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Background: Compared to day workers, shift workers face an elevated risk of cardiovascular disease. We reviewed the evidence to address the research question: Does acute exposure to shift work impact (blunt) the natural drop (dip) in Blood Pressure (BP) occurring during sleep and/or nighttime hours? (PROSPERO CRD42018110847). Methods: We performed a systematic review of five databases. We compared pooled estimates of mean BP stratified by periods of shift work, rest/leisure, and sleep, and evaluated the quality of evidence with the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) framework. Results: Our search covered 1/1/1980-10/24/2018 and yielded 1,636 records. Inter-rater agreement during screening was high (Kappa=0.87). We retained 44 studies described in 50 publications. We identified wide variation in shift worker type, shift schedules, and regularity of BP measurements. Most studies examined BP during one shift workday and one rest/leisure day. No study examined the impact of repeated exposure to shift work on the sleep-related dip in BP. Eighteen studies examined night shifts and one reported on BP during sleep post night shift. Compared to BP measured during shift work, BP measured during any sleep period separate from shift work was lower by 17.5 mmHg Systolic BP (95%CI 15.75, 19.27) and 15.4 mmHg lower for Diastolic BP (95%CI 14.38, 16.42) (p Conclusions: There is limited research exploring the acute and long-term impact of shift work on BP during sleep. The available evidence is heterogenous, low quality, and suggests that the mean dip in BP during sleep separate from shift work is not blunted.

Background: Cancer gene panels such as Foundation One are widely used clinically for aiding with cancer treatment decision-making since single point mutations in important genes and gene pathways in the tumor can point to tumor susceptibility that can be targeted with specific drugs. Tumor mutational load has more recently been proposed as a useful prognostic factor and indicator of clinical benefit in treatment with PD-1 and CTLA-4 blockade therapy. Breast cancer estrogen receptor positive patients (ER+) in particular appear to have a subset of high mutational load patients that could benefit if identified. However whole exome sequencing needed to directly measure mutational load is not yet widely available as a clinical tool. In this study we evaluate the predictive value of Foundation One cancer gene panel mutations for estimating tumor genome-wide mutational load and its use in identifying a clinically meaningful subset of breast cancer patients. Methods: The Cancer Genome Atlas breast cancer sequencing data on 569 ER+ patients was used to establish mutational load distribution. ER+ patients were divided into low mutational load and high mutational load groups according to 3 criteria: mean mutational load, the point of inflection in the mutational load distribution and the mutational load that optimally separates groups in terms of survival. Foundation One (FO) mutational load was then calculated as the number of mutations present within the 314 genes queried by the panel. FO mutational load was used to predict whether patients fell into the low or high mutational load groups found through analysis of the full exome data. Receiver Operating Characteristic (ROC) curves were constructed and optimal values for specificity and sensitivity of the FO mutational load classification were found. Results: Mean mutational load for ER+ patients was found to be 57 mutations, the point of inflection of the mutational load distribution was established at 100 mutations, and the number of mutations that best separated groups in terms of survival was 160, (HR = 6.6, p-value=0.004). FO mutational load was found to be a good predictor for the low and high classifications established by all three criteria, with areas under the curve of 0.74, 0.91 and 0.945 respectively. The optimal predictive value of the FO mutational load classification was found at 5 mutations as the cut-off, with 94.2% specificity and 88% sensitivity for predicting groups defined by survival and 95% specificity and 71% sensitivity for those defined by the mean. Conclusion: The Foundation One cancer gene panel can be used to effectively identify a clinically meaningful subgroup of ER+ patients with high mutational load. These patients may benefit from targeted treatments such as PD-1 inhibitors being offered through clinical trials. The compromise in sensitivity that results from the reduction in number of genes queried by a panel means an important proportion of patients with high mutational load will be missed but this still translates to a large improvement in the identification of these patients given the wide availability of gene panels in the clinic. Basic and clinical follow-up studies need to take place to clinically validate the high mutational load ER+ patient subgroup. Citation Format: Raska P, Abraham J, Budd T. Detecting high mutational load ER+ breast cancer patients through Foundation One cancer gene panel mutations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-22.

Clinical management of bacterial vaginosis (BV) is difficult owing to inaccurate diagnostic tests, limited drug choices, and a high rate of recurrence. To our knowledge, there has not been a previous study of antimicrobial resistance (AMR) genes in community practice using next-generation sequencing (NGS). A case–control study (1 : 1 age-matched with and without BV) was undertaken in a series of 326 nongravid women of reproductive age with symptoms of BV to determine the prevalence of AMR genes. NGS was used to describe the complete vaginal microbiota and identify bacterial genes associated with resistance to: macrolides and/or lincosamides – ermA, ermB, ermC, erM, ermTR and mefA; tetracyclines, β-lactams, streptomycin, gentamicin and/or tobramycin – acrA, acrB, mecA, tet, tetA, tolC and aac2; 5-nitroimadazoles – nim and nimB; and triazoles – cdr1 and mdr1. An evidence base was created to inform treatment decisions applicable to individual patients. AMR genes were identified in all drug classes: macrolides, 35.2 %; lincosamides, 35.6 %; tetracyclines, 21.8 %; aminoglycosides (streptomycin, gentamicin and tobramycin), 5.2 % each; 5-nitroimidazoles, 0.3 %; and triazoles, 18.7 %. There was more than a fourfold-higher frequency of AMR genes in pathogens from BV than from non-BV patients for macrolides (58.2 versus 12.3 %, respectively), lincosamides (58.9 versus 12.3 %) and tetracyclines (35.6 versus 8.0 %) (Fisher's exact test; all p < 0.001). For each patient with BV, the spectrum of resistance genes was matched to the pathogens present. AMR genes were present in the majority of vaginal microbiomes of patients with symptoms of BV.

Background: Endocrine therapy resistance in advanced breast cancer remains a significant clinical problem that may be overcome with use of histone deacetylase (HDAC) inhibitors such as entinostat. The ENCORE 301 randomized phase II study reported an improvement in progression-free (PFS) and overall survival (OS) with the addition of entinostat to the steroidal aromatase inhibitor (AI) exemestane in patients with advanced hormone receptor (HR)-positive, HER2-negative breast cancer. Protein lysine acetylation in peripheral blood mononuclear cells (PBMCs) was associated with prolonged PFS in the entinostat arm. Methods: E2112 is a multicenter randomized double-blind, placebo-controlled phase III study that enrolled men or women with advanced HR-positive, HER2-negative breast cancer whose disease had progressed on a non-steroidal AI in the adjuvant or metastatic setting (NCT02115282). Study participants were also required to have an ECOG performance status 0-1 with measurable or non-measurable (limited to 20% of the study population) disease. One prior chemotherapy for metastatic disease and prior treatment with fulvestrant and a CDK4/6 inhibitor was permitted but not required. Participants received exemestane 25mg po daily and entinostat (EE)/placebo (EP) 5mg po every week. Primary endpoints were PFS (central review) and OS. One-sided type 1 error 0.025 was split between two hypothesis tests: 0.001 for PFS test and 0.024 for OS. PFS tested in the first 360 pts, 88.5% power to detect 42% reduction in the hazard of PFS failure (median PFS, 4.1 to 7.1 months); OS tested in all 600 pts, 80% power to detect 25% reduction in the hazard of death (median OS, 22 to 29.3 months). Secondary endpoints included safety, objective response rate (ORR), and changes in protein lysine acetylation status in PBMCs (CD3+ T cells, CD14+ monocytes, CD19+ B cells, pan-leukocyte marker CD45+ cells, CD56+ NK cells) between C1D1 and C1D15 (integrated biomarker). Results: A total of 608 participants were randomized between March 2014 and October 2018 (305 EE, 303 EP), 98% enrolled in USA. Characteristics were well balanced between the arms. Median age was 63 years (range, 29-91), 99% female, 95% postmenopausal, 80% white and 15% black. A majority (84%) had disease resistant to AI in the metastatic setting at study entry, 78% had measurable disease and 60% visceral disease. Prior treatments included chemotherapy (60%), fulvestrant (30%), CDK4/6 inhibitor (35%), everolimus (3%). Median prior lines of chemotherapy was 1 (range, 0-4) and endocrine therapy was 2 (range, 1-7); in adjuvant/metastatic setting. Grade 3/4 adverse events in EE arm included neutrophil count decreased (20%), hypophosphatemia (14%), anemia (8%), white blood cell decreased (6%), fatigue (4%), diarrhea (4%), and platelet count decreased (3%). At final analysis, median PFS was 3.3 months (EE) versus 3.1 months (EP) (HR=0.87, 95% CI: 0.67, 1.13, p=0.30). Median OS was 23.4 months (EE) versus 21.7 months (EP) (HR=0.99, 95% CI: 0.82, 1.21, p=0.94). ORR was 4.6% (EE) and 4.3% (EP). The median fold change in lysine acetylation in PBMCs was approximately 1.5 in EE arm, and 1 in EP arm. Participants on EE had significantly higher increase in lysine acetylation by C1D15 than patients on EP (397 paired samples available for analysis, p Conclusion: The combination of exemestane and entinostat did not improve survival in AI resistant advanced HR-positive, HER2-negative breast cancer. Pharmacodynamic analysis confirmed target inhibition in entinostat-treated patients. Citation Format: Roisin M Connolly, Fengmin Zhao, Kathy D Miller, Min-Jung Lee, Richard L Piekarz, Karen L Smith, Ursa Brown-Glaberman, Jennifer S Winn, Bryan A Faller, Adedayo A Onitilo, Mark E Burkard, George T Budd, Ellis G Levine, Melanie E Royce, Peter A Kaufman, Alexandra Thomas, Jane B Trepel, Antonio C Wolff, Joseph A Sparano. E2112: Randomized phase 3 trial of endocrine therapy plus entinostat/placebo in patients with hormone receptor-positive advanced breast cancer. A trial of the ECOG-ACRIN cancer research group [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS4-02.

