved their balancing performance, they did so by making fewer destabilizing joystick movements and reducing the number and duration of joystick commands. The control strategy they acquired involved making more persistent short-term joystick movements, waiting longer before making changes to ongoing motion, and only intervening intermittently.

Purpose: We conducted a randomized phase III study to determine whether patients with early breast cancer would benefit from the addition of capecitabine (X) to a standard regimen of doxorubicin (A) plus cyclophosphamide (C) followed by docetaxel (T). Experimental Design: Treatment comprised eight cycles of AC→T (T dose: 100 mg/m2 on day 1) or AC→XT (X dose: 825 mg/m2 twice daily, days 1–14; T dose: 75 mg/m2 on day 1). The primary endpoint was 5-year disease-free survival (DFS). Results: Of 2,611 women, 1,304 were randomly assigned to receive AC→T and 1,307 to receive AC→XT. After a median follow-up of 5 years, the study failed to meet its primary endpoint [HR, 0.84; 95% confidence interval (CI), 0.67–1.05; P = 0.125]. A significant improvement in overall survival, a secondary endpoint, was seen with AC→XT versus AC→T (HR, 0.68; 95% CI, 0.51–0.92; P = 0.011). There were no unexpected adverse events. Of patients with estrogen receptor (ER)–positive/HER2-negative disease, 70% of whom were node-positive, 26% and 59% had tumors with a centrally assessed Ki-67 score of Conclusions: The very low event rate in patients with ER-positive, low Ki-67 cancers, regardless of nodal status, strongly suggests that these patients should not be enrolled in adjuvant trials that assess 5-year DFS rates and that central Ki-67 analyses can identify these patients. Clin Cancer Res; 21(19); 4305–11. ©2015 AACR.

Purpose: Up to one third of ovarian cancer patients are intrinsically resistant to platinum-based treatment. However, predictive and therapeutic strategies are lacking due to a poor understanding of the underlying molecular mechanisms. This study aimed to identify key molecular characteristics that are associated with primary chemoresistance in epithelial ovarian cancers. Experimental Design: Gene expression profiling was performed on a discovery set of 85 ovarian tumors with clinically well-defined response to chemotherapies as well as on an independent validation dataset containing 138 ovarian patients from the chemotreatment arm of the ICON7 trial. Results: We identified a distinct “reactive stroma” gene signature that is specifically associated with primary chemoresistant tumors and was further upregulated in posttreatment recurrent tumors. Immunohistochemistry (IHC) and RNA in situ hybridization (RNA ISH) analyses on three of the highest-ranked signature genes (POSTN, LOX, and FAP) confirmed that modulation of the reactive stroma signature genes within the peritumoral stromal compartments was specifically associated with the clinical chemoresistance. Consistent with these findings, chemosensitive ovarian cells grown in the presence of recombinant POSTN promoted resistance to carboplatin and paclitaxel treatment in vitro. Finally, we validated the reactive stroma signature in an independent dataset and demonstrated that a high POSTN expression level predicts shorter progression-free survival following first-line chemotherapy. Conclusions: Our findings highlight the important interplay between cancer and the tumor microenvironment in ovarian cancer biology and treatment. The identified reactive stromal components in this study provide a molecular basis to the further development of novel diagnostic and therapeutic strategies for overcoming chemoresistance in ovarian cancer. Clin Cancer Res; 21(13); 2941–51. ©2015 AACR.

Background: GDC-0941 is a potent and selective oral inhibitor of class I PI3K kinases that demonstrates broad activity in diverse xenograft cancer models and increases the anti-tumor activity of taxanes, associated with increased apoptotic cell death, in multiple BC xenografts. Methods: In the Phase Ib dose-escalation study, GDC4629g, GDC-0941 was administered in combination with paclitaxel dosed at 90 mg/m2 intravenous [IV] weekly for 3 out of 4 weeks, bevacizumab at 10 mg/kg IV biweekly, and trastuzumab 2−4 mg/kg IV weekly in 28-day cycles. The study was conducted in 2 parts: Part 1 evaluated oral doses of 60 and 100 mg GDC-0941 on a "21+7" (21 days on/7 days off) dosing schedule in combination with paclitaxel with and without bevacizumab. Part 2 evaluated GDC-0941 on a "5+2" schedule (5 days on/2 days off in a repeating pattern), at doses of 165 - 330 mg, in combination with paclitaxel (Arm A), paclitaxel plus bevacizumab (Arm B) or paclitaxel plus trastuzumab (Arm C). The primary objective was to evaluate safety, tolerability, and pharmacokinetics (PK), and to determine the maximum tolerated dose (MTD) for each combination. Anti-tumor activity was evaluated by RECIST v1.0 and available archival tumor samples were analyzed to assess PI3K related biomarkers. Results: 63 patients were enrolled: 20 in Part 1 and 43 in Part 2 (18 in Arm A, 16 in Arm B, and 9 in Arm C). The most frequent grade ≥3 drug-related adverse events (AEs) were: Part 1, neutropenia (45%), peripheral neuropathy and nail disorder (15% each); Part 2, Arm A, neutropenia (38%), pneumonia and rash (11% each); Arm B, neutropenia and rash (25% each); Arm C, rash and hypertension (11% each). The most common AEs assessed as being related to GDC-0941 were as follows: Most common AEs related to GDC-0941Adverse EventAll GradesGrade ≥ 3DIARRHEA45 (71.4%)2 (3.2%)NAUSEA33 (52.4%)0NEUTROPENIA29 (46.0%)17 (27.0%)RASH29 (46.0%)7 (11.1%)FATIGUE28 (44.4%)0STOMATITIS19 (30.2%)0DECREASED APPETITE15 (23.8%)1 (1.6%)VOMITING15 (23.8%)0 In Part 1, the MTD was not reached, the maximum administered dose (MAD) of GDC-0941 was 100 mg, a dose-limiting AE was subclavian vein thrombosis. In Part 2, Arm A, the MTD was 250 mg, Grade 3 febrile neutropenia, bacteremia, and rash were DLTs. In Arm B and Arm C, the MAD was 260 mg, Grade 3 rash was a DLT in both arms. No differences in the PK of either GDC 0941 or paclitaxel were observed when administered in combination compared to historical single-agent data. Best tumor response by RECIST was as follows: Part 1, 1 (5%) complete (CR) and 4 (21%) partial responses (PR); Arm A, 1 (6%) CR and 3 (17%) PR; Arm B, 7 (47%) PR; Arm C, 1 (11%) PR. Conclusions: GDC-0941 was generally well-tolerated in combination with paclitaxel with or without bevacizumab or trastuzumab at dose ranges of up to 330 mg at the "5+2" dosing schedule. Anti-tumor activity has been observed in combination with paclitaxel with and without bevacizumab or trastuzumab. Updated biomarker data and associations with clinical outcomes will be presented. Citation Format: Patrick Schöffski, Sara Cresta, Ingrid A Mayer, Hans Wildiers, Isabelle Rooney, Doris Apt, Steve Gendreau, Kari Morrissey, Mark Lackner, Jill Spoerke, Eric Winer. Tolerability and anti-tumor activity of the oral PI3K inhibitor GDC-0941 in combination with paclitaxel, with and without bevacizumab or trastuzumab in patients with locally recurrent or metastatic breast cancer [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 P5-19-10.

