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G. Wright



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    MO17 - Radiotherapy I: Stereotactic Ablative Body Radiotherapy (ID 106)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Radiation Oncology + Radiotherapy
    • Presentations: 1
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      MO17.01 - Response assessment of Stereotactic Ablative Body Radiotherapy (SABR) for pulmonary metastases: utility of 4D-FDG-PET and CT perfusion (ID 2225)

      16:15 - 17:45  |  Author(s): G. Wright

      • Abstract
      • Presentation
      • Slides

      Background
      Response assessment using conventional RECIST criteria after SABR of lung targets can be confounded by fibrotic response. The purpose of this study was to evaluate the utility of 4D-FDG-PET/CT and CT perfusion scans in the response assessment of single fraction SABR for inoperable pulmonary oligometastases.

      Methods
      This is a prospective ethics approved clinical study of patients undergoing single fraction SABR with 26Gy for pulmonary metastases. Eligible patients had 1-2 metastases with no extrathoracic disease on staging FDG-PET. Serial 3D / 4D-FDG-PET and CT perfusion studies were performed at baseline, 14 days and 70 days after therapy. Two radiologists independently reported CT perfusion scans.

      Results
      At a median follow-up of 16 months (range 3-27), 10 patients with 13 metastases received SABR. A further 7 patients (41%) were screened from the study due to interval progression of disease between the time of the original FDG-PET and trial 4D-FDG-PET / perfusion CT. The mean time between the original FDG-PET and trial scans was 62 days. No patient progressed locally, 7/10 patients progressed distantly of which 2/7 received subsequent SABR. At the end of study period, 5/10 patients are alive without disease. The median progression free survival was 14 months. The change in SUVmax from baseline was higher on 3D than 4D-PET by a mean of 20.6% (range 0.2%-47.2%) at 14 days and 14.8% (range 0-37.8%) at 70 days. Overall, the SUVmax increased at 14 days (mean 104.9%, p<0.01) and decreased at 70 days (mean=55.5%, p<0.01), despite persistent morphological lesions on the concurrent late timepoint CT. There was strong level of inter-observer agreement of CT perfusion interpretation with a median intraclass correlation coefficient of 89% (range 57%-98%). Perfusion parameters of Time to Peak Blood Flow and Blood Volume showed a median increase of 18.8% and 23.0% at 2 weeks post-therapy and decreased below baseline by a median 7.0% and 14.0% at 70 days (non-significant).

      Conclusion
      High rates of interval progression between staging scans indicates a need to expedite management of oligometastases in a timely fashion. Increased tumour perfusion and FDG-PET intensity at 2 weeks post-RT is likely due to an inflammatory response to large single dose SABR. Late PET response was associated with tumour control despite CT apparent morphological lesions. Conventional 3D PET may overestimate change in PET intensity post SABR as compared to 4D PET. These findings, in particular CT perfusion findings, require a larger patient cohort for validation.

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    O04 - Molecular Pathology I (ID 126)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      O04.01 - Identification of CD74-NRG1, a new recurrent fusion gene in invasive mucinous lung adenocarcinomas of never smokers (ID 4022)

      10:30 - 12:00  |  Author(s): G. Wright

      • Abstract
      • Presentation
      • Slides

      Background
      Lung adenocarcinoma (AD) of patients who have never smoked frequently bear targetable genome kinase alterations, such as EGFR mutations and translocations affecting ALK, ROS1, and RET genes. These mutations correlate with kinase inhibitor sensitivity in mouse models or in patients. Unfortunately, therapeutically relevant kinase alterations are not present in all lung cancer specimens. Thus, additional genome alterations need to be discovered in order to provide a therapeutic opportunity for the remaining patients.

      Methods
      We collected a cohort of 25 AD specimens of never smokers lacking mutations in KRAS or EGFR, in which we performed transcriptome sequencing with the aim of identifying new oncogenic driver genes.