Background: Clinical testing for HER2 amplification/over-expression is performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) as outlined by the ASCO/CAP guidelines. Although these guidelines standardize testing and reporting, in a subset of patients, HER2 is equivocal by both IHC and FISH (“Double Equivocal”). These double equivocal patients represent a clinically problematic sub-group that currently lack standardized management guidelines. In this study, we utilize Quantitative Real-Time PCR (Q-RT-PCR) to resolve HER2 status in invasive breast cancer cases that could not be resolved via IHC and FISH testing. Material and Methods: FISH for HER2 was performed on 2259 invasive breast carcinomas from 1/2008 to 12/2010. In accordance with ASCO/CAP, all equivocal HER2 FISH cases were reflex tested by IHC. In double equivocal cases, RNA extraction was performed following macro-dissection using High Pure RNA Paraffin Kit (Roche Applied Biosciences, Indianapolis, IN). Q-RT-PCR was carried out using TaqMan® RNA-to-CT™ 1-Step Kit with primers and probes for HER2, B2M, and GAPDH (Applied Biosystems, Foster City, CA). Q-RT-PCR results were expressed as the relative quantification of HER2 vs. two control genes, all normalized against calibrator RNA from the MCF7 cell line. Cut off for Q-RT-PCR HER2 overexpression was set using ROC curve analysis (MedCalc, Belgium). Results: In our cohort of 2259 patients, 124 (5.5%) had an equivocal HER2 result by primary FISH testing. Reflex HER2 testing by IHC was unable to resolve the HER2 status in 35 (1.5%) patients. Detection of HER2 overexpression by Q-RT-PCR was validated using 50 FISH confirmed amplified and 50 non-amplified cases. Q-RT-PCR performed on these 2 control populations generated two non-overlapping populations and ROC curve analysis using a cut off value of 7.0 showed 100% sensitivity and specificity in detection of HER2 overexpression. Application of Q-RT-PCR in the double equivocal sub-group resulted in resolution of HER2 status in all cases, 8 HER2 positive (test value ranging from 7.12 - 15.37) and 14 HER2 negative (test value ranging from 1.05 - 6.92). Conclusion: Application of Q-RT-PCR for HER2 represents a viable approach to resolve HER2 status in cases that fail classification by both FISH and IHC. Q-RT-PCR combines the precision and high sensitivity of real-time PCR with the morphological specificity of histological evaluation and ultimately allows definitive HER2 classification at the time of initial diagnosis. This knowledge of HER2 status at the time of diagnosis allows for comprehensive neoadjuvant treatment although, additional studies correlating response to anti-HER2 therapy and HER2 status by Q-RT-PCR are warranted. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-05.

Background Patients (pts) with (w/) node-negative HER2−positive BCA have a higher risk of recurrence than those with node-negative HER2−negative disease. Several randomized trials have shown the benefit of chemotherapy (most with an anthracycline) and trastuzumab (H) for pts with node-positive and high-risk node-negative BCA. However, the benefit of chemotherapy and H needs to be further explored in the node-negative group. Due to the 2–4% risk of symptomatic congestive heart failure (CHF) with an anthracycline-based treatment (Rx) followed by H, we set out to conduct a study of a taxane-based Rx with H in a node-negative population. Design: This is a single arm, multicenter, phase II study of paclitaxel (T) (80 mg/m2) and trastuzumab (H) x 12 weekly (w) (4 mg/kg load →2 mg/kg) → H x 52 w (2 mg/kg weekly or 6 mg/kg q 3 w). Pts with HER2−positive BCA (IHC 3 + or FISH amplified at ≥ 2.0) with negative nodes (micrometastasis later allowed) and with a tumor size ≤ 3 cm were enrolled. The primary endpoint is disease-free survival. Secondary endpoints include the incidence of G 3/4 left ventricular systolic dysfunction or congestive heart failure (CHF). Pts had serial left ventricular ejection fraction (LVEF) monitored at baseline (BSLN), and at month (mo) 3, mo 6, and mo 12 w/a multigated acquisition scan or echocardiogram. H was held for significant asymptomatic (Asx) LVEF ↓ (10-15% ↓ from BSLN and < lower limit of normal or ≥ 16% ↓ from BSLN), and H was stopped for CHF. Results: From 10-9-2007 to 9-3-2010, 406 pts were enrolled. The median (med) age was 55 years (range 23–84 years); 118/406 (29%) had hypertension and 30/406 (7%) had diabetes. As of 6-1-2011, 307 are reported as off Rx of which 261 have completed protocol therapy; 99 remain on therapy. To date, 100%, 94%, 84%, and 83% of pts had LVEF monitoring at BSLN, mo 3, mo 6, and mo 12. The med LVEF at BSLN was 65% (range 50%-81%), at mo 3 was 64% (range 45%-81%), at mo 6 was 64% (range 45%-81%), and at mo 12 was 65% (range 37%-90%). Two pts had CHF; 1 pt had CHF at mo 11 (LVEF was 55% at BSLN and 37% w/CHF event) and 1 pt had CHF at mo 6 (LVEF was 66% at BSLN and 49% w/CHF event). To date, 13 pts had H held for significant Asx LVEF ↓; details on those who had appropriate LVEF recovery and had H restarted will be provided. All patients will have completed 12 mo of Rx by October 2011, and an accurate report of the incidence of CHF will be available. Conclusion: This represents the cardiac report of pts receiving TH as adjuvant Rx for node-negative (micrometastasis allowed) HER2 positive BCA. Both the CHF events and number of pts w/H hold due to significant Asx ↓ LVEF appear low. Overall, the serial med LVEFs remain stable throughout the 12 mo of Rx. Final data on all 406 pts will be available in December 2011. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-18-02.

