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W.A. Cooper
Moderator of
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O04 - Molecular Pathology I (ID 126)
- Event: WCLC 2013
- Type: Oral Abstract Session
- Track: Pathology
- Presentations: 8
- Moderators:I.I. Wistuba, W.A. Cooper
- Coordinates: 10/28/2013, 10:30 - 12:00, Parkside Ballroom A, Level 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 - 10:40 | Author(s): L. Fernandez-Cuesta, D. Plenker, H. Osada, R. Sun, R. Menon, F. Leenders, M. Peifer, W. Vogel, I. Dahmen, G. Wright, P. Russell, Z. Wainer, B. Solomon, E. Brambilla, H.N. Mignotte, D. Moro-Sibilot, C.G. Brambilla, S. Lantuejoul, J. Altmüller, C. Becker, P. Nürnberg, J.M. Heuckmann, E. Stoelben, J.H. Clement, J. Sänger, M. Vingron, T. Zander, J. Wolf, S. Perner, S. Ansen, S.A. Haas, Y. Yatabe, R. Thomas
- Abstract
- Presentation
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-typeConclusion
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.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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O04.02 - Using NGS for Mutational Profiling of NSCLC in the Clinical Setting (ID 2571)
10:40 - 10:50 | Author(s): S. Roy Chowdhuri, M. Routbort, K. Patel, R. Singh, R. Broaddus, A. Lazar, K. Aldape, R. Luthra, I.I. Wistuba, G.R. Simon
- Abstract
- Presentation
Background
Recent advances in molecular characterization of lung cancer have led to the identification of potential therapeutic targets that play key roles in regulating cell growth and proliferation. With the introduction of new targeted therapies, it becomes increasingly important to accurately characterize mutation status in lung cancer patients to provide personalized care that define prognosis and predict response to therapy. The advent of next generation sequencing (NGS) platforms in the realm of clinical molecular diagnostics has made multi-gene mutational profiling an affordable and highly successful methodology for massively parallel sequencing using small quantities of DNA.Methods
Tumor specimens from 262 distinct samples of primary lung carcinoma including adenocarcinoma (n=228), squamous cell carcinoma (n=15), non small cell cancer not otherwise specified (NSC-NOS) (n=8), poorly differentiated carcinoma (n=4), neuroendocrine carcinoma (n=2), small cell carcinoma (n=1) and pleomorphic carcinoma (n=4) were tested by NGS. Tumor samples included formalin-fixed paraffin-embedded surgical core needle biopsies, resection specimens, cytopathology cell blocks, as well as cytopathology direct smears. Ten ng of DNA from each sample was tested for mutations in hotspot regions of 46 cancer related genes (Ion AmpliSeq Cancer Panel) using either a 316 chip or a 318 chip on an Ion Torrent Personal Genome Machine (PGM) Sequencer (Life Technologies, CA).Results
Mutations were detected in 222/240 (93%) patients with a histologic diagnosis of adenocarcinoma, NSC-NOS or PDC. EGFR mutations were detected in 47 (20%) of these patients and double EGFR mutations identified in 13 cases, including acquired resistance mutations T790M (n=6) and S768I (n=3). KRAS mutations were detected in 61 (25%) cases, most commonly involving codons 12 and 13 (n= 58) and less frequently involving codons 61 and 146 (n= 3). TP53 was most frequently mutated (n=65; 27%) and was often seen in conjunction with EGFR mutations (n=14; 5%) and KRAS mutations (n=15; 6%). Mutations were detected in 10/15 (67%) squamous cell carcinomas with mutations in TP53 (n=5), CDKN2A (n=3) and PIK3CA (n=2) most frequently seen. Additional mutations detected at a lower frequency from the entire dataset were STK11, ATM, BRAF, PIK3CA, CTNNB1, IDH1, NRAS, CDKN2A, KDR, RET, MET, FBXW7, APC, RB1, FLT3, GNAS, ABL1, HRAS, PTPN11, JAK3, NOTCH1, SMAD4, SMARCB1, SMO, MLH1, AKT1, and ERBB4.Conclusion
