Stephen Lam

The xc− cystine/glutamate antiporter as a potential therapeutic target for small-cell lung cancer: use of sulfasalazine

PurposeTo determine whether the xc− cystine transporter could be a useful therapeutic target for small-cell lung cancer (SCLC).MethodsHuman SCLC cell cultures were examined for growth dependence on extracellular cystine, xc− expression, glutathione levels and response to highly specific xc− inhibitors, i.e., monosodium glutamate (MSG) and the anti-inflammatory drug, sulfasalazine (SASP). In studying tumor growth inhibition by SASP, use was also made of a novel SCLC tissue xenograft model, LU6-SCLC, derived from a chemoresistant patient’s SCLC specimen.ResultsGrowth of NCI-H69 and NCI-H82 SCLC cells greatly depended on xc−-mediated uptake of cystine. SASP substantially reduced their glutathione levels (>70%; 0.3xa0mM SASP; 24xa0h) and growth (72xa0h) with IC50s of 0.21 and 0.13xa0mM, respectively; MSG also inhibited growth markedly. Both SASP- and MSG-induced growth arrests were largely prevented by cystine uptake-enhancing 2-mercaptoethanol (66xa0μM) indicating they were primarily due to cystine starvation. Without major side-effects, SASP (i.p.) restrained growth of NCI-H69 cell xenografts (~50%) and, importantly, substantially inhibited growth of the clinically more relevant LU6-SCLC tissue xenografts (~70% by stereological analysis), reducing tumor glutathione contents.ConclusionsThe xc− cystine/glutamate antiporter is potentially useful as a target for therapy of SCLC based on glutathione depletion. Sulfasalazine may be readily used for this approach, especially in combination chemotherapy.

Integrated endoscopy system for simultaneous imaging and spectroscopy for early lung cancer detection.

An integrated endoscopy system for simultaneous imaging and spectroscopy was developed to facilitate more accurate and convenient detection of early lung cancers. A specially designed three-CCD camera in combination with a dedicated light source permits capture of both white-light color images and tissue autofluorescence images without the need to switch between two different cameras. A mirror with an optical fiber at its center, placed at an interim imaging plane inside the camera unit, facilitates simultaneous imaging and spectroscopy measurements in either white-light reflectance mode or fluorescence mode. The system has been successfully tested in a clinic, demonstrating a practical approach to improve both diagnostic sensitivity and specificity at the same time.

Integrative genomic and gene expression analysis of chromosome 7 identified novel oncogene loci in non-small cell lung cancer

Lung cancer accounts for over a quarter of cancer deaths, with non-small cell lung cancer (NSCLC) accounting for approximately 80% of cases. Several genome studies have been undertaken in both cell models of NSCLC and clinical samples to identify alterations underlying disease behaviour, and many have identified recurring aberrations of chromosome 7. The presence of recurring chromosome 7 alterations that do not span the well-studied oncogenes EGFR (at 7p11.2) and MET (at 7q31.2) has raised the hypothesis of additional genes on this chromosome that contribute to tumourigenesis. In this study, we demonstrated that multiple loci on chromosome 7 are indeed amplified in NSCLC, and through integrative analysis of gene dosage alterations and parallel gene expression changes, we identified new lung cancer oncogene candidates, including FTSJ2, NUDT1, TAF6, and POLR2J. Activation of these key genes was confirmed in panels of clinical lung tumour tissue as compared with matched normal lung tissue. The detection of gene activation in multiple cohorts of samples strongly supports the presence of key genes involved in lung cancer that are distinct from the EGFR and MET loci on chromosome 7.

Elevated Expression of BIRC6 Protein in Non–Small-Cell Lung Cancers is Associated with Cancer Recurrence and Chemoresistance