Abstract #2018 Background: Overexpression of HER2 is associated with an aggressive cancer and poor prognosis for patients. HER2-positive patients are treated with trastuzumab; however resistance is common. Mechanisms that regulate activity of HER2 or downstream kinase AKT are involved in regulation of survival. Calpains are cysteine proteases involved in transmission of signals during growth, motility and transformation in cancer. Mechanisms by which calpain regulates transmission of signals in HER2-positive breast cancer are not known. Here we investigate possibility that calpain regulates survival in HER2-positive breast cancer. Material and Methods: Activity of calpain in parental or resistant skbr3 cells was measured alone or in presence of inhibitors under conditions of adhesion or stimulation with trastuzumab using a fluorescently labeled substrate. To investigate regulation of HER2, assays were performed by mixing purified proteins or cell lysates with calpain1. Activity of HER2 was analyzed using phosphotyrosine-specific antibodies in Western blots while cleavage using antibody specific to the C-terminus. HER2-positive cancer tissue or lysates of cell lines were analyzed for presence of C-terminal fragments. Cells were transfected with the plasmid expressing catalytically inactive mutant of calpain1, selected for resistance to G418 and used in experiments to analyze the activity of HER2 and AKT. Sensitivity to trastuzumab was determined by measuring survival in presence of trastuzumab in a 96 well plate assay. Results: In skbr3 cells, activity of calpain increased following adhesion or stimulation with trastuzumab and was inhibited by inhibitors. Calpain induced cleavage of C-terminal domains and reduced activity in full length HER2. C-terminal fragments were detectable in the lysates of HER2-positive breast cancer tissue, cell lines and also overproduced by overexpression of calpain1. Conversely, in cells expressing catalytically inactive calpain1 the activity of HER2 and AKT was increased correlating with increased survival on fibronectin in the presence of trastuzumab. As another model for calpain-induced regulation of HER2 in mechanisms of resistance, we analyzed regulation in trastuzumab-resistant skbr3 cells. Compared to parental in resistant cells adhesion- or trastuzumab-dependent activity of HER2 was increased whereas cleavage was reduced suggesting mechanisms that regulate generation of C-terminal fragments during transmission of signals were altered. Consistenly, adhesion-dependent activity of calpain was reduced whereas trastuzumab failed to stimulate activation. Conclusion: These data are consistent with possibilities 1) HER2 is a direct target of calpain, 2) calpain induces cleavage of the cytoplasmic region during transmission of signals to regulate activity and integrity of HER2, 3) calpain-dependent mechanisms are involved in coupling HER2 to survival and 4) inhibition of calpain during adhesion-dependent transmission of signal or in the presence of trastuzumab allows increased activity of HER2 contributing to resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2018.

There is an unacceptably high recurrence rate for bacterial vaginosis and vaginitis owing to multiple causes, including misdiagnosis and empirical under-treatment and over-treatment. A diagnostic test that provided complete knowledge of the vaginal microbiome in patients with bacterial vaginosis (BV) would fill an important void. In initial evaluations of Next-generation Sequencing (NGS), this new test offers great promise.As a clinical diagnostic for primary or recurrent bacterial vaginosis, NGS by simple vaginal swab offers heightened sensitivity and specificity compared to culture or limited three-organism genetic tests. NGS holds valuable potential in multiple BV quandary situations, including: (a) Recurrent BV in a patient that failed initial therapy, (b) Symptoms of BV in a patient with false-positive Amsel criteria, (c) Gardnerellavaginalis-negative BV with mixed infection. Multiplex next-generation sequencing (NGS) provides a complete and accurate description of the composition of the complex polymicrobial vaginal microbiome and associated antimicrobial resistance-determining genes, facilitating personalized diagnosis and therapy for patients with bacterial vaginosis, STIs and vaginitis.

Molecular-based Phylogenetic analyses, including the newly-completed Human Microbiomes Project and other next-generation sequencing (NGS) studies have revealed the complexity of the vaginal micro biome in health and disease. The healthy vagina tends to be dominated by hydrogen peroxide and lactic acid producing organisms, the most important of which are Lactobacillus species. The loss of these organisms and the rise of non-resident microorganisms can lead to a diseased state such as bacterial vaginosis (BV or vaginal bacteriosis). The new consensus generated by these studies is that BV is not a single disorder or caused by one etiological agent but rather it is a spectrum of symptoms clinically characterized by vaginal discharge, increased pH and malodor caused by an imbalance in the normal vaginal bacterial flora. NGS techniques allow for the capture of the entire vaginal micro biome in a single assay and create the ability not only to obtain a better understanding of the causes of BV but also lead to a viable clinical method for detecting it. In this study, we use next generation sequencing of 16S rRNA genes to obtain the vaginal micro biome from 270 vaginal swabs. Our analysis includes an in-depth characterization of bacterial communities identified, including quantitation and composition of the communities present in normal and BV samples. Our results show vaginal samples can be broken down into three distinct groups that segregate largely based on degree and type of Lactobacillus content and the presence or absence of G. vaginalis.

Over the past few years, the preparation of ITER-relevant plasma scenarios has been the main focus of experimental activity on tokamaks. The development of integrated, simultaneous, real-time controls of plasma shape, current, pressure, temperature, radiation, neutron profiles and also impurities, ELMs and MHD are now seen to be essential for further development of quasi-steady state conditions with feedback, or the stabilisation of transient phenomena with event-driven actions. For this thrust, the EFDA JET Real-Time Project has developed a set of real-time plasma measurements, experiment control and communication facilities. The plasma diagnostics used for real-time experiments are Far Infra Red interferometry, polarimetry, visible, UV and X-ray spectroscopy, LIDAR, bolometry, neutron and magnetics. Further analysis systems produce integrated results, such as temperature profiles on geometry derived from MHD equilibrium solutions. The actuators include toroidal, poloidal and divertor coils, gas and pellet fuelling, neutral beam injection, radio frequency (ICRH) waves and microwaves (LH). The heating/fuelling operators can either define a power or gas request waveform or select the real-time instantaneous power/gas request from the real-time experiment central control (RTCC) system. The real-time experiment control system provides both a high-level, control-programming environment and interlocks with the actuators. A MATLAB facility is being developed for the development of more complex controllers. The plasma measurement, controller and plant control systems communicate in ATM network. The EFDA Real-Time Project is essential groundwork for future reactors such as ITER. It involves many staff from several institutions. The facility is now frequently used in experiments. This work has been conducted under the European Fusion Development Agreement and is partly funded by Euratom and the UK Engineering and Physical Sciences Research Council.

Trace Tritium Experiments' (TTE) were successfully performed on JET in 2003. The Campaign marked the first use of tritium in JET plasmas since the Deuterium-Tritium Experiment (DTE1) Campaign in 1997, and was the first use of tritium in experiments under the EFDA organisation with the UKAEA as JET Operator. The safety and regulatory preparations for the experiment were extensive. Since JET has been operated by the UKAEA the operations have followed the model of a licensed nuclear site. The safe operation of the JET torus is demonstrated in a safety case. Key Safety Management Requirement (KSMR) and Key Safety Related Equipment (KSRE) are identified in the Safety Case for DT operation. The safe operation of the torus is within the bounds of, and under the control of, an Authority to Operate (ATO). New technical challenges were presented by the need to inject and account for small quantities of tritium in very short pulses (∼80ms), with an accurate time stamp. The safety and operational management of the campaign are described. Valuable lessons were learned which would help in running future experiments. It is concluded that JET is in a strong position to run future trace tritium and full DT discharges.