In summary, our results show that NGS-based mutational profiling using small amounts of DNA derived from FFPE as well as cytology smears can provide important information regarding mutation status of genes that play key roles in growth and progression of tumor in lung cancer patients and can provide insight into directing personalized cancer therapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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O04.03 - Oncogene Mutations and Novel Transcript Fusions in Lung Adenocarcinoma from Never Smokers (ID 2939)
10:50 - 11:00 | Author(s): J. Jen, J.S. Jang, A. Lee, J. Li, Y. Asmann, M. Erickson-Johnson, A.O. Bungum, V. Simon, E.S. Edell, K.J. Kopp, B. Eckloff, A. Oliveira, E. Wieben, M.C. Aubry, E. Yi, D.A. Wigle, P. Yang, R.B. Diasio
- Abstract
- Presentation
Background
Lung adenocarcinoma from never smoker represents a unique disease entity in that they often involve females of younger age and have a distinct mutation spectrum compared to those of smoker population. Mutations from the tumors of these patients often involve oncogenes that can be targeted for therapy by small molecule kinase inhibitors. We surveyed for tumor specific genetic changes in lung adenocarcinomas from never smokers for common oncogene mutations and transcript fusions.Methods
We first developed a multiplex assay detecting187 mutations in 10 actionable oncogenes frequently affected in lung cancer. We used this assay to examine 89 lung adenocarcinomas from never smokers identified through the Mayo Clinic Epidemiology and Genetics of Lung Cancer Program. NextGen sequencing (RNASeq) was used to identify transcript fusions affecting either a known kinase or an oncogene in 20 of 89 tumors. RT-PCR, FISH and IHC were used to verify the novel fusion identified in this study.Results
Sixty-four tumors had mutation in at least one of the tested oncogenes involving EGFR (49 cases, 55%), k-RAS (5 cases, 6%), MET (9 cases, 10%), BRAF (4 cases, 5%), PIK3CA (2 cases, 2%), and ERBB2 (4 cases, 5%). RNAseq identified five transcript fusions among the 20 tested tumors, involving known fusions of EZR- ROS1 or KIF5B-RET and three novel fusions involving SND1-BRAF, EML4-BIRC6, and GMEB2-TERT genes. We used RT-PCR to confirm the presence of the SND1-BRAF fusion transcript that involved exons 1-9 of SND1 with exon 2 to 3’ end of the BRAF on chromosome 7. Screening all 89 tumors by RT-PCR identified a total of three tumors with the identical fusion. Interestingly, two of these three tumors with a BRAF fusion also had a concurrent mutation in EGFR gene (S768I) and a third tumor had an additional mutation in the ERBB2 gene (M774_A775ins). Four additional samples were positive for EML4-ALK fusion by IHC and FISH.Conclusion
In our study of a primarily Caucasian population, a majority of lung adenocarcinomas from never smokers (70/89, or 78.6%) carry at least one genetic mutation in a targetable gene. For the first time, we report the presence of a transcript fusion involving SND1-BRAF in lung adenocarcinoma and that these fusions are present in tumors also having EGFR or ERBB2 mutations. Combined together, activation of BRAF by either point mutation or transcript fusion is one of the most frequent events in our study accounting for 7/89 (8%) cases. These findings support a rapid and targeted gene mutation testing strategy for lung adenocarcinoma from never smokers, as the knowledge of these mutations can be readily used to augment therapeutic management.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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O04.04 - DISCUSSANT (ID 3982)
11:00 - 11:15 | Author(s): I.I. Wistuba
- Abstract
- Presentation
Abstract not provided
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O04.05 - Epidemiology of PI3K pathway alterations in patients with metastatic non-small cell lung cancer (NSCLC): findings from the international BASALT-1 study (ID 1810)
11:15 - 11:25 | Author(s): G.K. Dy, J. Vansteenkiste, M. Thomas, T. De Pas, F. Grossi, J. De Grève, E. Felip, J. Canon, J. Gray, F. De Braud, M. Reck, S. Thongprasert, D. Planchard, C. Gridelli, P. Roussou, M. Gurnani, G. Atalla, D.W. Sternberg, J.A. Engelman
- Abstract
- Presentation
Background