Introduction: Non–small-cell lung cancer (NSCLC) is an aggressive, highly chemoresistant disease. Reliable prognostic assays and more effective treatments are critically required. BIRC6 (baculoviral inhibitors of apoptosis proteins repeat-containing 6) protein is a member of the inhibitors of apoptosis protein family thought to play an important role in the progression or chemoresistance of many cancers. In this study, we investigated whether BIRC6 expression can be used as a prognostic marker or potential therapeutic target for NSCLC. Methods: In a retrospective analysis, BIRC6 protein expression was determined for 78 resected primary NSCLCs and nine benign lung tissues. Twenty-nine chemoresistant or chemosensitive subrenal capsule NSCLC tissue xenografts were assessed for BIRC6 expression, using immunohistochemistry, and 13 of them for BIRC6 gene copy number, using array comparative genomic hybridization analysis. The effect of small interfering RNA–induced BIRC6 knockdown on the growth of human NSCLC cell cultures and apoptosis (in combination with cisplatin) was investigated. Results: Elevated BIRC6 protein expression in NSCLC tissues was associated with poor 3-year relapse-free patient survival, lymph node involvement, and advanced pathological tumor, node, metastasis stage. In patient-derived lung squamous cell carcinoma xenografts, chemoresistance was associated with elevated BIRC6 expression and increased gene copy number. Small interfering RNA–induced BIRC6 down-regulation inhibited growth of the NSCLC cells and sensitized the cells to cisplatin. Conclusions: BIRC6 may play an important role in the malignant progression and chemoresistance of NSCLC. Elevated BIRC6 protein expression may serve as a predictive marker for chemoresistance of NSCLCs and a poor prognostic factor for NSCLC patients. Down-regulation of the BIRC6 gene as a therapeutic approach may be effective, especially in combination with conventional chemotherapeutics.

Whole-Genome Sequencing Analysis Identifies a Distinctive Mutational Spectrum in an Arsenic-Related Lung Tumor

Introduction: Arsenic exposure is a significant cause of lung cancer in North America and worldwide. Arsenic-related tumors are structurally indistinguishable from those induced by other carcinogens. Because carcinogens, like tobacco, induce distinctive mutational signatures, we sought to characterize the mutational signature of an arsenic-related lung tumor from a never smoker with the use of whole-genome sequencing. Methods: Tumor and lung tissues were obtained from a never smoker with lung squamous cell carcinoma (LUSC), without familiar history of lung cancer and chronically exposed to high levels of arsenic-contaminated drinking water. The Illumina HiSeq-2000 platform was used to sequence each genome at approximately 30-fold haploid coverage. The mutational signature was compared with those observed in previously characterized lung tumors. Results: The arsenic-related tumor exhibited alterations common in LUSC, such as the increased number of copies at 3q26 (SOX2 locus). However, the arsenic-related genome not only harbored a lower number of point mutations, but also had a remarkably high fraction of T>G/A>C mutations and low fraction of C>A/G>T transversions, which is uncharacteristic of LUSCs. Furthermore, at the gene level, we identified a rare G>C mutation in TP53, which is uncommon in lung tumors in general (<0.2%) but has been observed in other arsenic-related malignancies. Conclusions: We generated the first whole-genome sequence of an LUSC from a never-smoker patient chronically exposed to arsenic, and identified a distinct mutational spectrum associated with arsenic exposure, providing novel evidence supporting the hypothesis that arsenic-induced lung tumors arise through molecular mechanisms that differ from those of the common lung cancer.

Multimodal tissue imaging: using coregistered optical tomography data to estimate tissue autofluorescence intensity change due to scattering and absorption by neoplastic epithelial cells

Abstract. Autofluorescence (AF) imaging provides valuable information about the structural and chemical states of tissue that can be used for early cancer detection. Optical scattering and absorption of excitation and emission light by the epithelium can significantly affect observed tissue AF intensity. Determining the effect of epithelial attenuation on the AF intensity could lead to a more accurate interpretation of AF intensity. We propose to use optical coherence tomography coregistered with AF imaging to characterize the AF attenuation due to the epithelium. We present imaging results from three vital tissue models, each consisting of a three-dimensional tissue culture grown from one of three epithelial cell lines (HCT116, OVCAR8, and MCF7) and immobilized on a fluorescence substrate. The AF loss profiles in the tissue layer show two different regimes, each approximately linearly decreasing with thickness. For thin cell cultures (<300u2009u2009μm), the AF signal changes as AF(t)/AF(0)=1−1.3t (t is the thickness in millimeter). For thick cell cultures (>400u2009u2009μm), the AF loss profiles have different intercepts but similar slopes. The data presented here can be used to estimate AF loss due to a change in the epithelial layer thickness and potentially to reduce AF bronchoscopy false positives due to inflammation and non-neoplastic epithelial thickening.