Background: Breast carcinomas that demonstrate a heterogeneous ERBB2 (HER2) status or equivocal results by both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) present diagnostic challenges for which there is neither a standard methodology to achieve resolution in the clinical laboratory nor a uniform approach to management. We assessed the feasibility of using a novel automated and quantitative HER2 mRNA bright field in situ hybridization (ISH) assay capable of single molecule detection to determine HER2 status in invasive breast carcinomas that demonstrated significant tumor heterogeneity or failed to be resolved by standard IHC and FISH algorithmic testing. Design: Formalin-fixed, paraffin-embedded (FFPE) breast carcinomas from a non-consecutive series of 163 patients were analyzed for HER2 mRNA using a fully automated bright field RNA ISH assay (RNAscope, Advanced Cell Diagnostics, Hayward, CA). Cases were assigned into either a training set (n = 34) or a validation set (n = 129) and analyzed by both Q-RT-PCR and RNAscope. automated image analysis was used to numerate the punctate signal dots per cell in RNAscope-stained slides. A HER2 mRNA score based on single-cell quantification by RNAscope was developed and correlated to HER2 FISH and HER2 mRNA Q-RT-PCR results. A simple cutoff value was derived using the training set and applied to the validation set. Results: Evaluable HER2 results were obtained for 154 cases (94.5%) by RNAscope and 163 cases (100%) by Q-RT-PCR. In the training set, both FISH/IHC positive and negative cases were definitively separated by both Q-RT-PCR and RNAscope. HER2 mRNA dots per cell correlated strongly to FISH (Spearman r=0.77) and Q-RT-PCR (r = 0.81). Application of both methods to the validation set resulted in correct identification of 31/31 positive cases and 41/43 negative (overall concordance=97.3%) for both RNAscope and Q-RT-PCR. RNAscope showed a significant advantage over Q-RT-PCR in correctly identifying cases equivocal by FISH that were resolved by reflex IHC testing. RNAscope classified 7 of 26 (26.9%) FISH/IHC double equivocal cases as positives. In cases with HER2 protein heterogeneity, RNAscope showed a 100% concordance with FISH results, whereas Q-RT-PCR showed a 42.9% concordance. Conclusion: RNAscope analysis of HER2 mRNA is an effective means to resolve HER2 status in double equivocal cases and cases that demonstrate heterogeneity. Automation and image analysis-based quantification minimize analytical and post-analytical variability. Quantification of single RNA transcripts in situ at single-cell level demonstrates superiority over qRTPCR and great potential in predictive biomarker analysis. Further studies of larger cohorts correlating clinical response with HER2 mRNA expression in situ are warranted. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD02-04.

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3'-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression.

Purpose: The present study was designed to determine the toxicity and maximum tolerated doses of oral intermittent oral capecitabine and subcutaneous (s.c.) rHuIFNα2a in patients with metastatic renal cell carcinoma (RCC). The pharmacokinetics of capecitabine and its metabolites were also investigated. Methods: A total of 27 patients were treated at four dose levels of capecitabine (825 or 1000 mg/m2 twice daily orally, days 1–14, 22–36) and rHuIFNα2a (1.5 or 3.0 MU/m2 s.c. three times weekly). Unchanged capecitabine and its metabolites were analyzed in plasma using liquid chromatography/mass spectrometry in ten patients. Results: The toxicity of combined capecitabine and rHuIFNα2a was moderate. Patients experienced mild nausea/vomiting (70%) and diarrhea (63%). The hand-foot syndrome was seen in 67% of patients and was generally mild, as was hematologic toxicity. Dose-limiting toxicity included diarrhea, mucositis, neutropenia and the hand-foot syndrome. The dose level recommended for further trials included capecitabine 1000 mg/m2 twice daily and rHuIFNα2a 3.0 MU/m2 three times weekly. One patient had a partial response of a liver lesion (duration >200 days). Pharmacokinetic parameters of capecitabine and its metabolites (5′-deoxy-5-fluorouridine, 5-fluorouracil and α-fluoro-β-alanine) were similar to those reported by other authors. There was rapid conversion to 5′-deoxyuridine. The peak plasma concentrations of capecitabine occurred between 0.5 and 3.0 h. Conclusions: The combination of capecitabine and rHuIFNα2a was well tolerated. The recommended dose levels for phase II trials are: rHuIFNα2a 3.0 MU/m2 s.c. three times weekly and oral capecitabine 1000 mg/m2 twice daily for 2 weeks. No evidence of an effect of rHuIFNα2a on the pharmacokinetics of capecitabine or its metabolites was apparent. A phase II trial in untreated patients with metastatic RCC is planned.

Joint European Torus (JET) discharges have plasma currents up to 6 MA and toroidal fields up to 4 T. The plasma parameters during discharges are determined by the action of a number of distinct systems. These systems can be divided into three groups; the toroidal and poloidal field systems; the fuelling systems; and the additional heating systems. The systems can either be programmed to follow pre-determined waveforms or they can be feedback controlled to achieve certain values of specific plasma parameters, as measured with JET diagnostics. This paper reviews the feedback control of plasma parameters using the three main digital control systems; the plasma position and current control system, the density feedback system and the real time central control system. It is planned to integrate these systems using the new asynchronous transfer mode (ATM) technology in the real time data network.

All major JET systems have been fully commissioned for D-T and the DTE1 series of experiments has started with the D-T fuel mixture and operating conditions foreseen for ITER. In the area of ITER physics, significant results have been produced in both D-D and D-T. In D-D, the LH threshold power database has been extended, the bounds on edge-electron temperature and density in ELMy H-modes have been defined and the advantages of Types I and III ELMy discharges have been compared. In D-T plasmas, the isotope effect on H-mode threshold power and transport has been determined so that a more accurate assessment can be made of the ignition margin and heating requirements for ITER. Trace tritium experiments have provided first particle transport measurements and an assessment of the ITER reference ion-cyclotron resonance-frequency heating scenarios has been started, In the area of fusion performance, record D-D neutron yields have been obtained by controlling the plasma and current profiles in hot ion ELM-free H-modes and optimized shear modes. In D-T, internal transport barriers have been readily established in optimized shear discharges and Alfven eigenmodes have been observed.

A phase II trial was conducted in patients with metastatic renal cell carcinoma, to assess the clinical efficacy and immunoregulatory effects of continuous-infusion recombinant interleukin-2 (rIL-2) (9.0 x 10(6) IU/m2/day on days 1-5, 8-12, 15-19, and 22-26) and subcutaneously administered recombinant human interferon-alpha 2b (rHuIFN alpha 2b) (10.0 x 10(6) U/m2/day TIW). Thirty-six patients with metastatic renal cell carcinoma, performance status of 0-1, and measurable disease who had not received prior rIL-2, rHuIFN alpha 2b, or chemotherapy were treated. Patients with CNS metastases, active infections, history of another malignancy within 3 years, and those requiring corticosteroids were ineligible. Cycles of rIL-2 and rHuIFN alpha 2b were administered in the outpatient department every 6-8 weeks in stable or responding patients until patient tolerance or a complete response were reached. Doses were modified for grade III or IV toxicity. Ancillary studies included three-color immunocytometric analysis of peripheral blood lymphocytes, repetitive tumor biopsies for immunohistologic analysis of infiltrating cells and proliferative responses of tumor infiltrating lymphocytes, and preliminary studies of changes in peripheral blood T-lymphocyte signal transduction molecules [T-cell receptor (TCR)-zeta, p56ick, p59fyn]. Thirty-six eligible patients were treated, with 6 of 36 patients (17%, 95% confidence interval 6-33%) responding (3 complete response, 3 partial response). In two of the partial responders, and in an additional three patients with either minimal tumor regression (one patient) or stable disease (two patients), surgical removal of residual disease was undertaken. The median survival of all patients was 14 months. The toxicity of this regimen was severe, but outpatient administration was possible in most instances. Immunoregulatory effects on T-cell subsets included increases in various CD3+ CD25+/- HLADr+/- subsets unrelated to response. Tumor biopsies before and/or during therapy were obtained in 17 patients, and no consistent alterations in the degree of T-lymphocyte or macrophage infiltrates could be detected. In a subset of patients, tumor infiltrating lymphocyte proliferative responses and levels of peripheral blood T-cell signal transduction molecules (TCR-zeta, p56lck, p59fyn) were investigated. Abnormalities were found in selected patients, which improved during rIL-2/rHuIFN alpha 2b therapy. This cytokine combination produces tumor regression in selected patients with metastatic renal cell carcinoma. Surrogate immunologic markers associated with response were not identified; however, preliminary studies demonstrate investigation of immune defects and their reversal with cytokine therapy is possible.

