Whole-genome analysis informs breast cancer response to aromatase inhibition.

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

Interrogation of inhibitor of nuclear factor κB α/nuclear factor κB (IκBα/NF-κB) negative feedback loop dynamics: from single cells to live animals in vivo.

Full understanding of the biological significance of negative feedback processes requires interrogation at multiple scales as follows: in single cells, cell populations, and live animals in vivo. The transcriptionally coupled IκBα/NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation, represents a model system for multiscalar reporters. Using a κB(5)→IκBα-FLuc bioluminescent reporter, we rigorously evaluated the dynamics of ΙκBα degradation and subsequent NF-κB transcriptional activity in response to diverse modes of TNFα stimulation. Modulating TNFα concentration or pulse duration yielded complex, reproducible, and differential ΙκBα dynamics in both cell populations and live single cells. Tremendous heterogeneity in the transcriptional amplitudes of individual responding cells was observed, which was greater than the heterogeneity in the transcriptional kinetics of responsive cells. Furthermore, administration of various TNFα doses in vivo generated ΙκBα dynamic profiles in the liver resembling those observed in single cells and populations of cells stimulated with TNFα pulses. This suggested that dose modulation of circulating TNFα was perceived by hepatocytes in vivo as pulses of increasing duration. Thus, a robust bioluminescent reporter strategy enabled rigorous quantitation of NF-κB/ΙκBα dynamics in both live single cells and cell populations and furthermore, revealed reproducible behaviors that informed interpretation of in vivo studies.

Toxicity and Survival After Intensity-Modulated Proton Therapy Versus Passive Scattering Proton Therapy for NSCLC.

OBJECTIVE: Although intensity-modulated radiation therapy (IMPT) is dosimetrically superior to passive scattering proton therapy (PSPT) for locally advanced NSCLC (LA-NSCLC), direct comparisons of clinical outcomes are lacking. Here, we compare toxicity profiles and clinical outcomes after IMPT versus PSPT for LA-NSCLC. METHODS: This is a nonrandomized, comparative study of two independent cohorts with LA-NSCLC (stage II-IIIB, stage IV with solitary brain metastasis) treated with concurrent chemotherapy and proton beam therapy. Toxicity (Common Terminology Criteria for Adverse Events version 4.0) and outcomes were prospectively collected as part of a clinical trial (ClinicalTrials.gov identifier NCT00915005) or prospective registry (ClinicalTrials.gov identifier NCT00991094). RESULTS: Of 139 patients, 86 (62%) received PSPT and 53 (38%) IMPT; median follow-up times were 23.9 and 29.0 months, respectively. IMPT delivered lower mean radiation doses to the lungs (PSPT 16.0 Gy versus IMPT 13.0 Gy, p < 0.001), heart (10.7 Gy versus 6.6 Gy, p = 0.004), and esophagus (27.4 Gy versus 21.8 Gy, p = 0.005). Consequently, the IMPT cohort had lower rates of grade 3 or higher pulmonary (17% versus 2%, p = 0.005) and cardiac (11% versus 0%, p = 0.01) toxicities. Six patients (7%) with PSPT and zero patients (0%) with IMPT experienced grade 4 or 5 toxicity. Lower rates of pulmonary (28% versus 3%, p = 0.006) and cardiac (14% versus 0%, p = 0.05) toxicities were observed in the IMPT cohort even after propensity score matching for baseline imbalances. There was also a trend toward longer median overall survival in the IMPT group (23.9 mo versus 36.2 mo, p = 0.09). No difference was found in the 3-year rates of local (25% versus 20%, p = 0.44), local-regional (29% versus 36%, p = 0.56) and distant (52% versus 51%, p = 0.71) recurrences. CONCLUSIONS: IMPT is associated with lower radiation doses to the lung, heart, and esophagus, and lower rates of grade 3 or higher cardiopulmonary toxicity; additional clinical studies will be needed to assess the potential differences in survival between the two techniques.

Altered shear stress stimulates upregulation of endothelial VCAM-1 and ICAM-1 in a BMP-4- and TGF-beta1-dependent pathway.