Background Epidermal growth factor receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K) are involved in the proliferation and survival of many cancer types. Enhanced antitumor activity may be achieved through combined inhibition of these pathways. We report results for pictilisib (GDC-0941, a class I pan-PI3K inhibitor) plus erlotinib (an EGFR tyrosine kinase inhibitor) in patients with advanced solid tumors. Materials and methods A 3 + 3 dose-escalation study was carried out at a starting daily dose of 60 mg pictilisib on days 1-21 of a 28-day cycle and 150 mg erlotinib from day 2 of cycle 1. The primary objectives of the study were to assess safety and tolerability, identify dose-limiting toxicities (DLTs), estimate the maximum tolerated dose, and identify the recommended phase II dose (RP2D). Evaluation of a dose-expansion cohort at the RP2D was performed. Results Fifty-seven patients were treated in the study. All patients experienced at least one adverse event (AE). Grade ≥3 AEs, serious AEs, and deaths were reported in 38 (66.7%), 19 (33.3%), and 4 (7.0%) patients, respectively. DLTs occurred in nine patients across eight cohorts and the RP2D was determined to be 340 mg pictilisib on a "5 days on, 2 days off" schedule plus 100 mg erlotinib. Two patients (3.5%) experienced partial response and 19 (33.3%) had stable disease. Conclusion Combining pictilisib with erlotinib in patients with advanced solid tumors is feasible; however, antitumor activity is limited. Additional studies may identify patients likely to benefit from combined inhibition of EGFR and PI3K pathways. Implications for practice Combining drugs targeting different signaling pathways in cancer growth and survival could overcome drug resistance and improve antitumor activity. In this first-in-human study for the combination, addition of the PI3K inhibitor pictilisib to the EGFR tyrosine kinase inhibitor erlotinib resulted in toxicity that led to dose and schedule modifications to identify a tolerable recommended phase II dose of 340 mg pictilisib on a "5 days on, 2 days off" schedule plus 100 mg erlotinib daily. The limited antitumor activity observed, however, suggests that additional studies are needed to identify patients most likely to benefit from combined EGFR and PI3K inhibition.

Clinical development of targeted anticancer therapies often involves interrogation of key driver alterations through diagnostic assessment of tumor DNA. However, predictive biomarker assessment of tumor tissue has substantial limitations. Recent clinical studies of circulating tumor DNA suggest such DNA may be a paradigm-changing medium for the diagnosis and management of cancer patients that can provide an up-to-date ‘liquid biopsy’ for use in clinical diagnostic assessment. The field is evolving rapidly and numerous studies have shown that highly sensitive technologies allow for detection of genomic alterations in circulating tumor DNA. Here, we consider how these advances have the potential to shape pharmacodynamic and predictive biomarker assessments in clinical trials by providing comprehensive, real-time molecular assessment in a minimally invasive manner.

Breast cancers are categorized into three subtypes based on protein expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/ERBB2). Patients enroll onto experimental clinical trials based on ER, PR, and HER2 status and, as receptor status is prognostic and defines treatment regimens, central receptor confirmation is critical for interpreting results from these trials. Patients enrolling onto experimental clinical trials in the metastatic setting often have limited available archival tissue that might better be used for comprehensive molecular profiling rather than slide-intensive reconfirmation of receptor status. We developed a Random Forests-based algorithm using a training set of 158 samples with centrally confirmed IHC status, and subsequently validated this algorithm on multiple test sets with known, locally determined IHC status. We observed a strong correlation between target mRNA expression and IHC assays for HER2 and ER, achieving an overall accuracy of 97 and 96 %, respectively. For determining PR status, which had the highest discordance between central and local IHC, incorporation of expression of co-regulated genes in a multivariate approach added predictive value, outperforming the single, target gene approach by a 10 % margin in overall accuracy. Our results suggest that multiplexed qRT-PCR profiling of ESR1, PGR, and ERBB2 mRNA, along with several other subtype associated genes, can effectively confirm breast cancer subtype, thereby conserving tumor sections and enabling additional biomarker data to be obtained from patients enrolled onto experimental clinical trials. Electronic supplementary material The online version of this article (doi:10.1007/s10549-014-3163-8) contains supplementary material, which is available to authorized users.

The phosphatidylinositol-3-kinase pathway plays an important role in proliferation, migration and survival in breast cancer and may play a role in resistance to endocrine therapy. Pathway activation occurs as a result of mutations in PIK3CA or loss of functional PTEN. Matched primary and recurrent samples from 120 breast cancer patients treated with endocrine therapy were profiled with a qPCR-based mutation assay covering eight mutational hotspots in PIK3CA. PTEN was assayed by immunohistochemistry. Samples were well characterized with respect to anatomic location of recurrence (metastatic nodal or local recurrence as opposed to contralateral or ipsilateral new primary cancers). In total, 43 % of patients had at least one PIK3CA mutation at diagnosis, and 41 % had a mutation at the time of recurrence. Only 8 % of patients with local recurrence, metastatic disease or progression on primary endocrine treatment changed their PIK3CA mutation status (four gains, two losses, total 76). The most common changes in PIK3CA mutation status were seen in patients who developed a new cancer either in the treated or contralateral breast (64 %, three gains, four losses, total 11). PIK3CA mutation status does not change in the majority of patients with breast cancer and the acquisition of mutations in PIK3CA is not responsible for the development of endocrine resistance. PTEN loss at diagnosis is associated with a significantly shorter time to progression compared with tumours in which PTEN was retained. These are the most comprehensive data currently available correlating PIK3CA status, site of recurrence and endocrine resistance.