Buparlisib (BKM120) is an oral PI3K inhibitor that inhibits all four isoforms of class I PI3K (α, β, γ, δ) and has demonstrated antiproliferative, proapoptotic, and antiangiogenic activity in multiple preclinical cancer models. NSCLC cell lines with PIK3CA mutations (muts) have demonstrated increased sensitivity to buparlisib in vitro. BASALT-1 – an ongoing, multicenter, open-label, two-stage Phase ll study (NCT01297491) – evaluates the safety and efficacy of single-agent buparlisib in patients (pts) with NSCLC and an activated PI3K pathway. Here we report data on the prevalence of PI3K pathway alterations in pts with squamous (sq) or non-squamous (non-sq) NSCLC prescreened for entry into BASALT-1.Methods
Pts prescreened for BASALT-1 were ≥18 years of age with previously treated metastatic NSCLC of sq or non-sq histology. PI3K pathway activation (defined as PIK3CA mut and/or PTEN mut and/or PTEN negative [neg; <10% protein expression at 1+ by immunohistochemistry]) was measured in archival or newly acquired tumor tissue collected at prescreening. PIK3CA (exons 1, 5, 7, 9, and 20) and PTEN (exons 1–9) muts were detected primarily using Sanger sequencing in a centralized fashion. Local analysis was permitted at selected sites where a SnapShot approach was most commonly used.Results
As of April 10, 2013, 1183 pts had submitted tumor samples to be assayed (1179 tumors had known histology). PI3K pathway activation was detected in 16.0% of sq and 11.3% of non-sq tumors. In sq tumors (N=612), loss of PTEN protein expression (8.2%) was the most common single alteration observed, followed by PIK3CA mut only (3.1%) and PTEN mut only (2.9%). In non-sq tumors (N=567), PTEN mut only was the most common alteration (4.9%), followed by PIK3CA mut only (2.6%) and PTEN neg only (2.1%). Frequencies of co-existing genetic alterations were: PTEN mut + PTEN neg only (1.0% sq vs 0.4% non-sq), PIK3CA mut + PTEN neg only (0.7% sq vs 0.4% non-sq), PIK3CA mut + PTEN mut only (0% sq vs 0.9% non-sq), and PIK3CA mut + PTEN mut + PTEN neg (0.2% sq vs 0% non-sq). No clear gender, age or ethnicity effects were observed (Table). Figure 1Conclusion
The findings from our large dataset indicate that genetic alterations in the PI3K pathway occur in a clinically significant proportion of pts with sq and non-sq relapsed NSCLC. An accurate characterization of PI3K pathway alteration frequencies in NSCLC will help guide the design of future clinical trials of PI3K inhibitors.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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O04.06 - An international standardization study using the ALK IHC antibody D5F3 and a sensitive detection kit demonstrates high concordance between ALK IHC and ALK FISH and between evaluators (ID 2875)
11:25 - 11:35 | Author(s): M.W. Wynes, M. Dietel, E. Schuuring, L.M. Sholl, M. Tsao, Y. Yatabe, R.R. Tubbs, F. Hirsch
- Abstract
- Presentation
Background
The goal of personalized medicine is treating patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or detrimental therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK positive non-small cell lung cancer (NSCLC) tumors, but to date the only US Food and Drug Administration approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate and standardize the interpretation of a new automated standardized ALK IHC assay.Methods
Archival NSCLC tumor specimens (n=103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody (Ventana Medical Systems, Inc) combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc). The evaluators went through an interpretation training session and scored the specimens as positive, if strong granular cytoplasmic brown staining was present in tumor cells, or negative. IHC results were compared to the FISH results and inter-evaluator agreement comparisons made.Results
Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%) and accurate (93%) relative to the ALK FISH results. Similar results were observed using a majority score. For the discrepant cases IHC negativity was scored by 7/7 on 3 FISH positive cases and 6/7 evaluators on 2 additional FISH positive cases. IHC positivity was scored on 2 FISH negative cases by 7/7 readers. There was agreement among 7/7 and 6/7 readers on 88% and 96%% of the cases before a consensus review, respectively, and following a review there was agreement among 7/7 and 6/7 on 95% and 97% of the cases, respectively.Conclusion
