FLASH X-RAY (FXR) LINEAR INDUCTION ACCELERATOR (LIA) OPTIMIZATION Upgrade of the OTR Emittance Diagnostic

Knowing the electron beam parameters at the exit of an accelerator is critical for several reasons. Foremost is to optimize the application of the beam, which is flash radiography in the case of the FXR accelerator. The beam parameters not only determine the theoretical dose, x-ray spectrum, and radiograph resolution (spot size), they are required to calculate the final transport magnetic fields that focus the beam on the bremsstrahlung converter to achieve the theoretical limits. Equally important is the comparison of beam parameters to the design specifications. This comparison indicates the ''health'' of the accelerator, warning the operator when systems are deteriorating or failing. For an accelerator of the size and complexity of FXR, a large suite of diagnostics is normally employed to measure and/or infer beam parameters. These diagnostics are distributed throughout the accelerator and can require a large number of ''shots'' (measurements) to calculate a specific beam parameter. The OTR Emittance Diagnostic, however, has the potential to measure all but one of the beam parameters simultaneous at a specific location. Using measurements from a scan of a few shots, this final parameter can also be determined. Since first deployment, the OTR Emittance Diagnostic has been limited to measuring only one of the seven desired parameters, the beam's divergence. This report describes recent upgrades to the diagnostic that permit full realization of its potential

BRAF inhibition is associated with increased clonality in tumor-infiltrating lymphocytes

There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) β chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10–14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones

Sex-Based Dimorphism of Anticancer Immune Response and Molecular Mechanisms of Immune Evasion

PURPOSE: We previously demonstrated that sex influences response to immune checkpoint inhibitors. In this article, we investigate sex-based differences in the molecular mechanisms of anticancer immune response and immune evasion in patients with NSCLC. EXPERIMENTAL DESIGN: We analyzed (i) transcriptome data of 2,575 early-stage NSCLCs from seven different datasets; (ii) 327 tumor samples extensively characterized at the molecular level from the TRACERx lung study; (iii) two independent cohorts of 329 and 391 patients, respectively, with advanced NSCLC treated with anti–PD-1/anti–PD-L1 drugs. RESULTS: As compared with men, the tumor microenvironment (TME) of women was significantly enriched for a number of innate and adaptive immune cell types, including specific T-cell subpopulations. NSCLCs of men and women exploited different mechanisms of immune evasion. The TME of females was characterized by significantly greater T-cell dysfunction status, higher expression of inhibitory immune checkpoint molecules, and higher abundance of immune-suppressive cells, including cancer-associated fibroblasts, MDSCs, and regulatory T cells. In contrast, the TME of males was significantly enriched for a T-cell–excluded phenotype. We reported data supporting impaired neoantigens presentation to immune system in tumors of men, as molecular mechanism explaining the findings observed. Finally, in line with our results, we showed significant sex-based differences in the association between TMB and outcome of patients with advanced NSCLC treated with anti–PD-1/PD-L1 drugs. CONCLUSIONS: We demonstrated meaningful sex-based differences of anticancer immune response and immune evasion mechanisms, that may be exploited to improve immunotherapy efficacy for both women and men. TRANSLATIONAL RELEVANCE: It is well known that sex (i.e., the biological differences between men and women) and gender (i.e., behavioral differences associated with being male or female) are variables that affect immune responses to both foreign and selfantigens. Such sex- and gender-based dimorphism of immune system function, in turn reflects complex interactions between genes, hormones, the environment, and commensal microbiome composition. In our previous works, we showed that patients' sex is significantly associated with effectiveness of immune checkpoint inhibitors (ICIs) in patients with several solid tumors, including NSCLC. Here, we identified meaningful differences in molecular mechanisms that drive anticancer immune response as well as in immune evasion mechanisms exploited by NSCLCs arising in men and women. Importantly, we showed that all the findings reported, were not related to other variables potentially associated with sex such as patients' age, stage of disease, tumor histotype, and smoking status. The findings reported in this our work explain our previous clinical observations and can open this area to different immunotherapy strategies in males and females with NSCLC to further improve prognosis of both

Serratamolide is a hemolytic factor produced by Serratia marcescens

Serratia marcescens is a common contaminant of contact lens cases and lenses. Hemolytic factors of S. marcescens contribute to the virulence of this opportunistic bacterial pathogen. We took advantage of an observed hyper-hemolytic phenotype of crp mutants to investigate mechanisms of hemolysis. A genetic screen revealed that swrW is necessary for the hyper-hemolysis phenotype of crp mutants. The swrW gene is required for biosynthesis of the biosurfactant serratamolide, previously shown to be a broad-spectrum antibiotic and to contribute to swarming motility. Multicopy expression of swrW or mutation of the hexS transcription factor gene, a known inhibitor of swrW expression, led to an increase in hemolysis. Surfactant zones and expression from an swrW-transcriptional reporter were elevated in a crp mutant compared to the wild type. Purified serratamolide was hemolytic to sheep and murine red blood cells and cytotoxic to human airway and corneal limbal epithelial cells in vitro. The swrW gene was found in the majority of contact lens isolates tested. Genetic and biochemical analysis implicate the biosurfactant serratamolide as a hemolysin. This novel hemolysin may contribute to irritation and infections associated with contact lens use. © 2012 Shanks et al

Prognostic model for patient survival in primary anorectal mucosal melanoma:stage at presentation determines relevance of histopathologic features