Motion sickness is a complex syndrome that includes many features besides nausea and vomiting. This review describes some of these factors and points out that under normal circumstances, many cases of motion sickness go unrecognized. Motion sickness can occur during exposure to physical motion, visual motion, and virtual motion, and only those without a functioning vestibular system are fully immune. The range of vulnerability in the normal population varies about 10,000 to 1. Sleep deprivation can also enhance susceptibility. Systematic studies conducted in parabolic flight have identified velocity storage of semicircular canal signals-velocity integration-as being a key factor in both space motion sickness and terrestrial motion sickness. Adaptation procedures that have been developed to increase resistance to motion sickness reduce this time constant. A fully adequate theory of motion sickness is not presently available. Limitations of two popular theories, the evolutionary and the ecological, are described. A sensory conflict theory can explain many but not all aspects of motion sickness elicitation. However, extending the theory to include conflicts related to visceral afferent feedback elicited by voluntary and passive body motion greatly expands its explanatory range. Future goals should include determining why some conflicts are provocative and others are not but instead lead to perceptual reinterpretations of ongoing body motion. The contribution of visceral afferents in relation to vestibular and cerebellar signals in evoking sickness also deserves further exploration. Substantial progress is being made in identifying the physiological mechanisms underlying the evocation of nausea, vomiting, and anxiety, and a comprehensive understanding of motion sickness may soon be attainable. Adequate anti-motion sickness drugs without adverse side effects are not yet available.

Purpose: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples. Experimental Design: We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer. Results: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA. Conclusions: MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice. Clin Cancer Res; 20(8); 2080–91. ©2014 AACR.

In a rotating environment, goal-oriented voluntary movements are initially disrupted in trajectory and endpoint, due to movement-contingent Coriolis forces, but accuracy is regained with additional movements. We studied whether adaptation acquired in a voluntary, goal-oriented postural swaying task performed during constant-velocity counterclockwise rotation (10 RPM) carries over to recovery from falling induced using a hold and release (H&R) paradigm. In H&R, standing subjects actively resist a force applied to their chest, which when suddenly released results in a forward fall and activation of an automatic postural correction. We tested H&R postural recovery in subjects ( n = 11) before and after they made voluntary fore-aft swaying movements during 20 trials of 25 s each, in a counterclockwise rotating room. Their voluntary sway about their ankles generated Coriolis forces that initially induced clockwise deviations of the intended body sway paths, but fore-aft sway was gradually restored over successive per-rotation trials, and a counterclockwise aftereffect occurred during postrotation attempts to sway fore-aft. In H&R trials, we examined the initial 10- to 150-ms periods of movement after release from the hold force, when voluntary corrections of movement path are not possible. Prerotation subjects fell directly forward, whereas postrotation their forward motion was deviated significantly counterclockwise. The postrotation deviations were in a direction consistent with an aftereffect reflecting persistence of a compensation acquired per-rotation for voluntary swaying movements. These findings show that control and adaptation mechanisms adjusting voluntary postural sway to the demands of a new force environment also influence the automatic recovery of posture.

Subjects exposed to a rotating environment that perturbs their postural sway show adaptive changes in their voluntary spatially directed postural motion to restore accurate movement paths but do not exhibit any obvious learning during passive stance. We have found, however, that a variable known to characterize the degree of stochasticity in quiet stance can also reveal subtle learning phenomena in passive stance. We extended Chow and Collins (Phys Rev E 52(1):909-912, 1995) one-dimensional pinned-polymer model (PPM) to two dimensions (2-D) and then evaluated the model's ability to make analytical predictions for 2-D quiet stance. To test the model, we tracked center of mass and centers of foot pressures, and compared and contrasted stance sway for the anterior-posterior versus medio-lateral directions before, during, and after exposure to rotation at 10 rpm. Sway of the body during rotation generated Coriolis forces that acted perpendicular to the direction of sway. We found significant adaptive changes for three characteristic features of the mean square displacement (MSD) function: the exponent of the power law defined at short time scales, the proportionality constant of the power law, and the saturation plateau value defined at longer time scales. The exponent of the power law of MSD at a short time scale lies within the bounds predicted by the 2-D PPM. The change in MSD during exposure to rotation also had a power-law exponent in the range predicted by the theoretical model. We discuss the Coriolis force paradigm for studying postural and movement control and the applicability of the PPM model in 2-D for studying postural adaptation.