Identification of dysregulated microRNAs (miRNAs) in prostate cancer is critical not only for diagnosis, but also differentiation between the aggressive and indolent forms of the disease. miR-9 was identified as an oncomiR through both miRNA panel RT-qPCR as well as high-throughput sequencing analysis of the human P69 prostate cell line as compared to its highly tumorigenic and metastatic subline M12, and found to be consistently upregulated in other prostate cell lines including DU-145 and PC3. While miR-9 has been characterized as dysregulated either as an oncomiR or tumour suppressor in a variety of other cancers including breast, ovarian, and nasopharyngeal carcinomas, it has not been previously evaluated and proven as an oncomiR in prostate cancer. miR-9 was confirmed an oncomiR when found to be overexpressed in tumour tissue as compared to adjacent benign glandular epithelium through laser-capture microdissection of radical prostatectomy biopsies. Inhibition of miR-9 resulted in reduced migratory and invasive potential of the M12 cell line, and reduced tumour growth and metastases in male athymic nude mice. Analysis showed that miR-9 targets e-cadherin and suppressor of cytokine signalling 5 (SOCS5), but not NF-ĸB mRNA. Expression of these proteins was shown to be affected by modulation in expression of miR-9.

An account is given of the high performance plasmas established by development of the H-mode regime in JET in the experimental campaigns up to 1992. High performance in this case is measured in terms of the confinement enhancement achieved over the L-mode scaling as measured using the plasma diamagnetism. Three JET H-mode regimes have achieved enhancement factors (H G DIA ) over Goldston L-mode scaling of 2.5 < H G DIA < 4.0. These are the Pellet Enhanced Performance (PEP) H-MODE, the high bootstrap fraction (high β POL ) H-mode and the Hot Ion (HI) H-mode. The phenomenology of these three regimes is reviewed and contrasts and common threads are elucidated

The therapeutic and biologic effects of murine monoclonal antibodies in patients with malignancies have been widely investigated. Attempts to enhance results by combining these agents with cytotoxic drugs are now under study. A Phase I trial was performed to assess the toxicity and biologic effects of escalating doses of R24 (0-40 mg/m2/day 1-5, 8-12), an antibody that binds to the ganglioside GD3 present on melanoma cells, administered in combination with cisplatin (120 mg/m2) and WR-2721 (740 mg/m2) on day 1. Twenty-three patients with metastatic malignant melanoma were treated and are evaluable. The true maximum tolerated dose of R24 given as part of this combination was not reached. The toxicity of the regimen was moderate and included fever and urticaria, which were attributed to R24. Severe but reversible renal failure was noted in six patients in subsequent (two or more) treatment cycles, but when cisplatin was administered in 3% saline, this toxicity was not seen. Responses were seen in 2 of 19 patients receiving all three agents and in 1 of 4 patients receiving only cisplatin and WR-2721. No significant enhancement of natural killer, lymphokine-activated killer, and antibody-dependent cellular cytotoxicity lytic activity or significant changes from baseline in lymphocyte subsets secondary to R24 were seen. In 4 of 10 patients tumor localization of mouse monoclonal antibody was found and appeared greatest at higher R24 doses and during week 1 of therapy. Human anti-mouse antibody responses developed by day 22 in 17 of 19 patients treated with R24, and the coadministration of cisplatin did not appear to abrogate this response. Finally, the half-life and Cmax of cisplatin were not affected by R24. In summary, the combination was well tolerated, responses were few, and significant biologic interactions or immunomodulation were not observed.

MicroRNAs (miRs) are a novel class of RNAs with important roles in regulating gene expression. To identify miRs controlling prostate tumor progression, we utilized unique human prostate sublines derived from the parental P69 cell line, which differ in their tumorigenic properties in vivo. Grown embedded in laminin-rich extracellular matrix (lrECM) gels these genetically-related sublines displayed drastically different morphologies correlating with their behaviour in vivo. The non-tumorigenic P69 subline grew as multicellular acini with a defined lumen and basal/polar expression of relevant marker proteins. M12, a highly tumorigenic, metastatic derivative, grew as a disorganized mass of cells with no polarization, whereas the F6 subline, a weakly tumorigenic, non-metastatic M12 variant, reverted to acini formation akin to the P69 cell line. These sublines also differed in expression of vimentin, which was high in M12, but low in F6 and P69 sublines. Analysis of vimentin’s conserved 3′-UTR suggested several miRs that could regulate vimentin expression. The lack of miR-17-3p expression correlated with an increase in vimentin synthesis and tumorigenicity. Stable expression of miR-17-3p in the M12 subline reduced vimentin levels 85% and reverted growth to organized, polarized acini in lrECM gels. In vitro motility and invasion assays suggested a decrease in tumorigenic behaviour, confirmed by reduced tumor growth in male athymic, nude mice dependent on miR-17-3p expression. Analysis of LCM-purified clinical human prostatectomy specimens confirmed that miR-17-3p levels were reduced in tumor cells. These results suggest that miR-17-3p functions as a tumor suppressor, representing a novel target to block prostate tumor progression.

This paper presents the main lessons that can be learned from two decades of Plasma Control development experience. It will present the general architectural choices and will provide examples from key systems, and, more importantly, will highlight the plasma operation requirements that have driven the developments. The aim is to present a meaningful set of functional and non- functional requirements that were derived from the JET experience and to discuss their potential applicability to future experimental devices, ITER in particular. ©2009 IEEE.

From June 1988 to November 1990 the Southwest Oncology Group initiated nine protocols for the phase II evaluation of recombinant human tumor necrosis factor alpha (rhuTNF alpha) in cancer patients. Patients with diverse metastatic malignancies including breast, colon, gastric, pancreatic, endometrial, and bladder cancers, as well as multiple myeloma and various sarcomas received 150 micrograms/m2 of rhuTNF alpha daily for 5 days every other week. Of 147 patients entered in the study, 127 were eligible and were evaluated for toxicity and response. Of 124 patients known to have completed treatment, 92 (74%) went off study for progression, 21 (17%) for toxicity, and 12 (10%) for other causes, mainly that of worsening medical condition. Thirteen percent of patients experienced grade 4 or fatal toxicity. The most serious toxicities were pulmonary failure and coagulopathies. The predominant grade 3 toxicities were symptomatic (chills, fever, malaise, headache, myalgia, and nausea or vomiting). Only one partial remission was seen in a patient with metastatic bladder cancer lasting 4 months (rate 0.8%, exact 95% confidence interval 0-4%). At the study dose and schedule, rhuTNF alpha does not appear to have significant antitumor activity. The biological basis for this finding is discussed.

A phase I trial was performed to assess the immunomodulatory activities, maximum tolerated doses, and the toxicity of recombinant interleukin-2 (rIL-2) administered in combination with doxorubicin to patients with refractory malignancies. Therapy was administered to successive cohorts of four to six patients who were treated at three different dose levels (1A, 1B, 2A). Levels 1-2 refer to doxorubicin (40 or 60 mg/m2) given as an intravenous (i.v.) bolus on day 1, and levels A-B refer to rIL-2 (1.0 or 3.0 x 10(6) U/m2) given as a continuous i.v. infusion on days 2-5, 9-12, and 16-19. Cycles were repeated every 28 days. Seventeen patients were entered in the trial. Dose limiting toxicity consisted of neutropenia, and the maximum tolerated dose (MTD) of the combination was doxorubicin 40 mg/m2 and rIL-2 3.0 x 10(6) U/m2. No objective responses were observed. Lymphocytosis related to rIL-2 occurred and flow cytometry demonstrated significant increases in the following subsets: CD3+CD25+HLADr+ and CD11b-CD16c+CD8-. Natural killer cell activity and lymphokine-activated killer (LAK) cell precursors were increased in patients treated at dose levels 1A and 1B (40 mg/m2 doxorubicin), but no consistent changes in LAK activity were noted. No clinical responses were seen and the overall toxicity of this combination was moderate to severe. Administration of doxorubicin prior to rIL-2 does not enhance the immunologic effects of rIL-2.