Improvements to a laser Raman spectroscopy system for reducing the false positives of autofluorescence bronchoscopies

Preneoplastic lesions of the bronchial tree have a high probability of developing into malignant tumours. Currently the best method for localizing them for further treatment is a combined white light and autofluorescence bronchoscopy (WLB+AFB). Unfortunately the average specificity from large clinical trials for this combined detection method is low at around 60%, which can result in many false positives. However a recent pilot study showed that adding a point laser Raman spectroscopy (LRS) measurement improved the specificity of detecting lesions with high grade dysplasia or carcinoma in situ to 91% with a sensitivity of 96% compared to WLB+AFB alone. Despite this success, there is still room for much improvement. One constant need is to find better ways to measure the inherently weak Raman emissions in vivo which will result in even better diagnostic sensitivity and specificity. With this aim in mind a new generation Raman system was developed. The system uses the latest charge coupled device (CCD) with low noise, and fast cool down times. A spectrometer was incorporated that was able to measure both the low and high frequency Raman emissions with high resolution. The Raman catheter was also redesigned to include a visible light channel to facilitate the accurate indication of the area being measured. Here the benefits in the adjunct use of LRS to WLB + AFB are presented, and description of the new system and the improvements it offers over the old system are shown.

Microlocalization of Photofrin in neoplastic lesions

Three tumor models were used to study the microlocalization of two photosensitizers, Photofrin and TPPS4 (mesotetra[p-sulphophenyl]porphyne). The three tumor types employed were subcutaneously and intramuscularly grown murine Lewis lung carcinoma, and lung micrometastases, grown on the same animal. The objective of the study was to determine whether these photosensitizers localize differently in these three tumor types. Common to all the tumors was the observation that no substantial photosensitizer fluorescence could be found in the tumor parenchyma. With Photofrin, most of the drug fluorescence was found in the periphery of the tumors, especially around blood vessels and possibly in some macrophages. On the other hand with TPPS4, most of the fluorescence was seen in the basement membrane of blood vessels and airways. The photosensitizer TPPS4 may therefore accumulate less specifically in tumor microenvironment than Photofrin. Both photosensitizers were also found to localize in necrotic areas.

Abstract C41: A phase 1 study of OMN54 in patients with advanced malignancies

Purpose: With the increasing interest in natural products as therapeutics, we performed a Phase I open label study of OMN54 in patients with advanced malignancies to determine toxicity, maximum tolerated dose (MTD), dose limiting toxicities (DLT), and pharmacokinetics (PK). OMN54 is a multitargeted agent prepared from three Chinese botanical sources: Ganoderma lucidum, Salvia miltiorrhiza, and Scutellaria barbata, each with long histories of use as single agents. Methods: Eligible patients (pts) were ≥ 18 years with advanced solid tumor malignancies, able to swallow oral capsules, ECOG performance status ≤ 2, measurable disease as defined by RECIST 1.0, and adequate organ function. Results: 22 pts were enrolled in 6 dose levels, 2 at daily and 4 with twice daily dosing ranging from 1 to 5 gm orally per day; all evaluable for toxicity and 20 for response. Most common cancers included colorectal (13 pts), non small cell lung (3 pts), and ovarian (2 pts). 5 pts patients completed Cycle 1, 9 pts Cycle 2, 3 pts Cycle 3 and 1 pt each completed Cycles 4, 5, and 8. 2 pt had Conclusion: OMN54 was well tolerated with no DLTs observed. Further studies at RP2D of 2.5 g bid orally should be done to assess activity. Citation Format: Daniel Renouf, Christian Kollmannsberger, Kim Chi, Stephen Chia, Anna Tinker, Teresa Mitchell, Stephen Lam, Teresa Joshi, David Kwok, John Ostrem, Simon Sutcliffe, Karen A. Gelmon. A phase 1 study of OMN54 in patients with advanced malignancies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C41.

Lung vasculature imaging using speckle variance optical coherence tomography

Architectural changes in and remodeling of the bronchial and pulmonary vasculature are important pathways in diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. However, there is a lack of methods that can find and examine small bronchial vasculature in vivo. Structural lung airway imaging using optical coherence tomography (OCT) has previously been shown to be of great utility in examining bronchial lesions during lung cancer screening under the guidance of autofluorescence bronchoscopy. Using a fiber optic endoscopic OCT probe, we acquire OCT images from in vivo human subjects. The side-looking, circumferentially-scanning probe is inserted down the instrument channel of a standard bronchoscope and manually guided to the imaging location. Multiple images are collected with the probe spinning proximally at 100Hz. Due to friction, the distal end of the probe does not spin perfectly synchronous with the proximal end, resulting in non-uniform rotational distortion (NURD) of the images. First, we apply a correction algorithm to remove NURD. We then use a speckle variance algorithm to identify vasculature. The initial data show a vascaulture density in small human airways similar to what would be expected.