Background: USON 01062 (O’Shaughnessy J, et al. Proc SABCS, 2010, abst S4-2) showed no improvement in the primary endpoint of disease-free survival (DFS) (median FU 5 yrs: HR 0.84, 95% CI: 0.67-1.05; p = 0.125) with the addition of capecitabine (X) to standard adjuvant chemotherapy, but showed improvement in OS (HR 0.68, 95% CI: 0.51-0.92; p = 0.011). Exploratory analysis of local pathology-assessed Ki67 suggested benefit from adjuvant X in pts with more highly proliferative cancers with Ki67 ≥ 10% (Pippen J et al. Proc ASCO, 2011, abst 500). The objective of this study is to determine whether centrally-performed Ki67 IHC results corroborate or refute this finding. Methods: 2610 pts with resected high risk EBC were randomized to receive 4 cycles of AC (doxorubicin 60mg/m2 and cyclophosphamide 600mg/m2) IV every 3 wks for 4 cycles followed by either docetaxel 100mg/m2 IV or docetaxel 75mg/m2 IV plus X 825mg/m2 PO bid for 14 days every 3 wks for 4 cycles. Archival primary breast cancer tissue was collected on 2000 pts for predictive biomarker analyses. Central Ki67 IHC was performed using the anti-Ki67 monoclonal antibody SP6 and was read by one pathologist (HK) according to published recommendations (Dowsett M, et al. JNCI 103:1-9, 2011). Results: Central Ki67 IHC has been performed on 1440 pts who had centrally-validated informed consents. The distribution of% Ki67-positive cells by locally-assessed ER/HER2 subtype is shown below. 45% of HR+ HER2- BCs had a Ki67 ≤ 10%, while 24% had a Ki67 11% to 20%, and 31% had a Ki67 > 20%. The concordance between the local vs central Ki67 results was low at 46% for Ki67 20%. The central Ki67 results tended to be higher than the local testing results. Central mRNA classifiers were developed for ER, PR, HER2 and Ki67 using Fluidigm Microfluidics Dynamic Arrays and correlate highly with central IHC assessment of these markers. Conclusions: HR+ HER2- EBC is enriched for cancers with a low proliferative rate, a group of pts unlikely to benefit from the cell cycle-specific cytotoxic agent, capecitabine. Analyses of the impact of adjuvant X added to AC/T in EBC pts according to ER status, and according to Ki67 (analyzed as a binary and continuous variable) will be performed prior to SABCS, 2013. Number of Patients% Ki67 Pos CellsTotal *HR+TNHER2+/HR+HER2+/ HR-0-104163622222711-151391066151016-20126871615821-3018411539201031-1005751403423555Total144081042510790*Totals do not equal sum of subtype categories due to missing HER2 information Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-09-01.

We tested an innovative method to estimate joint stiffness and damping during multijoint unfettered arm movements. The technique employs impulsive perturbations and a time-frequency analysis to estimate the arm's mechanical properties along a reaching trajectory. Each single impulsive perturbation provides a continuous estimation on a single-reach basis, making our method ideal to investigate motor adaptation in the presence of force fields and to study the control of movement in impaired individuals with limited kinematic repeatability. In contrast with previous dynamic stiffness studies, we found that stiffness varies during movement, achieving levels higher than during static postural control. High stiffness was associated with elevated reflexive activity. We observed a decrease in stiffness and a marked reduction in long-latency reflexes around the reaching movement velocity peak. This pattern could partly explain the difference between the high stiffness reported in postural studies and the low stiffness measured in dynamic estimation studies, where perturbations are typically applied near the peak velocity point.

We have previously shown that the Coriolis torques that result when an arm movement is performed during torso rotation do not affect movement trajectory. Our purpose in the present study was to examine whether torso motion-induced Coriolis and other interaction torques are counteracted during a turn and reach (T&R) movement when the effective mass of the hand is augmented, and whether the dominant arm has an advantage in coordinating intersegmental dynamics as predicted by the dynamic dominance hypothesis (Sainburg RL. Exp Brain Res 142: 241–258, 2002). Subjects made slow and fast T&R movements in the dark to just extinguished targets with either arm, while holding or not holding a 454-g object. Movement endpoints were equally accurate at both speeds, with either hand, and in both weight conditions, but subjects tended to angularly undershoot and produce more variable endpoints for targets requiring greater torso rotation. There were no changes in endpoint accuracy or trajectory deviation over repeated movements. The dominant right arm was more stable in its control of trajectory direction across targets, whereas the nondominant left arm had an improved ability to stop accurately on the target for higher levels of interaction torques. The trajectories to more eccentric targets were straighter when performed at higher speeds but slightly more deviated when subjects held the weight. Subjects did not slow their torso velocity or change the timing of the arm and torso velocities when holding the weight, although there was a slight decrease in their hand velocity relative to the torso. The delay between the onsets of torso and finger movements was almost twice as large for the right arm than the left, suggesting the right arm was better able to account for torso rotation in the arm movement. Holding the weight increased the peak Coriolis torque by 40% at the shoulder and 45% at the elbow and, for the most eccentric target, increased the peak net torque by 12% at the shoulder and 34% at the elbow. In accordance with Sainburg's dynamic dominance hypothesis, the right arm exhibited an advantage for coordinating intersegmental dynamics, showing a more stable finger velocity in relation to the torso across targets, decreasing error variability with movement speed, and more synchronized peaks of finger relative and torso angular velocities in conditions with greater joint torque requirements. The arm used had little effect on the movement path and the magnitude of the joint torques in any of the conditions. These results indicate that compensations for forthcoming Coriolis torque variations take into account the dynamic properties of the body and of external objects, as well as the planned velocities of the torso and arm.

ER-targeted therapeutics provide valuable treatment options for patients with ER+ breast cancer, however, current relapse and mortality rates emphasize the need for improved therapeutic strategies. The recent discovery of prevalent ESR1 mutations in relapsed tumors underscores a sustained reliance of advanced tumors on ERα signaling, and provides a strong rationale for continued targeting of ERα. Here we describe GDC-0810, a novel, non-steroidal, orally bioavailable selective ER downregulator (SERD), which was identified by prospectively optimizing ERα degradation, antagonism and pharmacokinetic properties. GDC-0810 induces a distinct ERα conformation, relative to that induced by currently approved therapeutics, suggesting a unique mechanism of action. GDC-0810 has robust in vitro and in vivo activity against a variety of human breast cancer cell lines and patient derived xenografts, including a tamoxifen-resistant model and those that harbor ERα mutations. GDC-0810 is currently being evaluated in Phase II clinical studies in women with ER+ breast cancer. DOI: http://dx.doi.org/10.7554/eLife.15828.001

Breast cancer is a heterogeneous disease and patients are managed clinically based on ER, PR, HER2 expression, and key risk factors. We sought to characterize the molecular landscape of high-risk breast cancer patients enrolled onto an adjuvant chemotherapy study to understand how disease subsets and tumor immune status impact survival. DNA and RNA were extracted from 861 breast cancer samples from patients enrolled onto the United States Oncology trial 01062. Samples were characterized using multiplex gene expression, copy number, and qPCR mutation assays. HR+ patients with a PIK3CA mutant tumor had a favorable disease-free survival (DFS; HR 0.66, P=0.05), however, the prognostic effect was specific to luminal A patients (Luminal A: HR 0.67, P=0.1; Luminal B: HR 1.01, P=0.98). Molecular subtyping of triple-negative breast cancers (TNBCs) suggested that the mesenchymal subtype had the worst DFS, whereas the immunomodulatory subtype had the best DFS. Profiling of immunologic genes revealed that TNBC tumors (n=280) displaying an activated T-cell signature had a longer DFS following adjuvant chemotherapy (HR 0.59, P=0.04), while a distinct set of immune genes was associated with DFS in HR+ cancers. Utilizing a discovery approach, we identified genes associated with a high risk of recurrence in HR+ patients, which were validated in an independent data set. Molecular classification based on PAM50 and TNBC subtyping stratified clinical high-risk patients into distinct prognostic subsets. Patients with high expression of immune-related genes showed superior DFS in both HR+ and TNBC. These results may inform patient management and drug development in early breast cancer.