Background: Induction of apoptosis is a common effect of many new and existing drugs, and Bcl-2 expression is associated with resistance to apoptosis. CTCs are a minimally invasive blood test and are a source of tumor cells that can be obtained serially during therapy. Monitoring Bcl-2, apoptosis, and other markers in CTCs may be useful endpoints in trials of new and targeted agents. The goal of this pilot study is to estimate Bcl-2 expression and apoptosis in CTCs from pts being treated for metastatic breast cancer (MBC). Methods: Whole blood was collected from pts initiating therapy for MBC at baseline, post-treatment (24, 48, or 72 hours), and 3–4 weeks. Samples were split into 3 × 7.5ml fractions. CTCs were isolated and enumerated using the CellTracks and CellSpotter systems. CTCs were defined as DAPI+, cytokeratin+, CD45-, with a cellular morphology. Apoptosis defined by positive staining with MAb M30. Bcl-2 status defined by staining with MAb Bcl-2–100. Results: 39/81 patients (49%) had ≥5 CTCs at baseline. 25% had ≥5 CTCs at 3–4 weeks. 35% of pts with CTCs had positive Bcl-2 staining in 91–100% of their CTCs (highest decile). Other pts were equally distributed across the remaining Bcl-2 deciles. At baseline, apoptosis correlates with CTC number. In samples with 1–4 CTCs, 80% of cells were apoptotic. For 5–49 CTCs, 48% were apoptotic; for 50–99, 34% were apoptotic; for 100–500, 28% were apoptotic; and for >500, 22% were apoptotic. Bcl-2 staining was inversely correlated with apoptosis staining. Pts were risk stratified by changes in CTC number after one cycle of therapy, and apoptosis increased with improving prognostic groups. One pt had markedly elevated CTCs of >20,000. The cells were remarkably 100% Bcl-2 positive and M30 negative (1% staining). Conclusions: These data confirm the prevalence of CTCs seen in the pivotal trial of CTCs in MBC (Cristofanilli NEJM 2004) and provide novel observations for Bcl-2 expression and apoptosis in CTCs. Higher Bcl-2 expression appears to correlate with decreased apoptosis. High fractions of apoptotic cells correlate with lower numbers of CTCs, which is associated with a better prognosis. Analysis of the post-treatment (24, 48, or 72 hour) samples and correlation of markers with progression free survival are ongoing and will be presented.

B-cell chronic lymphocytic leukemia (CLL) accounts for 95% of chronic leukemia cases and 25% of all leukemia. Despite the prevalence of CLL, progress in its treatment has been only modest over the past three decades. Based upon the ability of fludarabine to produce high-grade remissions especially among patients with low initial tumor mass, and the ability of alkylators to reduce tumor mass, we hypothesized that sequential administration of a limited number of cycles of intermediate-dose cyclophosphamide followed by fludarabine could result in a larger percentage of patients with complete remissions (CRs). In all, 27 of the 49 eligible patients achieved overall responses of CR, unconfirmed complete remission (UCR), or PR, for a total response rate of 55% (95% confidence interval (CI) 40–69%). Considering the confounding medical issues of this patient population with advanced aggressive disease, the regimen was generally well tolerated. This study demonstrates that high-dose cyclophosphamide followed by fludarabine was relatively well tolerated in this group of advanced CLL patients. The study's criterion for testing whether the regimen is sufficiently effective to warrant further investigation was met: 14 (32%) of the first 44 eligible patients achieved CR or UCR.

The design of the systems for delivering active and non-active gas to the JET torus during plasma pulses is outlined. Emphasis is given to the novel engineering aspects, which include radiation resistant pressure gauges, a matrix gas distribution system and an automated system for verifying, the purity of gas species. The performance of the system is reviewed and future enhancement for tritium introduction are proposed.

Background: Preoperative chemotherapy is an effective approach for “downstaging” some breast cancer patients, some of whom achieve a pathologic complete response (pCR), especially for non-luminal B HER2 positive molecular class (HNL). Determination of which HNL patients are more or not likely to achieve pCR would allow a more personalized, predictive approach to neoadjuvant management. Methods and Materials: Formalin-fixed biopsy specimens from 34 patients with invasive ductal carcinoma treated with trastuzumab-based therapy prior to definitive resection and pathologic staging were evaluated by dual color dual hapten bright field in situ hybridization (dual ISH) using repeat depleted locus specific probes (designation based on the name of a gene included within the targeted region) and a reference centromeric locus probe (CEN) on the same chromosome. Probe pairs included MET+CEN7, TOP2A+CEN17, PTEN+CEN10, and PIK3CA+CEN3. Only invasive carcinoma tumor cells were scored. In addition to assessing genomic gains and losses by the average number of gene loci or centromeres per cell and the ratio of gene loci to CEN, the percentage of cells with >2 gene or CEN signals (gene locus gain or CEN gain), CEN signals (gene locus/CEN gain), and gene locus signals < CEN signals (gene locus/CEN loss) were calculated for each parameter and cut point. The percentage of cells with either gene locus/CEN gain or loss was also evaluated. Sensitivities and specificities for detecting patients with pCR, based on either the high parameter being positive for pCR, and conversely based on the low parameter being positive for pCR, were calculated for each parameter and each cutoff. Receiver Operator Characteristics (ROC) curves were generated as sensitivity versus 1-specificity over all cutoffs tested, and Area Under the Curve (AUC) was calculated as one measure of a parameter's ability to distinguish patients with pCR from patients without pCR, with AUC = 1 being ideal and progressively lower values being less favorable. 2×2 contingency tables were evaluated at each cutoff to provide chi square probabilities as another measure of a parameter's ability to differentiate patients with pCR. Combinations of parameters were also evaluated by ROC and contingency table analyses. Results: MET/CEN7 gain or loss was predictive of pCR (AUC = 0.824, N=24) achieving 100% sensitivity and 69% specificity at a cutoff of 50% (X2 p < 0.00089). Combining this parameter with PIK3CA/CEN3 gain provided further improvement (AUC 0.937, N=24) with 89% sensitivity and 93% specificity achieved at respective cutoffs of 50 and 55%. Conclusion: Genomic signatures of moderate complexity generated from dual ISH evaluation of invasive breast carcinoma predict pathologic complete response in HER2 molecular class breast carcinoma patients treated with trastuzumab-based preoperative therapy. These findings require validation in additional patient cohorts. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-45.

Variations in practice patterns are markers for the quality of patient care in general medicine, but little is known about variation in care delivered to cancer patients. This study's purpose was to describe chemotherapy use, variations in chemotherapy delivery, and the incidence of complications in community practice settings.Data describing adjuvant chemotherapy for patients with early-stage breast carcinoma (ESBC) were collected from an ongoing Oncology Practice Pattern Study at 13 large managed care, academic, and community practices (1111 patients). Data collection included information about diagnoses and adjuvant chemotherapy treatments, laboratory results, supportive care, complications, and treatment modifications.The median patient age was 50 years, and most patients had zero to three positive lymph nodes. Chemotherapy regimens consisting of cyclophosphamide, methotrexate, and 5-fluororacil (CMF) and of doxorubicin and cyclophosphamide (AC) accounted for 76% of the adjuvant therapies used. Overall, 30% of patients had delivered average relative dose intensities/= 85% of the referenced targets. Delivered summation dose intensities (SDIs) frequently were well below targeted SDIs. Neutropenia-related dose modifications occurred for 27.6% of patients and recurred with a 60.7% rate. AC was the regimen delivered with a dose intensity closest to the referenced target. However, patients who were treated with AC regimens and with regimens consisting of cyclophosphamide, doxorubicin, and 5-fluorouracil had significantly higher rates of chemotherapy-related complications compared with patients who were treated with CMF regimens in the most recent treatment years.Adjuvant chemotherapy for patients with ESBC frequently is not administered as referenced in off-protocol community settings. Variation in the delivered SDI raises concerns about potential treatment outcomes and warrants strategies to identify patients who are at risk for complications early in therapy.