Pathological staging of primary anorectal mucosal melanoma is often performed according to the American Joint Commission on Cancer (AJCC) guidelines for cutaneous melanoma, as an anorectal melanoma-specific staging system does not exist. However, it remains unknown whether prognostic factors derived for cutaneous melanoma also stratify risk in anorectal melanoma. We retrospectively determined correlations between clinicopathological parameters and disease-specific survival in 160 patients. Patients were grouped by clinical stage at presentation (localized disease, regional or distant metastases). Cox proportional hazards regression models determined associations with disease-specific survival. We also summarized the somatic mutations identified in a subset of tumors analyzed for hotspot mutations in cancer-associated gene panels. Most of the patients were white (82%) and female (61%). The median age was 62 years. With a median follow-up of 1.63 years, median disease-specific survival was 1.75 years, and 121 patients (76%) died of anorectal melanoma. Patients presenting with regional (34%) or distant metastases (24%) had significantly shorter disease-specific survival compared to those with disease localized to the anorectum (42%). Of the 71 anorectal melanoma tumors analyzed for hotspot genetic alterations, somatic mutations involving the KIT gene (24%) were most common followed by NRAS (19%). Increasing primary tumor thickness, lymphovascular invasion, and absence of regression also correlated with shorter disease-specific survival. Primary tumor parameters correlated with shorter disease-specific survival in patients presenting with localized disease (tumor thickness) or regional metastases (tumor thickness, absence of regression, and lymphovascular invasion), but not in patients presenting with distant metastases. Grouping of patients according to a schema based on modifications of the 8th edition AJCC cutaneous melanoma staging system stratified survival in anorectal melanoma. Our findings support stage-specific associations between primary tumor parameters and disease-specific survival in anorectal melanoma. Moreover, the AJCC cutaneous melanoma staging system and minor modifications of it predicted survival among anorectal melanoma patients

Systematic identification of signaling pathways with potential to confer anticancer drug resistance

Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)–mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF[superscript V600E] melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.National Institutes of Health (U.S.) (Grant CA103866)National Institutes of Health (U.S.) (Grant AI07389

Does the drug sensitivity of malaria parasites depend on their virulence?

<p>Abstract</p> <p>Background</p> <p>Chemotherapy can prompt the evolution of classical drug resistance, but selection can also favour other parasite traits that confer a survival advantage in the presence of drugs. The experiments reported here test the hypothesis that sub-optimal drug treatment of malaria parasites might generate survival and transmission advantages for virulent parasites.</p> <p>Methods</p> <p>Two <it>Plasmodium chabaudi </it>lines, one derived from the other by serial passage, were used to establish avirulent and virulent infections in mice. After five days, infections were treated with various doses of pyrimethamine administered over 1 or 4 days. Virulence measures (weight and anaemia), parasite and gametocyte dynamics were followed until day 21.</p> <p>Results</p> <p>All treatment regimes reduced parasite and gametocyte densities, but infections with the virulent line always produced more parasites and more gametocytes than infections with the avirulent line. Consistent with our hypothesis, drug treatment was disproportionately effective against the less virulent parasites. Treatment did not affect the relative transmission advantage of the virulent line. Neither of the lines contained known mutations conferring classical drug resistance.</p> <p>Conclusion</p> <p>Drug-sensitivity of malaria parasites can be virulence-dependent, with virulent parasites more likely to survive sub-optimal treatment. If this proves to be general for a variety of drugs and parasite species, selection imposed by sub-optimal drug treatment could result in the evolution of more aggressive malaria parasites.</p

Ionic immune suppression within the tumour microenvironment limits T cell effector function.

Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy

Short-term treatment with multi-drug regimens combining BRAF/MEK-targeted therapy and immunotherapy results in durable responses in Braf-mutated melanoma

Targeted and immunotherapy regimens have revolutionized the treatment of advanced melanoma patients. Despite this, only a subset of patients respond durably. Recently, combination strategies of BRAF/MEK inhibitors with immune checkpoint inhibitor monotherapy (α-CTLA-4 or α-PD-1) have increased the rate of durable responses. Based on evidence from our group and others, these therapies appear synergistic, but at the cost of significant toxicity. We know from other treatment paradigms (e.g. hematologic malignancies) that combination strategies with multi-drug regimens (\u3e4 drugs) are associated with more durable disease control. To better understand the mechanism of these improved outcomes, and to identify and prioritize new strategies for testing, we studied several multi-drug regimens combining BRAF/MEK targeted therapy and immunotherapy combinations in a Braf-mutant murine melanoma model (BrafV600E/Pten−/−). Short-term treatment with α-PD-1 and α-CTLA-4 monotherapies were relatively ineffective, while treatment with α-OX40 demonstrated some efficacy [17% of mice with no evidence of disease, (NED), at 60-days]. Outcomes were improved in the combined α-OX40/α-PD-1 group (42% NED). Short-term treatment with quadruplet therapy of immunotherapy doublets in combination with targeted therapy [dabrafenib and trametinib (DT)] was associated with excellent tumor control, with 100% of mice having NED after combined DT/α-CTLA-4/α-PD-1 or DT/α-OX40/α-PD-1. Notably, tumors from mice in these groups demonstrated a high proportion of effector memory T cells, and immunologic memory was maintained with tumor re-challenge. Together, these data provide important evidence regarding the potential utility of multi-drug therapy in treating advanced melanoma and suggest these models can be used to guide and prioritize combinatorial treatment strategies