A comparison of immunohistochemical assays and FISH in detecting the ALK translocation in diagnostic histological and cytological lung tumor material.

Introduction: Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH), but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives. 
Methods: Fifteen FISH-positive cases from patients, seven with data on crizotinib therapy and clinical response, were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3, using the proprietary automated system (Ventana), ALK1 (Dako), and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (<15% rearrangement detected) cases were also retrieved. Of the total 32 specimens, 17 were excisions and 15 were computed tomography-guided biopsies or cytological specimens. All three antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity, and the proportion of malignant cells stained was documented. Cutoffs were set by receiver operating curve analysis for positivity to optimize correct classification. 
Results: All three IHC assays were 100% specific but sensitivity did vary: D5F3 86%, ALK 79%, 5A4 71%. Intensity was the most discriminating measure overall, with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene.
Conclusions: IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%), specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative, suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein.

Lung stereotactic body radiation therapy (SBRT) delivered over 4 or 11days: A comparison of acute toxicity and quality of life.

Abstract
PURPOSE:
The optimal duration over which lung SBRT should be delivered is unknown. We conducted a randomized pilot study in patients treated with four fractions of lung SBRT delivered over 4 or over 11days.
METHODS:
Patients with a peripheral solitary lung tumor (NSCLC or pulmonary metastasis) ?5cm were eligible. For NSCLC lung tumors ?3cm, a dose of 48Gy in 4 fractions was used, otherwise 52Gy in 4 fractions was delivered. Patients were randomized to receive treatment over 4 consecutive days or over 11days. The primary end-point was acute grade ?2 toxicity. Secondary end-points included quality of life (QOL) assessed using the EORTC QLQ-C30 and QLQ-LC13 questionnaires.
RESULTS:
Fifty four patients were enrolled. More patients in the 11day group had respiratory symptoms at baseline. 55.6% patients treated over 4days and 33.3% of patients treated over 11days experienced acute grade ?2 toxicity (p=0.085). Dyspnea, fatigue and coughing domains were worse in the 11day group at baseline. At 1 and 4months, more patients in the 4day group experienced a clinically meaningful worsening in the dyspnea QOL domain compared to the 11day group (44.5% vs 15.4%, p=0.02; 38.5% vs 12.0%, p=0.03, respectively). However, raw QOL scores were not different at these time-points between treatment groups.
CONCLUSIONS:
Grade 2 or higher acute toxicity was more common in the 4day group, approaching statistical significance. More patients treated on 4 consecutive days reported a clinically meaningful increase in dyspnea, although interpretation of these results is challenging due to baseline imbalance between treatment groups. Larger studies are required to validate these results.

Automated tumor analysis for molecular profiling in lung cancer

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics.

Ischemic preconditioning before lower limb ischemia-reperfusion protects against acute lung injury

Objective: Prolonged limb ischemia followed by reperfusion (I/R) is associated with a systemic inflammatory response syndrome and remote acute lung injury. Ischemic preconditioning (IPC), achieved with repeated brief periods of I/R before the prolonged ischemic period, has been shown to protect skeletal muscle against ischemic injury. The aim of this study was to ascertain whether IPC of the limb before I/R injury also attenuates systemic inflammation and acute lung injury in a fully resuscitated porcine model of hind limb I/R. Methods: This prospective, randomized, controlled, experimental animal study was performed in a university-based animal research facility with 18 male Landrace pigs that weighed from 30 to 35 kg. Anesthetized ventilated swine were randomized (n = 6 per group) to three groups: sham-operated control group, I/R group (2 hours of bilateral hind limb ischemia and 2.5 hours of reperfusion), and IPC group (three cycles of 5 minutes of ischemia/5 minutes of reperfusion immediately preceding I/R). Plasma was separated and stored at -70° C for later determination of plasma tumor necrosis factor-a and interleukin-6 with bioassay as markers of systemic inflammation. Circulating phagocytic cell priming was assessed with a whole blood chemiluminescence assay. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were markers of edema and neutrophil sequestration, respectively. The alveolar-arterial oxygen gradient and pulmonary artery pressure were indices of lung function. Results: In a porcine model, bilateral hind limb (I/R) injury significantly increased plasma interleukin-6 concentrations, circulating phagocytic cell priming, and pulmonary leukosequestration, edema, and impaired gas exchange. Conversely, pigs treated with IPC before the onset of the ischemic period had significantly reduced interleukin-6 levels, circulating phagocytic cell priming, and experienced significantly less pulmonary edema, leukosequestration, and respiratory failure. Conclusion: Lower limb IPC protects against systemic inflammation and acute lung injury in lower limb I/R injury.