OBJECTIVE: Hemodynamics has been associated with aortic valve (AV) inflammation, but the underlying mechanisms are not well understood. Here we tested the hypothesis that altered shear stress conditions stimulate the expression of cytokines and adhesion molecules in AV leaflets via a bone morphogenic protein (BMP)- and transforming growth fact (TGF)-beta1-dependent pathway. METHODS AND RESULTS: The ventricularis or aortic surface of porcine AV leaflets were exposed for 48 hours to unidirectional pulsatile and bidirectional oscillatory shear stresses ex vivo. Immunohistochemistry was performed to detect expressions of the 4 inflammatory markers VCAM-1, ICAM-1, BMP-4, and TGF-beta1. Exposure of the aortic surface to pulsatile shear stress (altered hemodynamics), but not oscillatory shear stress, increased expression of the inflammatory markers. In contrast, neither pulsatile nor oscillatory shear stress affected expression of the inflammatory markers on the ventricularis surface. The shear stress-dependent expression of VCAM-1, ICAM-1, and BMP-4, but not TGF-beta1, was significantly reduced by the BMP inhibitor noggin, whereas the TGF-beta1 inhibitor SB431542 blocked BMP-4 expression on the aortic surface exposed to pulsatile shear stress. CONCLUSIONS: The results demonstrate that altered hemodynamics stimulates the expression of AV leaflet endothelial adhesion molecules in a TGF-beta1- and BMP-4-dependent manner, providing some potential directions for future drug-based therapies for AV diseases.

Clinical outcomes after intensity-modulated proton therapy with concurrent chemotherapy for inoperable non-small cell lung cancer.

BACKGROUND & PURPOSE: We report disease control, survival, and toxicity in patients with advanced inoperable non-small cell lung cancer (NSCLC) receiving concurrent chemotherapy and intensity-modulated proton therapy (IMPT) at a single institution. MATERIAL AND METHODS: All patients were treated with IMPT with concurrent chemotherapy. Endpoints assessed were local, regional, and distant control, disease-free survival (DFS), and overall survival (OS). RESULTS: Fifty-one patients were enrolled with a median follow-up time of 23.0 months; 39 (76%) were treated with a simultaneous integrated boost to the gross tumor volume (GTV). The median GTV dose was 67.3 CGE and the median CTV dose was 60.0 CGE. Median OS and DFS times were 33.9 months and 12.6 months. The 3-year local control rate was 78.3%. Treatment was well tolerated, with a grade 3 toxicity rate of 18% (9 events: 4 esophagitis, 3 dermatitis, 1 esophageal stricture, and 1 fatigue) and no grade 4 or 5 toxicity. The most common grade 2 toxic effects were esophagitis (22 [43%]), dermatitis (16 [31%]), pain (15 [29%]), and fatigue (14 [27%]). CONCLUSIONS: Treatment of inoperable NSCLC with IMPT and concurrent chemotherapy achieves excellent disease control with tolerable toxicity.

Postoperative Radiotherapy for Multiple Myeloma of Long Bones: Should the Entire Rod Be Treated?

PURPOSE: To characterize local relapse after surgical fixation and postoperative radiotherapy (RT) for multiple myeloma (MM) with cortical involvement of long bones. PATIENTS AND METHODS: We retrospectively identified patients with MM involving cortical long bones treated with surgical fixation followed by postoperative RT at our institution. Local failures, defined as radiographic recurrence along the surgical hardware, were documented, and potential associations of independent variables (RT dose, fractionation, and extent of hardware coverage) with local failure were assessed by univariate Cox regression. RESULTS: We identified 33 patients with 40 treated sites with a median follow-up of 25.7 months; 68% of treatments were for pathologic fracture, and 32% were for impending fracture. The most common dose and fractionation were 20 to 25 Gy in 8 to 12 fractions. On average, 76% of the surgical hardware was covered by the postoperative RT field (median, 80%; range, 28%-100%). Local failure was observed in 5 cases (12.5%), 2 within the RT field and 3 out of field. None of the relapses resulted in hardware failure, and 2 were retreated with RT. The extent of hardware coverage predicted disease relapse along the hardware (hazard ratio = 6.44; 95% confidence interval, 1.09-37.97; P = .04); however, total RT dose, biologically effective dose, and number of fractions did not. CONCLUSION: After internal fixation of long bones with MM, full hardware coverage with the RT field could reduce the risk, though small, of disease developing in the future in the proximate hardware. Postoperative RT doses of 20 to 25 Gy in 8 to 10 fractions can achieve excellent local control.