Based on expert evaluation the ALK IHC assay using the D5F3 antibody combined with Optiview Detction and Optiview amplification is sensitive, specific and accurate, relative to FISH, and a majority score of multiple readers does not improve these results over an individual reader’s score. Excellent inter-reader agreement was observed for the IHC assay. These data support the algorithmic use of ALK IHC as a screening procedure for ALK protein expression in NSCLC.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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- Abstract
- Presentation
Background
Owing to use of high-resolution computed tomography (CT) scan for lung cancers, small nodules could be detected, so that more patients are identified carrying more than one lesion in lung synchronously at the time of diagnosis. Under this circumstance, precisely distinguish multiple primary lung cancers from intrapulmonary metastasis has important significance on clinical staging and appropriate therapy design.Methods
We comprehensively compared genomic aberration profiles of each tumor in the patients with multifocal pulmonary lesions, assuming that metastasis shared a certain portion of genetic aberrations with the lesion it was originated. Therefore, whole-genome/exome sequencing were applied on 15 intrapulmonary tumors that had highly similar histological diagnosis, and 1 lymph node metastasis derived from six patients with synchronous multifocal lung cancers. The somatic nucleotide variations (SNVs) detected by whole-genome/exome sequencing were validated by either mass spectrometry or the Sanger sequencing.Results
A total of 344 non-synonymous somatic point mutations were detected in whole genome sequencing analysis (3 lesions and 1 lymph node metastasis in 1 patient), corresponding to 306 unique mutation sites. Among the 70 mutations detected in the lymph node metastasis, 36 (51.4%) were also found in lesion 1 of the 3 intrapulmonary lesions, whereas no shared mutation were detected between the metastasis and either of the other two lesions. Meanwhile, there was only 1 common mutation between lesion 2 and 3, while no shared mutations were observed between lesion 1 and 2 or lesion 1 and 3. These results suggested that the metastasis was originated from lesion 1 and the 3 lesions were independent primary tumors. In whole exome sequencing analysis (12 lesions from 5 patient), among the 389 somatic non-synonymous mutations detected, we observed a similarity between each pair of tumors within one patient ranged from 0% to 5.3%, suggesting that all the lesions were independent primary tumors rather than intrapulmonary metastasis. We also reached a same conclusion when we included all somatic mutations found across the genome/exome in the analysis. These genomic abnormality profile-based diagnosis were consistent with the diagnosis based on histological examination except one lesion in patient 5, which had been considered as an intrapulmonary metastasis by histological judgment. At the same time, EGFR or KRAS mutations, which are therapy targets for adenocarcinoma in lung were detected in 7 or 3 out of the 15 tumors in 3 or 2 patients, respectively. Heterogeneity was also observed in mutation status of these two genes among different lesions in a single patient.Conclusion
Comprehensive genomic aberration profiling is powerful for identification of multiple primary lung cancers. The lymph node metastasis in the study stands as a positive control, compared with which we could tell that lesions from multifocal primary lung cancers shared too few somatic mutations to be intrapulmonary metastasis. Considering the heterogeneous mutation status of EGFR or KRAS among different tumors derived from a single patient with multifocal primary lung cancer, molecular diagnosis should be taken for each accessible lesion when targeted adjuvant therapy is under consideration.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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O04.08 - DISCUSSANT (ID 3983)