Summary Background Inhibition of phosphatidylinositol 3-kinase (PI3K) is a promising approach to overcome resistance to endocrine therapy in breast cancer. Pictilisib is an oral inhibitor of multiple PI3K isoforms. The aim of this study is to establish if addition of pictilisib to fulvestrant can improve progression-free survival in oestrogen receptor-positive, endocrine-resistant breast cancer. Methods In this two-part, randomised, double-blind, placebo-controlled, phase 2 study, we recruited postmenopausal women aged 18 years or older with oestrogen receptor-positive, HER2-negative breast cancer resistant to treatment with an aromatase inhibitor in the adjuvant or metastatic setting, from 123 medical centres across 21 countries. Part 1 included patients with or without PIK3CA mutations, whereas part 2 included only patients with PIK3CA mutations. Patients were randomly allocated (1:1 in part 1 and 2:1 in part 2) via a computer-generated hierarchical randomisation algorithm to daily oral pictilisib (340 mg in part 1 and 260 mg in part 2) or placebo starting on day 15 of cycle 1, plus intramuscular fulvestrant 500 mg on day 1 and day 15 of cycle 1 and day 1 of subsequent cycles in both groups. In part 1, we stratified patients by presence or absence of PIK3CA mutation, primary or secondary aromatase inhibitor resistance, and measurable or non-measurable disease. In part 2, we stratified patients by previous aromatase inhibitor treatment for advanced or metastatic disease or relapse during or within 6 months of an aromatase inhibitor treatment in the adjuvant setting and measurable or non-measurable disease. All patients and those administering treatment and assessing outcomes were masked to treatment assignment. The primary endpoint was progression-free survival in the intention-to-treat population for both parts 1 and 2 and also separately in patients with PIK3CA -mutated tumours in part 1. Tumour assessment (physical examination and imaging scans) was investigator-assessed and done at screening and after 8 weeks, 16 weeks, 24 weeks, and 32 weeks of treatment from day 1 of cycle 1 and every 12 weeks thereafter. We assessed safety in as-treated patients who received at least one dose of study medication. This trial is registered with ClinicalTrials.gov, number NCT01437566. Findings In part 1, between Sept 27, 2011, and Jan 11, 2013, we randomly allocated 168 patients to the pictilisib (89 [53%]) or placebo (79 [47%]) group. In part 2, between March 18, 2013, and Jan 2, 2014, we randomly allocated 61 patients to the pictilisib (41 [67%]) or placebo (20 [33%]) group. In part 1, we found no difference in median progression-free survival between the pictilisib (6·6 months [95% CI 3·9–9·8]) and placebo (5·1 months [3·6–7·3]) group (hazard ratio [HR] 0·74 [95% CI 0·52–1·06]; p=0·096). We also found no difference when patients were analysed according to presence (pictilisib 6·5 months [95% CI 3·7–9·8] vs placebo 5·1 months [2·6–10·4]; HR 0·73 [95% CI 0·42–1·28]; p=0·268) or absence (5·8 months [3·6–11·1] vs 3·6 months [2·8–7·3]; HR 0·72 [0·42–1·23]; p=0·23) of PIK3CA mutation. In part 2, we also found no difference in progression-free survival between groups (5·4 months [95% CI 3·8–8·3] vs 10·0 months [3·6–13·0]; HR 1·07 [95% CI 0·53–2·18]; p=0·84). In part 1, grade 3 or worse adverse events occurred in 54 (61%) of 89 patients in the pictilisib group and 22 (28%) of 79 in the placebo group. 19 serious adverse events related to pictilisib treatment were reported in 14 (16%) of 89 patients. Only one (1%) of 79 patients reported treatment-related serious adverse events in the placebo group. In part 2, grade 3 or worse adverse events occurred in 15 (36%) of 42 patients in the pictilisib group and seven (37%) of 19 patients in the placebo group. Four serious adverse events related to pictilisib treatment were reported in two (5%) of 42 patients. One treatment-related serious adverse event occurred in one (5%) of 19 patients in the placebo group. Interpretation Although addition of pictilisib to fulvestrant did not significantly improve progression-free survival, dosing of pictilisib was limited by toxicity, potentially limiting its efficacy. For future assessment of PI3K inhibition as an approach to overcome resistance to hormonal therapy, inhibitors with greater selectivity than that of pictilisib might be needed to improve tolerability and potentially increase efficacy. No further investigation of pictilisib in this setting is ongoing. Funding F Hoffmann-La Roche.

Mutations in ESR1 have been associated with resistance to aromatase inhibitor (AI) therapy in patients with ER+ metastatic breast cancer. Little is known of the impact of these mutations in patients receiving selective oestrogen receptor degrader (SERD) therapy. In this study, hotspot mutations in ESR1 and PIK3CA from ctDNA were assayed in clinical trial samples from ER+ metastatic breast cancer patients randomized either to the SERD fulvestrant or fulvestrant plus a pan-PI3K inhibitor. ESR1 mutations are present in 37% of baseline samples and are enriched in patients with luminal A and PIK3CA-mutated tumours. ESR1 mutations are often polyclonal and longitudinal analysis shows distinct clones exhibiting divergent behaviour over time. ESR1 mutation allele frequency does not show a consistent pattern of increases during fulvestrant treatment, and progression-free survival is not different in patients with ESR1 mutations compared with wild-type patients. ESR1 mutations are not associated with clinical resistance to fulvestrant in this study., Fulvestrant degrades the oestrogen receptor. Here, the authors report on a clinical trial using fulvestrant and show that mutations in the oestrogen receptor alpha gene are prevalent in circulating tumour DNA and do not influence the clinical outcome of patients to fulvestrant.