Bactericidal/permeability-increasing protein attenuates systemic inflammation and acute lung injury in porcine lower limb ischemia-reperfusion injury.

OBJECTIVE: To investigate the role of recombinant bactericidal/permeability-increasing protein (rBPI21) in the attenuation of the sepsis syndrome and acute lung injury associated with lower limb ischemia-reperfusion (I/R) injury. SUMMARY BACKGROUND DATA: Gut-derived endotoxin has been implicated in the conversion of the sterile inflammatory response to a lethal sepsis syndrome after lower torso I/R injury. rBPI21 is a novel antiendotoxin therapy with proven benefit in sepsis. METHODS: Anesthetized ventilated swine underwent midline laparotomy and bilateral external iliac artery occlusion for 2 hours followed by 2.5 hours of reperfusion. Two groups (n = 6 per group) were randomized to receive, by intravenous infusion over 30 minutes, at the start of reperfusion, either thaumatin, a control-protein preparation, at 2 mg/kg body weight, or rBPI21 at 2 mg/kg body weight. A control group (n = 6) underwent laparotomy without further treatment and was administered thaumatin at 2 mg/kg body weight after 2 hours of anesthesia. Blood from a carotid artery cannula was taken every half-hour for arterial blood gas analysis. Plasma was separated and stored at -70 degrees C for later determination of plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 by bioassay, and IL-8 by enzyme-linked immunosorbent assay (ELISA), as a markers of systemic inflammation. Plasma endotoxin concentration was measured using ELISA. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were used as markers of edema and neutrophil sequestration, respectively. Bronchoalveolar lavage protein concentration was measured by the bicinclinoic acid method as a measure of capillary-alveolar protein leak. The alveolar-arterial gradient was measured; a large gradient indicated impaired oxygen transport and hence lung injury. RESULTS: Bilateral hind limb I/R injury increased significantly intestinal mucosal acidosis, intestinal permeability, portal endotoxemia, plasma IL-6 concentrations, circulating phagocytic cell priming and pulmonary leukosequestration, edema, capillary-alveolar protein leak, and impaired gas exchange. Conversely, pigs treated with rBPI21 2 mg/kg at the onset of reperfusion had significantly reduced intestinal mucosal acidosis, portal endotoxin concentrations, and circulating phagocytic cell priming and had significantly less pulmonary edema, leukosequestration, and respiratory failure. CONCLUSIONS: Endotoxin transmigration across a hyperpermeable gut barrier, phagocytic cell priming, and cytokinemia are key events of I/R injury, sepsis, and pulmonary dysfunction. This study shows that rBPI21 ameliorates these adverse effects and may provide a novel therapeutic approach for prevention of I/R-associated sepsis syndrome.

(18)F-FDG PET-CT simulation for non-small-cell lung cancer: effect in patients already staged by PET-CT.

Purpose: Positron emission tomography (PET), in addition to computed tomography (CT), has an effect in target volume definition for radical radiotherapy (RT) for non–small-cell lung cancer (NSCLC). In previously PET-CT staged patients with NSCLC, we assessed the effect of using an additional planning PET-CT scan for gross tumor volume (GTV) definition. Methods and Materials: A total of 28 patients with Stage IA-IIIB NSCLC were enrolled. All patients had undergone staging PET-CT to ensure suitability for radical RT. Of the 28 patients, 14 received induction chemotherapy. In place of a RT planning CT scan, patients underwent scanning on a PET-CT scanner. In a virtual planning study, four oncologists independently delineated the GTVon the CT scan alone and then on the PET-CTscan. Intraobserver and interobserver variability were assessed using the concordance index (CI), and the results were compared using the Wilcoxon signed ranks test. Results: PET-CT improved the CI between observers when defining the GTVusing the PET-CT images compared with using CTalone for matched cases (median CI, 0.57 for CTand 0.64 for PET-CT, p = .032). The median of the mean percentage of volume change from GTVCT to GTVFUSED was 5.21% for the induction chemotherapy group and 18.88% for the RT-alone group. Using the Mann-Whitney U test, this was significantly different (p = .001). Conclusion: PET-CT RT planning scan, in addition to a staging PET-CT scan, reduces interobserver variability in GTV definition for NSCLC. The GTV size with PET-CT compared with CT in the RT-alone group increased and was reduced in the induction chemotherapy group.