Breast tumors of BRCA1 mutation carriers and those of early onset breast cancer cases share similar histological features, being generally high-grade, highly proliferative, aneuploid tumors that are predominantly estrogen- and progesterone-receptor negative. Because histological features of tumors of premenopausal women differ from those of tumors of older women, we sought to determine whether the immunophenotype of breast tumors of BRCA1 mutation carriers was influenced by age at diagnosis.We examined 31 breast tumors from BRCA1 mutation carriers and compared them with 81 tumors of age-matched (plus or minus 5 years) breast cancer patients unselected for family history. Tumors were further matched for histology, grade, and size. Paraffin-embedded tumor tissues were examined for protein expression of estrogen receptor (ER), PR, Ki-67, cyclin D1, TP53, HER2, beta-catenin, and cyclin E using immunohistochemical approaches.ER (P = 0.01), PR (P = 0.06), and cyclin D1 (P = 0.002) were less frequently expressed and Ki-67 (P = 0.01) and beta-catenin (P = 0.04) were more frequently expressed in tumors of BRCA1 mutation carriers than controls. After age stratification, we found a significant difference in the frequency of tumors of BRCA1 mutation carriers diagnosed before 50 years of age compared with age-matched controls that stained positive for ER (P = 0.01), PR (P = 0.03), Ki-67 (P = 0.008), cyclin D1 (P0.001), HER2 (P = 0.04), and beta-catenin (P = 0.05). However, no significant differences were observed in tumors of BRCA1 mutation carriers diagnosed at age 50 or older compared with age-matched controls.These data suggest that age at diagnosis, possibly related to menopausal status, may be an important factor in the expression of specific proteins in breast tumors of BRCA1 mutation carriers.

The authors evaluated the clinical and biologic effects of human recombinant interleukin-6 (rhIL-6) in patients with refractory cancer. A phase 1 trial using escalating doses of rhIL-6 (1-50 microg x kg(-1) x d(-1), Monday through Friday for 4 weeks) was performed in 30 patients. Toxicity was moderate and the maximum tolerated dose was determined to be 25 microg x kg(-1)x d(-1) based on cardiac and neurocortical toxicity in one patient each and thrombocytosis (platelets800,000/microL) in three patients. One patient with non-small-cell lung cancer had a partial response after three cycles of therapy. The biologic effects of rhIL-6 included anemia and dose-related thrombocytosis. Various proinflammatory activities were induced and included dose-related cyclical increases in peripheral blood monocytes and the CD14+/CD45RB+ +/- CD16C+ mononuclear cell populations. These increases were accompanied by increased levels of C-reactive protein, serum neopterin, and type I soluble tumor necrosis factor receptor. In contrast, rhIL-6 did not affect lymphocyte numbers or function (cytotoxicity, cytokine levels, immunoglobulin levels), with the possible exception of IL-2Ralpha mRNA induction in peripheral blood lymphocytes. rhIL-6 has pleiotropic proinflammatory actions in vivo and moderate toxicity when administered as long-term therapy.

Sulindac sulfone (Exisulind), a metabolite of the non-steroidal anti-inflammatory drug, sulindac, was evalauted for its effects on the development of rectal polyps in patients with familial adenomatous polyposis. Three cohorts of 6 patients each were given doses of 200, 300, or 400 mg Exisulind twice daily. Hepatotoxicity, shown by elevation in blood transaminase levels, was the dose-limiting toxicity and occurred at the 400 mg bid dose. Due to this toxicity, all patients treated with the 400 mg dose were subsequently reduced to the 200 mg dose level. Subsequently, 2 of the 6 patients were dose-escalated to 400 mg bid dose. The patients were treated with Exisulind for a period of six months. Sixteen of 18 patients had regression of small polyps (or = 6 mm in diameter) characterized by a flattening of the polyps and a macular "halo" appearance. Histopathologic examination of the polyp biopsy specimens showed a marked increase in the proportion of mucin producing cells in the glands after treatment with Exisulind at all dose levels. Ki-67 staining, a measure of cell proliferation, was higher in the polyps than in normal mucosa. There was no significant change in the proliferation index between baseline and six month values in any of the groups treated with Exisulind or in normal tissues. The median apoptotic labeling index, as determined by the TUNEL technique, was higher in the polyps than in normal-appearing mucosa. Overall, there was no significant change in the apoptotic labeling index between base-line and 6 months in normal-appearing mucosa however, the index in polyps was increased. These results suggest that treatment of FAP patients with Exisulind for a period of six months may lead to regression of small polyps, and that the mechanisms of Exisulind--induced regression appear to be through stimulation of mucus differentiation and apoptosis in glandular epithelium.

Exisulind (sulindac sulfone; FGN-1), a metabolite of sulindac without known effects on prostaglandin synthesis, can promote apoptosis and inhibit tumorigenesis in preclinical systems. We performed a Phase I trial of this compound in patients with familial adenomatous polyposis (FAP) to examine the tolerability and safety of this drug in the cancer chemoprevention setting. Six patients each were treated with exisulind at doses of 200, 300, and 400 mg p.o. twice a day. Reversible hepatic dysfunction was noted in four of six patients treated at the 400-mg p.o., twice-a-day dose level, but in only one to two of six patients treated at each of the lower dose levels. The serum half-life of exisulind was 6-9 h; little drug accumulation was noted over time. A nonsignificant trend toward increased apoptosis in polyps was noted at the maximum tolerated dose, but no decrease in polyp numbers or significant effects on cellular proliferation was noted. After treatment, polyps tended to display a "halo" appearance grossly and mucinous differentiation histologically. The maximum safe dose of exisulind is 300 mg p.o. twice a day in patients with subtotal colectomies. Reversible hepatic dysfunction limits further dose escalation. A decrease in polyp numbers could not be demonstrated, but the trend toward increased apoptosis at the MTD and the observation of mucinous change histologically suggest that further investigation of drugs of this class might be warranted.

Purpose: We performed a phase I/II dose-escalation trial of cyclophosphamide, mitoxantrone, and 5-fluorouracil (CNF) in combination with human recombinant granulocyte-colony-stimulating factor (G-CSF, filgrastim) in patients with advanced breast cancer. The objectives of this trial were (1) to gain experience with filgrastim given to patients with advanced breast cancer and receiving standard-dose CNF, and (2) to determine the maximum tolerated dose of CNF that could be given with filgrastim support by incremental dose escalation of two components of the CNF regimen, cyclophosphamide and mitoxantrone. Methods: Four patients who had received prior therapy for advanced disease received standard-dose CNF with filgrastim support. Sequentially enrolled patients who had received no prior chemotherapy for advanced disease were treated with standard-dose CNF without filgrastim (5 patients), standard-dose CNF with filgrastim (15 patients), or were entered into sequential cohorts of 3–6 patients to be treated with increasing doses of CNF with filgrastim support (29 patients). Results: The maximum tolerated doses that could be given with filgrastim support were 1500 mg/m2 cyclophosphamide, 20 mg/m2 mitoxantrone, and 500 mg/m2 5-FU. Overall, 7 complete (14%) and 13 partial responses (26%) were observed. Despite the use of filgrastim, repeated cycles of CNF at doses of 2000 mg/m2 cyclophosphamide, 25 mg/m2 mitoxantrone, and 500 mg/m2 5-FU could not be given because of neutropenia and thrombopenia. Among 18 patients with bidimensionally measurable disease there were 3 complete (17%) and 5 partial (28%) responses. The median progression-free survival of all patients was 236 days (34 weeks). Conclusion: The use of filgrastim allows CNF to be given at approximately twice the dose intensity of “standard”-dose CNF. Because non-hematopoietic toxicity was not dose-limiting, further dose escalation of this regimen might be possible with more effective hematopoietic support. The response rate and survival of patients treated in this study were within the range expected with standard-dose chemotherapy.