      Results
      We were able to identify known kinase fusions affecting ALK, ROS1 and RET genes in 3 cases each. Moreover, we detected one sample carrying a novel chimeric transcript fusing the first six exons of CD74 to the EGF-like domain of the NRG1 III-β3 isoform, leading to the expression of its EGF-like domain in an otherwise NRG1-negative tumor tissue. The fusion gene was further detected in four additional cases out of 94 pan-negative* ADs of never smokers. In total, all 5 cases were identified in stage I invasive mucinous lung adenocarcinomas (IMA) of never smoker females. This tumor type frequently presents with multifocal unresectable disease, for which no effective treatment has been yet established. IMA is highly associated with KRAS mutations; indeed, out of 15 IMA analysed, 6 carried a KRAS mutation (40%), and 4 the CD74-NRG1 fusion (27%). Given the fact that NRG1 signals through ERBB3 and ERBB4 receptors, we aimed to determine which receptor CD74-NRG1 provides the ligand for. We observed that ERBB4 was not expressed in the index case, while ERBB3 was relatively highly expressed and this expression also correlated with a positive phospho-ERBB3 (p-ERBB3) signal in the tumoral tissue of all 5 CD74-NRG1 positive cases. In order to test if this phosphorylation of ERBB3 was statistically significant, we stained a cohort of 241 ADs and found that p-ERBB3 was only positive in 6 of them (p-value<0.0001). Additionally, although both EGFR and ERBB2 were expressed in the index case, only ERBB2 expression correlated with a p-ERBB2 positive signal. These data suggest that CD74-NRG1 might provide the ligand for ERBB3, which may form heterodimers with ERBB2, since ERBB3 is devoid of intrinsic kinase activity and cannot support linear signaling in isolation. This is in line with previous studies showing that NRG1 induces an oncogenic signal through ERBB2-ERBB3 heterodimers engaging the PI3K-AKT pathway. This was further supported by the activation of the PI3K-AKT, but not the MAPK pathway, in CD74-NRG1 transduced H2052 lung cells, after 24h starvation. *pan-negative: EGFR, KRAS, ALK, HER2, BRAF, ROS1 and RET wild-type

      Conclusion
      Altogether, these data shows that CD74-NRG1 is a new recurrent oncogenic fusion gene, highly associated with IMA of never smokers. It also suggests that CD74-NRG1 fusion protein signals through the ERBB2-ERBB3 receptors complex leading to the activation of the PI3K-AKT pathway, providing a therapeutic opportunity for a tumor type with, so far, no effective treatment.

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    O17 - Anatomical Pathology I (ID 128)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      O17.07 - Prevalence, morphology and natural history of FGFR1-amplified lung cancer detected by FISH and SISH (ID 2776)

      10:30 - 12:00  |  Author(s): G. Wright

      • Abstract
      • Presentation
      • Slides

      Background
      Fibroblast growth factor receptor 1 (FGFR1), which codes for a receptor tyrosine kinase, was recently reported to be amplified in 20% of lung squamous cell carcinoma (SqCC). In vitro and preclinical tests suggest that FGFR1 amplification is a therapeutic target. Our aims were to investigate the prevalence of FGFR1 amplification by fluorescence in situ hybridization (FISH) and determine correlation with outcome in an Australian cohort of resected lung cancer. We also correlated results of FGFR1 FISH with silver in situ hybridization (SISH).

      Methods
      A clinically-annotated tissue microarray was constructed from resected lung cancer tissue collected from 1996-2012. FGFR1 FISH and SISH were performed according to manufacturer’s protocols, with SISH performed on Ventana benchmark XT platform. FGFR1 FISH and SISH were scored by one pathologist, with high level amplification defined as ratio of FGFR1/centromere 8 ≥ 2, or tumor cell percentage with ≥ 15 signals ≥ 10%, or average number of FGFR1 signals/tumor cell nucleus ≥ 6, and low level amplification as tumor cell percentage with ≥ 5 signals ≥ 50%. Results of FGFR1 FISH and SISH were compared. Patient outcome related to FGFR1-amplified tumors was assessed and compared to patients with SqCC, or with a morphologic component of, or immunoprofile of SqCC, but normal FGFR1 copy number.

      Results
      Of 406 tumors tested, there were 191 pure SqCC, 28 carcinomas with a SqCC component, 24 large cell carcinomas with an immunoprofile of SqCC, 115 adenocarcinomas, 22 pulmonary neuroendocrine tumors, and 28 other carcinomas without a morphologic component or immunoprofile of SqCC. FGFR1 amplification was assessable in 368 tumors. FGFR1 amplification was identified with FISH in 50 tumors, 48 (48/225; 21.3%) of which were either pure SqCC or a carcinoma with morphologic component or immunoprofile of SqCC. Only two cases were completely of non-squamous origin (2/143; 1.4%, p<0.00001). FGFR1 SISH was performed in 385 tumors, with 347 tumors assessable. Of 46 FGFR1 FISH-amplified tumors assessed with FGFR1 SISH, all showed FGFR1 amplification with SISH, whilst all other tumors tested were negative. Survival from radically treated FGFR1-amplified tumors was similar to all others with a squamous component (73% versus 60% 5-yr survival, HR 0.68, p=0.25; Figure 1).Figure 1

      Conclusion
      FGFR1 amplification with FISH was identified in 21.3% of pure SqCC or carcinomas with a morphologic component or immunoprofile of SqCC, but only 1.4% of completely non-squamous tumors. All adenocarcinomas and neuroendocrine tumors were negative. FGFR1 SISH showed 1:1 correlation to FGFR1 FISH.

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    P1.18 - Poster Session 1 - Pathology (ID 175)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P1.18-013 - Extracting High quality RNA from FFPE samples for gene expression studies (ID 2092)

      09:30 - 16:30  |  Author(s): G. Wright

      • Abstract

      Background
      The use of targeted therapies in the treatment of non-small cell lung cancer is still limited to a relatively small fraction of patients. Chemotherapy remains the mainstay of treatment for most patients today. So far, the best predictors for chemotherapeutic success are based on the expression of certain nucleotide metabolism genes or DNA damage response and repair related genes. However, the samples available for study most commonly comprise FFPE samples, which are characterised by a high degree of RNA fragmentation or degradation.