Radioresistant Cervical Cancers Are Sensitive to Inhibition of Glycolysis and Redox Metabolism.

Highly glycolytic cervical cancers largely resist treatment by cisplatin and coadministered pelvic irradiation as the present standard of care. In this study, we investigated the effects of inhibiting glycolysis and thiol redox metabolism to evaluate them as alternate treatment strategies in these cancers. In a panel of multiple cervical cancer cell lines, we evaluated sensitivity to inhibition of glycolysis (2-deoxyglucose, 2-DG) with or without simultaneous inhibition of glutathione and thioredoxin metabolism (BSO/AUR). Intracellular levels of total and oxidized glutathione, thioredoxin reductase activity, and indirect measures of intracellular reactive oxygen species were compared before and after treatment. Highly radioresistant cells were the most sensitive to 2-DG, whereas intermediate radioresistant cells were sensitive to 2-DG plus BSO/AUR. In response to 2-DG/BSO/AUR treatment, we observed increased levels of intracellular oxidized glutathione, redox-sensitive dye oxidation, and decreased glucose utilization via multiple metabolic pathways including the tricarboxylic acid cycle. 2-DG/BSO/AUR treatment delayed the growth of tumors composed of intermediate radioresistant cells and effectively radiosensitized these tumors at clinically relevant radiation doses both in vitro and in vivo Overall, our results support inhibition of glycolysis and intracellular redox metabolism as an effective alternative drug strategy for the treatment of highly glycolytic and radioresistant cervical cancers.Significance: This study suggests a simple metabolic approach to strike at an apparent Achilles' heel in highly glycolytic, radioresistant forms of cervical cancers, possibly with broader applications in cancer therapy. Cancer Res; 78(6); 1392-403. ©2018 AACR.

Clinical outcomes and differential effects of PI3K pathway mutation in obese versus non-obese patients with cervical cancer.

The purpose of this study was to evaluate the effect of obesity and obesity-associated factors on the outcomes of patients with cervical cancer. Outcomes were evaluated in 591 patients with FIGO Ib to IV cervical cancer treated uniformly with definitive radiation. Patients were stratified into 3 groups based upon pretreatment Body Mass Index (BMI): A ≤ 18.5; B 18.6 - 34.9; and C ≥ 35. The 5-year freedom from failure rates were 58, 59, and 73% for BMI groups A, B, and C (p = 0.01). Overall survival rates were 50, 59, and 68%, respectively (p = 0.02). High expression of phosphorylated AKT (pAKT) was associated with poor outcomes only in non-obese patients. Obese patients with PI3K pathway mutant tumors had a trend toward favorable outcomes, while a similar effect was not observed in non-obese patients. Compared to similar tumors from non-obese hosts, PIK3CA and PTEN mutant tumors from obese patients failed to express high levels of phosphorylated AKT and its downstream targets. These results show that patients with obesity at the time of diagnosis of cervical cancer exhibit improved outcomes after radiation. PI3K/AKT pathway mutations are common in obese patients, but are not associated with activation of AKT signaling.

Late gastrointestinal tissue effects after hypofractionated radiation therapy of the pancreas.

BACKGROUND: To consolidate literature reports of serious late gastrointestinal toxicities after hypofractionated radiation treatment of pancreatic cancer and attempt to derive normal tissue complication probability (NTCP) parameters using the Lyman-Kutcher-Burman model. METHODS: Published reports of late grade 3 or greater gastrointestinal toxicity after hypofractionated treatment of pancreatic cancer were reviewed. The biologically equivalent dose in 1.8 Gy fractions was calculated using the EQD model. NTCP parameters were calculated using the LKB model assuming 1-5% of the normal tissue volume was exposed to the prescription dose with α/ß ratios of 3 or 4. RESULTS: A total of 16 human studies were examined encompassing a total of 1160 patients. Toxicities consisted of ulcers, hemorrhages, obstructions, strictures, and perforations. Non-hemorrhagic and non-perforated ulcers occurred at a rate of 9.1% and were the most commonly reported toxicity. Derived NTCP parameter ranges were as follows: n = 0.38-0.63, m = 0.48-0.49, and TD50 = 35-95 Gy. Regression analysis showed that among various study characteristics, dose was the only significant predictor of toxicity. CONCLUSIONS: Published gastrointestinal toxicity reports after hypofractionated radiotherapy for pancreatic cancer were compiled. Median dose was predictive of late grade ≥ 3 gastrointestinal toxicity. Preliminary NTCP parameters were derived for multiple volume constraints.