The current single-arm, open-label trial was designed to evaluate the activity of apitolisib (GDC-0980), a dual phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor, in patients with advanced endometrial cancer (EC).Patients with recurrent or persistent EC who were treated with 1 to 2 prior lines of chemotherapy but no prior PI3K/mTOR inhibitor received oral apitolisib at a dose of 40 mg daily during 28-day cycles until disease progression or intolerable toxicity occurred. Patients with type I/II diabetes who required insulin were excluded. The primary endpoints were progression-free survival (PFS) at 6 months and objective response rate.A total of 56 women were enrolled, including 13 (23%) with well-controlled diabetes. Reasons for discontinuation were disease progression (24 patients; 43%), adverse events (13 patients; 23%), and withdrawal by subject (12 patients; 21%). Grade 3/4 apitolisib-related adverse events were hyperglycemia (46%), rash (30%), colitis (5%), and pneumonitis (4%) (toxicities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.0]). The PFS rate at 6 months was 20% (Kaplan-Meier estimate; 95% confidence interval [95% CI], 7%-33%). The objective response rate was 6% (confirmed). The median PFS was 3.5 months (95% CI, 2.7-3.7 months) and the median overall survival was 15.7 months (95% CI, 9.2-17.0 months). Nineteen patients discontinued the study before the first tumor assessment. Dose reductions were required for 4 diabetic (31%) and 18 nondiabetic (42%) patients. Comprehensive molecular profiling of 46 evaluable archival tumor samples demonstrated that 57% of patients had at least 1 alteration in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphatase and tensin homolog (PTEN), or AKT1. All 3 patients with a confirmed response had at least 1 alteration in a PI3K pathway gene.The antitumor activity noted with the use of a dose of 40 mg of apitolisib daily was limited by tolerability, especially in diabetic patients. Patients with PI3K pathway mutations may have derived enhanced benefit from apitolisib. Cancer 2016;122:3519-28. © 2016 American Cancer Society.

The solution structure of the cold-shock-like protein from R. rickettsii, the causative agent of Rocky Mountain spotted fever, is reported., Rocky Mountain spotted fever is caused by Rickettsia rickettsii infection. R. rickettsii can be transmitted to mammals, including humans, through the bite of an infected hard-bodied tick of the family Ixodidae. Since the R. rickettsii genome contains only one cold-shock-like protein and given the essential nature of cold-shock proteins in other bacteria, the structure of the cold-shock-like protein from R. rickettsii was investigated. With the exception of a short α-helix found between β-strands 3 and 4, the solution structure of the R. rickettsii cold-shock-like protein has the typical Greek-key five-stranded β-barrel structure found in most cold-shock domains. Additionally, the R. rickettsii cold-shock-like protein, with a ΔG of unfolding of 18.4 kJ mol−1, has a similar stability when compared with other bacterial cold-shock proteins.

A custom-made 228Th source of several MBq activity was produced for the Borexino experiment for studying the external background of the detector. The aim was to reduce the unwanted neutron emission produced via (alpha,n) reactions in ceramics used typically for commercial 228Th sources. For this purpose a ThCl4 solution was converted chemically into ThO2 and embedded into a gold foil. The paper describes the production and the characterization of the custom-made source by means of gamma-activity, dose rate and neutron source strength measurements. From gamma-spectroscopic measurements it was deduced that the activity transfer from the initial solution to the final source was >91% (at 68% C.L.) and the final activity was (5.41+-0.30) MBq. The dose rate was measured by two dosimeters yielding 12.1 mSv/h and 14.3 mSv/h in 1 cm distance. The neutron source strength of the 5.41 MBq 228Th source was determined as (6.59+-0.85)/sec., 8 pages, 5 figures

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor–resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients. Mol Cancer Ther; 11(5); 1143–54. ©2012 AACR.

Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non–small cell lung cancer (NSCLC). Experimental Design: Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-d-glucose–positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints. Results: CTCs were detected (≥1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation detection was observed in ctDNA than in CTCs and detected mutations were strongly concordant with mutation status in matched tumor. Higher baseline CTC counts were associated with response to treatment by Response Evaluation Criteria in Solid Tumors (RECIST, P = 0.009) and decreased CTC counts upon treatment were associated with FDG-PET and RECIST response (P = 0.014 and P = 0.019) and longer progression-free survival (P = 0.050). Conclusion: These data provide evidence of a correlation between decreases in CTC counts and radiographic response by either FDG-PET or RECIST in patients with advanced NSCLC. These findings require prospective validation but suggest a potential role for using CTC decreases as an early indication of response to therapy and ctDNA for real-time assessment of mutation status from blood. Clin Cancer Res; 18(8); 2391–401. ©2012 AACR.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog (PTEN), and deregulation of mammalian target of rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceutical properties. GDC-0980 potently inhibits signal transduction downstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980 was potent across a broad panel of cancer cell lines, with the greatest potency in breast, prostate, and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistance markers. Treatment of cancer cell lines with GDC-0980 resulted in G1 cell-cycle arrest, and in contrast to mTOR inhibitors, GDC-0980 induced apoptosis in certain cancer cell lines, including those with direct pathway activation via PI3K and PTEN. Low doses of GDC-0980 potently inhibited tumor growth in xenograft models including those with activated PI3K, loss of LKB1 or PTEN, and elicited an exposure-related decrease in PD biomarkers. These preclinical data show that GDC-0980 is a potent and effective dual PI3K/mTOR inhibitor with promise for the clinic. Mol Cancer Ther; 10(12); 2426–36. ©2011 AACR.