11:45 - 12:00 | Author(s): S. Dacic
- Abstract
- Presentation
Abstract not provided
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Author of
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O17 - Anatomical Pathology I (ID 128)
- Event: WCLC 2013
- Type: Oral Abstract Session
- Track: Pathology
- Presentations: 1
- Moderators:K. Jones, K.F. To
- Coordinates: 10/29/2013, 10:30 - 12:00, Bayside 105, Level 1
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O17.08 - DISCUSSANT (ID 3988)
11:45 - 12:00 | Author(s): W.A. Cooper
- Abstract
- Presentation
Abstract not provided
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P2.18 - Poster Session 2 - Pathology (ID 176)
- Event: WCLC 2013
- Type: Poster Session
- Track: Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P2.18-009 - Mutation Testing in Non-Small Cell Lung Cancer - Suitability of Small Biopsy and Cytology Specimens (ID 1750)
09:30 - 09:30 | Author(s): W.A. Cooper
- Abstract
Background
Patients with non-small cell lung cancer (NSCLC) harboring sensitizing mutations in epidermal growth factor receptor (EGFR) benefit from treatment with EGFR tyrosine kinase inhibitors. As most patients with NSCLC present with advanced-stage disease and are not candidates for surgical resection, somatic mutation testing is often performed on small biopsy and cytology specimens. Compared to resection specimens, the suitability of these specimens is not well established. We aimed to explore the suitability of small biopsy and cytology specimens for mutation testing in NSCLC.Methods
We undertook a retrospective review of NSCLC mutation testing cases performed at Royal Prince Alfred Hospital, Sydney, from March 2012 to May 2013. Mutation testing was requested by the treating physician. DNA was extracted from formalin-fixed, paraffin embedded tissue and a multiplex PCR assay (OncoCarta Panel v1.0) used to identify mutations in 19 oncogenes including EGFR, KRAS, and BRAF. The results were analyzed on the Sequenom MassArray platform. Fragment analysis was also undertaken to assess for exon 19 deletions.Results
Mutation testing was undertaken on 151 NSCLC specimens, including 44 (29.1%) resection specimens (27 lung resection specimens and 17 metastatic site resections), 67 (44.4%) small biopsy specimens, and 40 (26.5%) cytology specimens. Overall, EGFR mutations were detected in 32/151 (21.2%) cases, KRAS mutations in 29/151 (19.2%) cases, and BRAF mutations in 3/151 (2%) cases. Mutations were detected in 25/44 (56.8%) resection specimens, principally lung resection specimens (19/27, 70.4%), 26/67 (38.8%) small biopsies and 13/40 (32.5%) cytology specimens. The mutation rate was significantly lower in small biopsies (p=0.006) and cytology specimens (p=0.002), compared to lung resection specimens. Specifically, EGFR mutations were identified in 13/44 (29.5%) resection specimens, again mainly in lung resection specimens (10/27, 37%), 9/67 (13.4%) small biopsies and 10/40 (25%) cytology specimens. Compared to lung resection specimens, the proportion of EGFR mutation positive cases was significantly lower in small biopsy (p=0.01), but not in cytology specimens (p=0.29). One paired cytology and lung resection specimen from a single patient was available and both specimens confirmed the presence of an L858R EGFR mutation.Conclusion
Mutations, including EGFR mutations, were most frequently detected in lung resection specimens. Compared to lung resection specimens, the EGFR mutation rate was significantly lower in small biopsy, but not in cytology specimens. This suggests that cytology specimens are more likely to be adequate for mutation testing than small biopsies such as core and bronchial biopsies. However, we cannot exclude bias in this study from differing referral patterns which may affect these results. Careful assessment of DNA quality and quantity is important for all specimens, particularly small biopsy specimens, to reduce the risk of false positive or negative results.