A high-throughput fluorimetric assay for 2-hydroxyglutarate identifies Zaprinast as a glutaminase inhibitor.

UNLABELLED: Recently identified isocitrate dehydrogenase (IDH) mutations lead to the production of 2-hydroxyglutarate (2HG), an oncometabolite aberrantly elevated in selected cancers. We developed a facile and inexpensive fluorimetric microplate assay for the quantitation of 2HG and performed an unbiased small-molecule screen in live cells to identify compounds capable of perturbing 2HG production. Zaprinast, a phosphodiesterase 5 inhibitor, was identified as an efficacious modulator of 2HG production and confirmed to lower 2HG levels in vivo. The mechanism of action was not due to cGMP stabilization, but rather, profiling of metabolites upstream of mutant IDH1 pointed to targeted inhibition of the enzyme glutaminase (GLS). Zaprinast treatment reversed histone hypermethylation and soft-agar growth of IDH1-mutant cells, and treatment of glutamine-addicted pancreatic cancer cells reduced growth and sensitized cells to oxidative damage. Thus, Zaprinast is efficacious against glutamine metabolism and further establishes the therapeutic linkages between GLS and 2HG-mediated oncogenesis. SIGNIFICANCE: Gain-of-function IDH mutations are common events in glioma, acute myelogenous leukemia, and other cancer types, which lead to the accumulation of the oncometabolite 2HG. We show that the drug Zaprinast is capable of reducing cellular 2HG levels by inhibiting the upstream enzyme GLS, thus identifying a new strategy to target 2HG production in selected IDH-mutant cancers.

Dual-color click beetle luciferase heteroprotein fragment complementation assays.

Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically relevant time scales. Herein, we describe a set of reversible multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discrete pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to ß-TrCP, an E3-ligase common to the regulation of both ß-catenin and IκBα, GSK3ß was identified as a candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells.

Design of an ex vivo culture system to investigate the effects of shear stress on cardiovascular tissue.

Mechanical forces are known to affect the biomechanical properties of native and engineered cardiovascular tissue. In particular, shear stress that results from the relative motion of heart valve leaflets with respect to the blood flow is one important component of their mechanical environment in vivo. Although different types of bioreactors have been designed to subject cells to shear stress, devices to expose biological tissue are few. In an effort to address this issue, the aim of this study was to design an ex vivo tissue culture system to characterize the biological response of heart valve leaflets subjected to a well-defined steady or time-varying shear stress environment. The novel apparatus was designed based on a cone-and-plate viscometer. The device characteristics were defined to limit the secondary flow effects inherent to this particular geometry. The determination of the operating conditions producing the desired shear stress profile was streamlined using a computational fluid dynamic (CFD) model validated with laser Doppler velocimetry. The novel ex vivo tissue culture system was validated in terms of its capability to reproduce a desired cone rotation and to maintain sterile conditions. The CFD results demonstrated that a cone angle of 0.5 deg, a cone radius of 40 mm, and a gap of 0.2 mm between the cone apex and the plate could limit radial secondary flow effects. The novel cone-and-plate permits to expose nine tissue specimens to an identical shear stress waveform. The whole setup is capable of accommodating four cone-and-plate systems, thus concomitantly subjecting 36 tissue samples to desired shear stress condition. The innovative design enables the tissue specimens to be flush mounted in the plate in order to limit flow perturbations caused by the tissue thickness. The device is capable of producing shear stress rates of up to 650 dyn cm(-2) s(-1) (i.e., maximum shear stress rate experienced by the ventricular surface of an aortic valve leaflet) and was shown to maintain tissue under sterile conditions for 120 h. The novel ex vivo tissue culture system constitutes a valuable tool toward elucidating heart valve mechanobiology. Ultimately, this knowledge will permit the production of functional tissue engineered heart valves, and a better understanding of heart valve biology and disease progression.