We studied the contribution of vestibular and somatosensory/proprioceptive stimulation to the perception of the apparent zenith (AZ). Experiment 1 involved rotation on a centrifuge and settings of the AZ. Subjects were supine on the centrifuge, and their body position was varied in relation to the rotation axis so that the gravitoinertial resultant force at the otoliths was 1 or 1.2 g with the otolith organs positioned 50 or 100 cm from the axis of rotation. Their legs were also positioned in different configurations, flexed and elevated or extended, to create different distributions of blood and lymph. Experiment 2 involved (a) settings of the AZ for subjects positioned supine with legs fully extended or legs flexed and elevated to create a torsoward shift of blood and (b) settings of the subjective visual vertical for subjects horizontally positioned on their sides with legs extended or bent. Experiment 3 had subjects in the same body configurations as in Experiment 2 indicate when they were horizontal as they were rotated in pitch or roll about an inter-aural or naso-occipital axis. The experimental results for all three experiments demonstrated that both visual localization and apparent body horizontal are jointly determined by multimodal combinations of otolithic and somatosensory/proprioceptive stimulation. No evidence was found for non-overlapping or exclusive mechanisms determining one or the other. The subjective postural horizontal and AZ were affected in similar ways by comparable manipulations.

Gastric cancer (GC) is the fifth most common cancer diagnosis with roughly 60% of patients with metastatic disease. Tumor heterogeneity represents a major challenge to personalized cancer therapy. Intratumor heterogeneity can result in missing critical DNA genomic alterations using conventional tumor biopsies. Circulating cell-free DNA (cfDNA) in the peripheral blood has the potential to capture DNA alterations in a more dynamic manner in particular types of advanced tumors where feasibility of repeat tissue biopsies is limited. Using low-pass whole-genome sequencing (LP-WGS) data from 329 blood plasma samples collected from 63 1L metastatic (mGC) patients enrolled in phase II trial NCT01590719, receiving either onartuzumab or placebo in combination with mFOLFOX6, we evaluated the global percent genome change (%GC) and copy number (CN) gain at baseline and throughout the course of treatment. cfDNA samples were isolated with the automated QIAsymphony system and analyzed on the Illumina NextSeq 500 system with a novel LP-WGS protocol at 0.5x coverage. Seven chromosomes (chr1, 7, 8, 14, 15, 20, and 21) showed the most frequent CN gains of evaluated patients (≥15%). Of these seven chromosomes, chr8 and 20 featured the largest number of genes at baseline with CN gains with 30.2% (19/63) at chr8 and 25.4% (16/63) at chr20. 81% of patients (13/16) with chr20 CN gain also showed an increase in CN at chr8. Patients with gains at chr8 and/or 20 have higher %GC compared to patients with normal copy status. Recurrent CN gains at chr8 and 20 in tumor tissues were previously reported in GC patients and correlated with tumor progression. Copy number gains at chr8 and 20 in cfDNA from mGC patients, reported here for the first time, suggests a potential utility for early detection of disease progression. Moreover, patients with CN gains at chr8 and/or 20 had higher circulating tumor cells detected at baseline (p=0.003) and also showed the most decrease in %GC post-treatment. 75% (9/12) of the patients with CN gains at chr8 and/or 20 were responders and showed a mean 3-fold decrease (range of 1.6-4.7 fold change) in tumor size post-treatment. This suggests CN gain at chr8 or 20 in cfDNA could be used as a noninvasive measure to identify a novel subset of mGC patients sensitive to chemo-based treatment. When comparing %GC pre- and post-treatment, the %GC decreased significantly at C2D1 (p=0.006). This decrease in %GC correlates with a decrease in tumor burden in patients with response, while %GC and tumor burden remain unchanged or increased in nonresponders. Monitoring %GC pre- and post-treatment demonstrates the ability to assess the depth of the treatment and early detection of disease progression. Findings from this study suggest that plasma genomic abnormality monitoring may be used for treatment decision-making in mGC and should be evaluated further in clinical trials. Citation Format: An D. Do, Charles D. Baudo, Samir Seshadri, Xiaoji Chen, Rebecca Suttmann, David S. Shames, Mark R. Lackner, Shih-Min A. Huang, Teiko Sumiyoshi. Changes in plasma genomic abnormalities at chromosome 8 and 20 predict treatment response and monitor disease progression in advanced gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5571.

To assess vestibular influences on recovery of balance during sudden falls, we measured the postural responses of five healthy subjects to a hold and release perturbation coupled with galvanic vestibular stimulation (GVS). Two electrode pairs were located with the anterior electrode of each pair over the mastoid process and the posterior electrode over the trapezius muscle on the same side. Bilateral unipolar GVS was generated 60 ms after a holding force against the sternum was released by individually driving left and right electrode pairs as cathode or anode at 1 mA for 12 s or 2 mA for 6 s. We computed the frequency and damping parameters of a multi-link inverted pendulum model of the body which best fit the transient postural oscillations after release for each subject. These parameters did not differ significantly across conditions indicating the GVS did not modify the preset overall strategy of postural recovery. The intensity and polarity of GVS significantly biased both the postural lean during the oscillatory period and the resting postural stance achieved during stimulation, deviating them forward for cathodal stimulation and backward for anodal. The residuals of the multi-link fit, the frequency spectra of the actual body sway ripples about the modeled sway, were different across conditions. Because GVS affected postural bias but not dynamics, it is likely that it provided erroneous velocity signals leading to vestibulospinal compensations in segmental stiffness and damping mechanisms. Our findings are consistent with theoretical analyses of the influence of GVS on the semicircular canals and otolith organs of the inner ear.

Signaling through the insulin-like growth factor receptor (IGF-1R) is required for neoplastic transformation by a number of oncogenes, and preclinical validation studies have suggested IGF-1R is an attractive target for anticancer therapy. A number of small molecules and antibodies targeting IGF-1R have entered clinical development, and early results have suggested that these agents have generally acceptable safety profiles as single agents. Some evidence of antitumor activity has also been reported. This review highlights key aspects of the IGF-1R signaling pathway that implicate it as an attractive therapeutic target in the management of cancer, as well as some key lessons that have emerged from early clinical development of anti-IGF-1R targeting agents. In addition, we consider the importance of selecting indications characterized by pathological alterations in the signaling pathway, rational selection of combinations based on signaling pathway interactions, and strategies for patient selection based on analysis of predictive biomarkers. Clin Cancer Res; 16(9); 2512–7. ©2010 AACR.