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P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 2
- Moderators:
- Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P3.06-017 - Distinctive Insulin-Like Growth Factor 1 gene copy number and protein expression in non-small cell lung cancer (ID 1753)
09:30 - 09:30 | Author(s): W.A. Cooper
- Abstract
Background
The insulin-like growth factor (IGF) pathway is involved in the development and progression of many tumours and there is growing preclinical evidence that blockade of this pathway has anti-tumour effects in NSCLC. IGF receptors (IGFR) are another potential target for targeted treatment of NSCLC and a number of agents are already undergoing clinical trial. Biomarkers are needed to select patients most likely to derive clinical benefit from these agents. The downstream pathway components of IGF1R and MET activation include PI3K and AKT, which are other potential biomarkers currently being investigated in this patient cohort. IGF1R has also been implicated in acquired resistance to EGFR-TKI treatments. Only a few small retrospective studies have investigated the prognostic role of IGF1R in NSCLC and the relationship with EGFR mutations is not known.Methods
IGF1R status was evaluated by chromogenic silver in situ hybridization (ISH) and immunohistochemistry (IHC) in tissue microarray sections from a retrospective cohort of 264 surgically resected NSCLCs and results were compared to clinicopathological features and patient survival. Patients were classified as IGF1R gene amplification (either presence of tight gene clusters, IGF1R to CEN15 ratio ≥ 2, or ≥ 15 copies of IGF1R per cell in ≥ 10% of analysed cells); high polysomy (≥ 4 copies of IGF1R in ≥ 40% of tumour cells); low copy number (< 4 copies of IGF1R in < 40% of cells). Patients were also grouped as IGF1R-positve (amplification or high polysomy) or IGF1R-negative (low copy number).Results
High IGF1R gene copy number was identified in 77 cases (29.2%) in which there were 32 amplified IGF1R cases (12.1%) and 45 high-polysomy IGF1R cases (17%). Increased copy number of IGF1R was more common in squamous cell carcinomas (SCC) compared to large cell carcinomas (LCC) or adenocarcinomas (ADC) (p<0.05). There was no correlation between IGF1R gene copy number status and other clinicopathological features including patient age, gender, smoking status, tumour size, vessel, perineural or lymphatic invasion, grade or stage. IGF1R copy number alteration in primary tumours was highly correlated with IGF1R copy number status in metastatic tumours (p<0.01). High IGF1R protein expression was observed in 61/259 (23.6%) primary tumours and 14/215 (6.5%) normal adjacent bronchial mucosae. High expression of IGF1R protein was significantly associated with SCC in comparison with non-SCC primary tumours, as well as with lymphatic and vessel invasion. There was a moderate correlation between IGF1R copy number status (positive versus negative) and IGF1R protein expression (high versus low) (Cramer’s V=0.3, p-value <0.001). Both IGF1R copy number status and protein expression were not associated with patient overall survival in univariate analyses (p>0.05).Conclusion
High IGF1R gene copy number and its protein expression are frequent in NSCLC, particularly in SCC. However, alterations of IGF1R are not associated with patient prognosis. IGF1R gene copy number can readily be assessed in formalin fixed paraffin embedded tissue and warrants further investigation as a potential biomarker of targeted therapy in NSCLC. -
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P3.06-032 - Evaluation of Anaplastic lymphoma kinase (ALK) rearrangements using ALK/EML4 TriCheck Fluorescence In Situ Hybridisation (FISH) in Non-Small Cell Lung Cancers (NSCLC) and its utility for equivocal cases. (ID 2487)
09:30 - 09:30 | Author(s): W.A. Cooper
- Abstract
Background
Accurate assessment of ALK gene rearrangement in NSCLCs is critical to identify patients likely to respond to crizotinib. Currently, the gold standard for identifying ALK gene rearrangements is FISH and the Abbott Molecular ALK break apart probe is commonly used. We evaluated a new ALK/EML4 TriCheck FISH Probe for the detection of ALK rearrangements and confirmation of EML4 as the inversion partner. In addition, we evaluated its use as an ancillary FISH probe for use in cases with subtle, equivocal or atypical ALK FISH patterns.Methods