The Deubiquitinating Enzyme USP17 Is Highly Expressed in Tumor Biopsies, Is Cell Cycle Regulated, and Is Required for G1-S Progression

Ubiquitination is a reversible posttranslational modification that is essential for cell cycle control, and it is becoming increasingly clear that the removal of ubiquitin from proteins by deubiquitinating enzymes (DUB) is equally important. In this study, we have identified high levels of the DUB USP17 in several tumor-derived cell lines and primary lung, colon, esophagus, and cervix tumor biopsies. We also report that USP17 is tightly regulated during the cell cycle in all the cells examined, being abundantly evident in G1 and absent in S phase. Moreover, regulated USP17 expression was necessary for cell cycle progression because its depletion significantly impaired G1-S transition and blocked cell proliferation. Previously, we have shown that USP17 regulates the intracellular translocation and activation of the GTPase Ras by controlling Ras-converting enzyme 1 (RCE1) activation. RCE1 also regulates the processing of other proteins with a CAAX motif, including Rho family GTPases. We now show that USP17 depletion blocks Ras and RhoA localization and activation. Moreover, our results confirm that USP17-depleted cells have constitutively elevated levels of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, known downstream targets of Ras and RhoA signaling. These observations clearly show that USP17 is tightly regulated during cell division and that its expression is necessary to coordinate cell cycle progression, and thus, it may be considered a promising novel cancer therapeutic target. Cancer Res; 70(8); 3329–39. ©2010 AACR.

Raman microscopy in the diagnosis and prognosis of surgically resected nonsmall cell lung cancer

The main curative therapy for patients with nonsmall cell lung cancer is surgery. Despite this, the survival rate is only 50%, therefore it is important to more efficiently diagnose and predict prognosis for lung cancer patients. Raman spectroscopy is useful in the diagnosis of malignant and premalignant lesions. The aim of this study is to investigate the ability of Raman microscopy to diagnose lung cancer from surgically resected tissue sections, and predict the prognosis of these patients. Tumor tissue sections from curative resections are mapped by Raman microscopy and the spectra analzsed using multivariate techniques. Spectra from the tumor samples are also compared with their outcome data to define their prognostic significance. Using principal component analysis and random forest classification, Raman microscopy differentiates malignant from normal lung tissue. Principal component analysis of 34 tumor spectra predicts early postoperative cancer recurrence with a sensitivity of 73% and specificity of 74%. Spectral analysis reveals elevated porphyrin levels in the normal samples and more DNA in the tumor samples. Raman microscopy can be a useful technique for the diagnosis and prognosis of lung cancer patients receiving surgery, and for elucidating the biochemical properties of lung tumors. (C) 2010 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3323088]

18F-fluorodeoxyglucose positron emission tomography/computed tomography-based radiotherapy target volume definition in non-small-cell lung cancer: delineation by radiation oncologists vs. joint outlining with a PET radiologist?

Purpose: F-18-Fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) has benefits in target volume (TV) definition in radiotherapy treatment planning (RTP) for non small-cell lung cancer (NSCLC); however, an optimal protocol for TV delineation has not been determined. We investigate volumetric and positional variation in gross tumor volume (GTV) delineation using a planning PET/CT among three radiation oncologists and a PET radiologist.

RUNX3 downregulation in human lung adenocarcinoma is independent of p53, EGFR or KRAS status

RUNX3 aberrations play a pivotal role in the oncogenesis of breast, gastric, colon, skin and lung tissues. The aim of this study was to characterize further the expression of RUNX3 in lung cancers. To achieve this, a lung cancer tissue microarray (TMA), frozen lung cancer tissues and lung cell lines were examined for RUNX3 expression by immunohistochemistry, while the TMA was also examined for EGFR and p53 expression. RUNX3 promoter methylation status, and EGFR and KRAS mutation status were also investigated. Inactivation of RUNX3 was observed in 70% of the adenocarcinoma samples, and this was associated with promoter hypermethylation but not biased to EGFR/KRAS mutations. Our results suggest a central role of RUNX3 downregulation in pulmonary adenocarcinoma, which may not be dependent of other established cancer-causing pathways and may have important diagnostic and screening implications.

Time and Cell Type Dependency of Survival Responses in Co-cultured Tumor and Fibroblast Cells after Exposure to Modulated Radiation Fields

Advanced radiotherapy techniques such as intensity-modulated radiation therapy (IMRT) achieve high levels of conformity to the target volume through the sequential delivery of highly spatially and temporally modulated radiation fields, which have been shown to impact radiobiological response. This study aimed to characterize the time and cell type dependency of survival responses to modulated fields using single cell type (SCT) and mixed cell type (MCT) co-culture models of transformed fibroblast (AG0-1522b) cells, and prostate (DU-145) and lung (H460) cancer cells. In SCT cultures, in-field responses showed no significant time dependency while out-of-field responses occurred early, and plateaued 6 h after irradiation in both DU-145 and H460 cells. Under modulated beam configurations MCT co-cultures showed cell-specific, differential out-of-field responses depending on the irradiated in-field and responding out-of-field cell type. The observed differential out-of-field responses may be due to the genetic background of the cells, in particular p53 status, which has been shown to mediate radiation-induced bystander effects (RIBEs). These data provide further insight into the radiobiological parameters that influence out-of-field responses, which have potential implications for advanced radiotherapy modalities and may provide opportunities for biophysical optimization in radiotherapy treatment planning.