Individuals exposed to both an increase in magnitude and a rotation of the gravitoinerital acceleration vector experience changes in visual and auditory localization and apparent body orientation. These effects are known as the oculogravic, audiogravic and somatogravic illusions, respectively. In our main experiment, we measured the magnitude, direction, and time course of the audiogravic and oculogravic illusions in recumbent subjects (n = 6) exposed to an increase to 2 g and a simultaneous 60 degrees rotation of the gravitoinertial acceleration (GIA) vector in their azimuthal plane (around their z-axis) in the Brandeis slow rotation room. In separate runs, subjects used a pointer to indicate the apparent azimuthal location of auditory and visual targets and of their head midline. Parallel, time linked changes in auditory, visual, and haptic localization of comparable magnitude and direction occurred. Two additional subjects adjusted the onset asynchrony of a pair of pulsing tactors on the forehead until they experienced a fused, midline sensation. A vibrotactile shift occurred during exposure to altered GIA in the same direction as the auditory, visual, and haptic shifts. These parallel multimodal results point to a GIA-induced remapping of a peripersonal spatial referent as underlying the changes in localization of auditory, visual, and somatosensory stimuli. Vestibular, somatosensory, and oculomotor signals could contribute to the proposed shift in the reference system.

The insulin-like growth factor-I receptor (IGF-IR) pathway is required for the maintenance of the transformed phenotype in neoplastic cells and hence has been the subject of intensive drug discovery efforts. A key aspect of successful clinical development of targeted therapies directed against IGF-IR will be identification of responsive patient populations. Toward that end, we have endeavored to identify predictive biomarkers of response to an anti-IGF-IR-targeting monoclonal antibody in preclinical models of breast and colorectal cancer. We find that levels of the IGF-IR itself may have predictive value in these tumor types and identify other gene expression predictors of in vitro response. Studies in breast cancer models suggest that IGF-IR expression is both correlated and functionally linked with estrogen receptor signaling and provide a basis for both patient stratification and rational combination therapy with antiestrogen-targeting agents. In addition, we find that levels of other components of the signaling pathway such as the adaptor proteins IRS1 and IRS2, as well as the ligand IGF-II, have predictive value and report on the development of a pathway-focused panel of diagnostic biomarkers that could be used to test these hypotheses during clinical development of IGF-IR-targeting therapies. [Mol Cancer Ther 2009;8(8):2110–21]

Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease. We investigated the role of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer. Experimental Design: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models. Results: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes. We also showed that loss of PTEN is a negative predictor of response to MEK inhibition, that treatment with a selective MEK inhibitor caused up-regulation of PI3K pathway signaling, and that dual blockade of both PI3K and MEK/extracellular signal–regulated kinase signaling synergized to potently impair the growth of basal-like breast cancer models in vitro and in vivo. Conclusions: Our studies suggest that single-agent MEK inhibition is a promising therapeutic modality for basal-like breast cancers with intact PTEN, and also provide a basis for rational combination of MEK and PI3K inhibitors in basal-like cancers with both intact and deleted PTEN.

Molecular profiling of human cancer is complicated by both stromal contamination and cellular heterogeneity within samples from tumor biopsies. In this study, we developed a tissue-processing protocol using mechanical dissociation and flow cytometric sorting that resulted in the respective enrichment of stromal and tumor fractions from frozen pancreatic adenocarcinoma samples. Molecular profiling of DNA from the sorted populations using high-density single nucleotide polymorphism arrays revealed widespread chromosomal loss of heterozygosity in tumor fractions but not in either the stromal fraction or unsorted tissue specimens from the same sample. Similarly, a combination of KRAS mutations and chromosomal copy number changes at key pancreatic cancer loci, such as CDK2NA and TP53, was detected in a substantial proportion of the tumor fractions but not in matched stromal fractions from the same sample. This approach to tissue processing could greatly expand the amount of archived tissue that is available for molecular profiling of human cancer and enable a more accurate diagnosis of genetic alterations in patient samples.

We had recumbent subjects (n = 7) indicate the amplitude of imposed, passive yaw-axis body rotations in the 0, 1, and 1.8 g background force levels generated during parabolic flight maneuvers. The blindfolded subject, restrained in a cradle, aligned a gravity-neutral pointer with the subjective vertical while in an initial position and then tried to keep it aligned with the same external direction during a body rotation, lasting less than 1.5 s about the z-axis 30 degrees, 60 degrees, or 120 degrees in amplitude. All the rotations were above semicircular threshold levels for eliciting perception of angular displacement under terrestrial test conditions. In 1 and 1.8 g test conditions, subjects were able to indicate both the subjective vertical and the amplitude of the body rotation reasonably accurately. By contrast in 0 g, when indicating the subjective vertical, they aligned the pointer with the body midline and kept it nearly aligned with their midline during the subsequent body tilts. They also reported feeling supine throughout the 0 g test periods. The attenuation of apparent self-displacement in 0 g is discussed in terms of (1) a possible failure of integration of semicircular canal velocity signals, (2) a contribution of somatosensory pressure and contact cues, and (3) gravicentric versus body-centric reference frames. The significance of the findings for predicting and preventing motion sickness and disorientation in orbital space flight and in rotating artificial gravity environments is discussed.

Breast cancers can be divided into subtypes with important implications for prognosis and treatment. We set out to characterize the genetic alterations observed in different breast cancer subtypes and to identify specific candidate genes and pathways associated with subtype biology. mRNA expression levels of estrogen receptor, progesterone receptor, and HER2 were shown to predict marker status determined by immunohistochemistry and to be effective at assigning samples to subtypes. HER2+ cancers were shown to have the greatest frequency of high-level amplification (independent of the ERBB2 amplicon itself), but triple-negative cancers had the highest overall frequencies of copy gain. Triple-negative cancers also were shown to have more frequent loss of phosphatase and tensin homologue and mutation of RB1, which may contribute to genomic instability. We identified and validated seven regions of copy number alteration associated with different subtypes, and used integrative bioinformatics analysis to identify candidate oncogenes and tumor suppressors, including ERBB2, GRB7, MYST2, PPM1D, CCND1, HDAC2, FOXA1, and RASA1. We tested the candidate oncogene MYST2 and showed that it enhances the anchorage-independent growth of breast cancer cells. The genome-wide and region-specific differences between subtypes suggest the differential activation of oncogenic pathways. (Mol Cancer Res 2009;7(4):511–22)