ALK FISH was prospectively performed on 29 routine diagnostic cases using the ALK/EML4 TriCheck Probe (ZytoVision) and the Vysis ALK Break Apart FISH Probe (Abbott Molecular). ALK immunohistochemistry (IHC) was performed using the 5A4 clone antibody (Novocastra) and D5F3 clone antibody (Cell Signaling Technology).Results
Both probes were concordant in all cases, except for one case which showed an atypical signal pattern using the Abbott Molecular ALK probe. This case was technically negative using standard scoring criteria for the Abbott probe, despite positive ALK IHC, but was confirmed as positive using the ZytoVision TriCheck probe. Two additional cases which were equivocal (10-16% ALK rearrangement), were confirmed to be positive for ALK rearrangement using the ALK/EML4 TriCheck probe. Of the 10 ALK rearranged cases, 4 showed evidence of EML4 translocation.Conclusion
The ALK/EML4 TriCheck FISH Probe is useful for the detection of ALK gene rearrangements, including those involving EML4 as the translocation partner, especially for borderline cases or cases displaying atypical signal patterns, where an additional unique ALK FISH probe can provide further confirmation of rearrangement.
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P3.18 - Poster Session 3 - Pathology (ID 177)
- Event: WCLC 2013
- Type: Poster Session
- Track: Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P3.18-003 - ROS1 Gene Rearrangements in Non-Small Cell Lung Carcinoma - A New Genetic Target that can be Identified by Immunohistochemistry and FISH (ID 1482)
09:30 - 09:30 | Author(s): W.A. Cooper
- Abstract
Background
Targeted therapies aimed at specific molecular genetic alterations are revolutionizing cancer treatment, particularly in non-small cell lung cancer (NSCLC). ROS1 is an oncogene that encodes a transmembrane tyrosine kinase receptor that has high homology with the intracellular kinase domain of ALK. Driver mutations involving translocation of the ROS1 gene have recently been identified in NSCLC and show promise as a target for tyrosine kinase inhibitors. In this study we aimed to: (1) Investigate the incidence and clinicopathological features of NSCLCs harbouring ROS1 rearrangements in an Australian population. (2) Investigate the accuracy of immunohistochemistry (IHC) compared to FISH at identifying tumours with ROS1 rearrangements.Methods
We tested for ROS1 translocations using both a FISH breakapart probe (Zytovision and Abbott Molecular) (≥15% cells with split signals or single green 3' signal considered positive for rearrangement), and immunohistochemistry (D4D6 clone, Cell Signaling Technology). Testing was undertaken on both (1) A retrospective cohort of 316 early stage lung adenocarcinomas in tissue microarrays. (2) A prospective cohort of 42 NSCLC, selected on clinical grounds for mutation testing (eg EGFR/KRAS/ALK negative samples and young age or never/light smoker).Results
In the retrospective cohort, only 1 case was positive for ROS1 gene rearrangement by FISH (0.3% incidence). ROS1 IHC identified positive staining in 7 (2.0%) cases, including the FISH+ case. ROS1 IHC had a sensitivity of 100% and specificity of 98% for identifying ROS1 gene rearrangements. In the prospective cohort of 42 cases, 4 cases with ROS1 gene rearrangement were identified by FISH and all 4 cases showed positive ROS1 immunohistochemical staining. Of the total 5 cases with ROS1 gene rearrangement, all occurred in adenocarcinomas from female patients with an age range of 33-81 years (mean 58). Four of the five patients were non-smokers and two were of Asian ethnicity. All 5 cases were negative for ALK rearrangements and in the 4 cases where EGFR status was known, they were all wild type.Conclusion
ROS1 gene rearrangements occur in a very small percentage of lung adenocarcinomas with distinctive clinicopathological features and appear to be mutually exclusive with other driver mutations in the small number of positive cases available for evaluation. Screening with IHC may be a suitable method of reducing the number of cases requiring FISH testing.