      Methods
      To address this problem, we have developed a protocol to reliably extract reasonable-quality RNA from FFPE samples. The protocol includes pathology review of the FFPE block, removal of a 2mm core, followed by RNA extraction. Next, the total RNA amount is quantified and a small proportion is accessed for fragmentation e.g. by TapeStation technology and/or a multiplex RT-PCR to determine the amount and size of amplifiable templates. We then assessed the extracted total RNA by various RNA based methodologies.

      Results
      To this end, we prepared core punches from 118 different lung adenocarcinomas and successfully extracted sufficient amounts of total RNA (> 50ng /ul in a 20ul elution) from 111 of the cores (average is 307ng/ul ranging from 53ng to 1.1ug/ul). Fragmentation assessment of 26 of these RNAs showed that all samples contained sufficient amounts of fragments with at least >100 nt. We first tested single gene expression by RT-qPCR. Of 26 samples tested, 24 samples showed robust amplification of a 161 bp fragment of the TBP housekeeping mRNA. We next assessed our RNA using gene expression analysis by NanoString®. We interrogated 150ng total RNA from 10 samples for the expression levels of 45 genes. Data analysis showed robust expression values and no quality control problems in all samples. Finally, we tested whether the RNA was of sufficient quality for next-generation RNA sequencing. We used 100 and 50 bp paired end sequencing on un-size-selected RNA, and 100 bp paired end sequencing after one round of size selection. On average, we obtained 23 million reads per sample, of which 70% mapped to reference sequences after either extensive read clipping or size selection.

      Conclusion
      In conclusion, our extraction protocol enables us to reliably extract total RNA from FFPE samples, which can be used for single-gene expression by RT-qPCR and gene expression of limited gene sets by NanoString® technology. However, the amount of samples and genes tested here were not sufficient to allow identification significant differences between samples, but shows the possibility to use the RNA extracted following our extraction protocol. RNAseq, however, poses a larger problem. The amount of mapped reads is significantly lower compared to high quality RNA from e.g. fresh frozen material or cell lines. The reason for these problems and possible solutions remain elusive. Overall, we present a simple and fast way to accurately extract RNA from FFPE material and show that after QC, single or small gene panels can successfully be assessed. However, large-scale sequencing efforts remain problematic and further optimization is needed.

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    P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.06-002 - RLIP76 expression is prognostic and predictive of chemotherapy benefit in resected NSCLC (ID 247)

      09:30 - 16:30  |  Author(s): G. Wright

      • Abstract

      Background
      Despite adjuvant chemotherapy improving overall survival of resected Stage II and III non-small cell lung cancer (NSCLC), acute and late toxicities of chemotherapy have highlighted the need to better predict which patients will benefit from treatment. RLIP76 is a ubiquitously expressed multi-functional transporter that is associated with cisplatin and vinorelbine resistance. Our aim was to analyse the prognostic and predictive value of RLIP76 expression in NSCLC.

      Methods
      We identified 367 NSCLC patients resected between 1996 and 2009. A tissue microarray was created and immunohistochemistry (IHC) performed with an anti-human RLIP76 rabbit polycloncal antibody (Millipore, Temecula, CA). The intensity (0-3) and proportion of tumour cells (0-100) with staining was scored. The product of RLIP76 intensity and proportion of tumour cells staining was calculated (range 0-300) and divided into high (>100) and no/low expression (≤100). RLIP76 expression was correlated with clinical features and patient outcome.

      Results
      IHC was available for 285 patients, 173(60.7%) of which were male. Number of patients according to stage I, II, III and IV was 150(52.6%), 83(29%), 44(15.4%) and 8(3%), respectively. RLIP76 was overexpressed in 117(41%) specimens. There was no relationship between RLIP76 expression and stage, histology, sex or age. High RLIP76 expression was associated with an improved prognosis on univariate (HR 0.62, CI 9.44-0.90, p=0.012,Figure 1) and multivariate analysis (HR 0.57, CI 0.39-0.83, p=0.003). Adjuvant chemotherapy was also associated with an improved survival on multivariate analysis (HR 0.52, CI 0.33-0.82, p=0.005). When stratifying by RLIP76 expression, the benefit of chemotherapy was limited to patients with no/low expression (HR =0.44, CI 0.24-0.8, p=0.008)(Figure 2). No benefit of chemotherapy was seen in patients with high RLIP76 expression (HR=0.75, CI 0.34-1.63, p=0.5). Figure 1 Figure 2

      Conclusion
      High RLIP76 expression is associated with an improved prognosis in resected NSCLC.Interestingly no/low RLIP76 expression may predict for benefit of adjuvant chemotherapy. Further studies are needed to confirm these results.