PET/CT imaging for target volume delineation in curative intent radiotherapy of non-small cell lung cancer: IAEA consensus report 2014

This document describes best practice and evidence based recommendations for the use of FDG-PET/CT for the purposes of radiotherapy target volume delineation (TVD) for curative intent treatment of non-small cell lung cancer (NSCLC). These recommendations have been written by an expert advisory group, convened by the International Atomic Energy Agency (IAEA) to facilitate a Coordinated Research Project (CRP) aiming to improve the applications of PET based radiation treatment planning (RTP) in low and middle income countries. These guidelines can be applied in routine clinical practice of radiotherapy TVD, for NSCLC patients treated with concurrent chemoradiation or radiotherapy alone, where FDG is used, and where a calibrated PET camera system equipped for RTP patient positioning is available. Recommendations are provided for PET and CT image visualization and interpretation, and for tumor delineation using planning CT with and without breathing motion compensation.

Inhibition of dUTPase induces synthetic lethality with thymidylate synthase-targeted therapies in non-small cell lung cancer

Chemotherapies that target thymidylate synthase (TS) continue to see considerable clinical expansion in non-small cell lung cancer (NSCLC). One drawback to TS-targeted therapies is drug resistance and subsequent treatment failure. Novel therapeutic and biomarker-driven strategies are urgently needed. The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is reported to protect tumor cells from aberrant misincorporation of uracil during TS inhibition. The goal of this study was to investigate the expression and significance of dUTPase in mediating response to TS-targeted agents in NSCLC. The expression of dUTPase in NSCLC cell lines and clinical specimens was measured by quantitative real-time reverse transcriptase PCR and immunohistochemistry. Using a validated RNA interference approach, dUTPase was effectively silenced in a panel of NSCLC cell lines and response to the fluoropyrimidine fluorodeoxyuridine (FUdR) and the antifolate pemetrexed was analyzed using growth inhibition and clonogenic assays. Apoptosis was analyzed by flow cytometry. Significant variation in the quantity and cellular expression of dUTPase was observed, including clear evidence of overexpression in NSCLC cell line models and tumor specimens at the mRNA and protein level. RNA interference-mediated silencing of dUTPase significantly sensitized NSCLC cells to growth inhibition induced by FUdR and pemetrexed. This sensitization was accompanied by a significant expansion of intracellular dUTP pools and significant decreases in NSCLC cell viability evaluated by clonogenicity and apoptotic analyses. Together, these results strongly suggest that uracil misincorporation is a potent determinant of cytotoxicity to TS inhibition in NSCLC and that inhibition of dUTPase is a mechanism-based therapeutic approach to significantly enhance the efficacy of TS-targeted chemotherapeutic agents.

Prognostic significance of combining VEGFA, FLT1 and KDR mRNA expression in lung cancer

Angiogenesis is important in cancer progression. Promising results in clinical trials have indicated that targeting vascular epidermal growth factor (VEGF) signaling may prolong lung cancer patient survival. In particular, various studies have implicated VEGFA as a potential prognostic marker in lung cancer, although prognostication using the expression of VEGF receptors (VEGFRs), such as fms-related tyrosine kinase 1 (FLT1; also known as VEGFR1) and kinase insert domain receptor (KDR; also known as VEGFR2), has produced varied results in different lung cancer studies. The present study aimed to investigate the prognostic significance of these three factors, alone or in combination. mRNA expression data were extracted from four independent lung cancer cohorts totaling 583 patients, and the association between mRNA expression and survival was investigated by performing statistical analyses. When VEGFA, FLT1 and KDR expression were considered alone, only VEGFA demonstrated a significant association with patient survival consistently across all four datasets (P<0.05). Patients with a high expression of VEGFA and one of the two receptors were associated with significantly worse survival than patients expressing low levels of VEGFA and the particular receptor (P<0.05). Notably, patients with a high level expression of all three genes in their tumor specimens were associated with a significantly shorter survival time compared with patients exhibiting a low level expression of one, two or all three genes (P<0.05). The results indicate that a high level of VEGFA expression and its receptors may be required for cancer progression. Therefore, these three factors should be considered together as a prognostic indicator for lung cancer patients.