Mature results are reported from a phase II trial of accelerated induction chemoradiotherapy and surgical resection for stage III non-small-cell lung cancer whose prognosis is poor. Surgically staged patients with poor prognosis stage III non-small-cell lung cancer were eligible for this study. Four-day continuous intravenous infusions of cisplatin 20 mg/m 2 / day, 5-fluorouracil 1,000 mg/m 2 /day, and etoposide 75 mg/m 2 / day were given concurrently with accelerated fractionation radiation therapy, 1.5 Gy twice a day, to a total dose of 27 Gy. Surgical resection followed in 4 weeks. Identical postoperative chemotherapy and concurrent radiation to a total dose of 40 to 63 Gy was subsequently given. Between February 1991 and June 1994, 42 eligible and evaluable patients, 23 with stage IIIA disease and 19 with stage IIIB disease, were entered in this trial. Treatment was well tolerated. The pathologic response rate was 40%. This response was complete in 5%. With a median follow-up of 54 months, the Kaplan-Meier 4-year survival estimate is 19%: 26% for stage IIIA and 11% for stage IIIB patients. Patients with a pathologic response, resectable disease, or pathologic downstaging to stage 0, 1, or II had a better survival. The 4-year estimates of locoregional and distant disease control are 70% and 19%, respectively. It is concluded that although the ultimate role of concurrent chemoradiotherapy and surgery in stage III non-small-cell lung cancer must await the results of phase III clinical trials, survival and locoregional control in this study appear improved in comparison with historical experience. There is a subset of patients, able to undergo resection with pathologic downstaging, who have a projected survival equivalent to that of patients with more limited disease. Clinical or pathologic tools to identify these patients before treatment would be highly useful.

Thrombocytopenia is a significant problem for patients receiving prolonged or aggressive chemotherapy for malignancy. For carboplatin, it is the predominant dose-limiting toxicity and it is cumulative in nature. A number of agents have been evaluated for efficacy in reducing the problem of thrombocytopenia. Some have proved valueless and have been discarded. Others (eg, recombinant thrombopoietin) are under current study, and one (interleukin-11 or oprelvekin) is now commercially available. In addition, the currently available cytoprotectant, amifostine (Ethyol; Alza Pharmaceuticals, Palo Alto, CA/US Bioscience, West Conshohocken, PA), has been shown to reduce the severity and duration of thrombocytopenia caused by carboplatin. Because of the short half-life of amifostine relative to that of carboplatin, multiple doses of amifostine have been administered in conjunction with carboplatin. The optimal dosing regimen with amifostine and carboplatin needs to be further evaluated in clinical studies. Future trials will also expand these observations to carboplatin-containing combination chemotherapy regimens and will further define the role of amifostine as a multilineage bone marrow protectant. The ability of amifostine to demonstrate multilineage bone marrow protection differentiates it from currently available growth factors and fulfills a medical need, including reducing the need for platelet transfusions and maintaining the desired chemotherapy dose intensity.

Studies have demonstrated abnormalities of the CD3/T-cell antigen receptor (TCR) and pathways of signal transduction in T lymphocytes from animals and patients with advanced malignancy. Diminished expression of TCRzeta and p56(lck) that are associated with the TCR and reduced nuclear localization of RelA containing nuclear factor kappaB (NFkappaB) complexes have been noted. These defects have been described in T cells from patients with malignant melanoma, renal cell carcinoma (RCC), ovarian cancer, and colorectal cancer. Preliminary observations also indicate possible correlation with clinical variables such as stage in selected instances. To further characterize altered expression of TCRzeta, p56(lck), and impaired activation of NFkappaB, T lymphocytes were obtained from 65 patients with RCC, the majority of whom were receiving combination cytokine therapy [interleukin (IL)-2, IFN alpha-containing regimens] and 37 control individuals. In 29 of these patients, levels of TCRzeta and p56(lck) were determined by Western blots of T-cell lysates and semiquantitated using densitometry. Relative levels were then correlated with a series of clinical variables including response to therapy, performance status, survival, disease sites, age, and others. In another group of 28 patients (three individuals from the first group), the frequency of abnormal NFkappaB activation was studied using electrophoretic mobility shift assays after activation of T cells with phorbol myristate acetate/ionomycin or anti-CD3 monoclonal antibody. Changes in these signaling molecules during cytokine treatment were also investigated. TCRzeta and p56(lck) were detected in the peripheral blood T cells in 27 of 29 patients, and overall, reduced levels were noted visually in 12 of 29 (41%) and 13 of 29 (45%) individuals, respectively. When levels were semiquantitated using densitometry, significant decreases of TCRzeta (P = 0.029) and p56(lck) (P = 0.029) but not CD3epsilon (P = 0.131), compared with control levels, were found. In patients treated with IL-2/IFN alpha-based therapy, relative levels of TCRzeta increased significantly (P = 0.002) on day 15 of cycle one compared with the baseline. Correlations of TCRzeta or p56(lck) levels with response or disease variables, except for lower TCRzeta levels (P0.001) in the presence of bone metastases, were not found. Abnormal NFkappaB activation after stimulation with phorbol myristate acetate/ionomycin and/or anti-CD3 monoclonal antibody was found in 59% of patients (17 of 28) and was not accounted for by the advanced age of the study cohort. Activation of NFkappaB in peripheral blood T cells was inducible during cytokine therapy in four of six individuals who displayed impaired NFkappaB activity prior to therapy. Moreover, impaired activation of NFkappaB does not appear linked to a reduction of TCRzeta expression, because in five patients, normal TCRzeta levels were present although kappaB binding was not inducible. In the majority of patients with advanced RCC, peripheral blood T cells express TCRzeta and p56(lck), and in a subset, reduced levels of these TCRzeta associated molecules are seen that may increase during cytokine-based therapy. Abnormal activation of NFkappaB is also present in50% of patients and may also revert to normal during IL-2/IFN alpha-based treatment. This alteration in NFkappaB activation occurred in the presence of normal expression of TCRzeta-associated signaling elements. The clinical significance of these findings remains unclear.

Because tamoxifen and all-trans-retinoic acid (ATRA) have additive antitumor effects in preclinical systems, we performed a Phase I/II clinical trial of this combination in patients with advanced breast cancer. Patients with potentially hormone-responsive advanced breast cancer were enrolled. All received 20 mg of tamoxifen by mouth daily. Consecutive cohorts of 3-6 patients were treated on odd-numbered weeks with ATRA at doses of 70, 110, 150, 190, or 230 mg/m2/day. Twenty-six patients were entered in this trial; 25 were evaluable. A dose of 230 mg/m2 ATRA produced unacceptable headache and dermatological toxicity, but dosesor = 190 mg/m2 were tolerable. Two of 7 patients with measurable disease responded. Seven of 18 patients with evaluable, nonmeasurable disease achieved disease stability for more than 6 months. Plasma AUCs on day 1 of successive weeks of treatment were stable over time. A nonsignificant decrease in serum insulin-like growth factor I levels was noted during treatment, but this trend was similar to that observed in three "control" patients treated with tamoxifen alone. When given with daily tamoxifen, the maximum tolerated dose of ATRA that could be given on alternate weeks was 190 mg/m2/day. This schedule of ATRA resulted in repeated periods of exposure to potentially therapeutic concentrations of ATRA. Declines in the serum insulin-like growth factor I concentrations observed in patients treated with tamoxifen and ATRA were similar to those observed in patients treated with tamoxifen alone. Objective responses were observed, some in patients who had previously progressed while receiving tamoxifen, suggesting that further studies would be of interest.