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Stephen P Finn
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MA 04 - Advocacy: Listen to the Patients (ID 655)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Patient Advocacy
- Presentations: 1
- Moderators:Rudolf M Huber, C.L. Dégi
- Coordinates: 10/16/2017, 11:00 - 12:30, Room 313 + 314
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MA 04.10 - An Assessment of the Willingness to Provide Serial Bio-Specimens: Experience from an Irish Tertiary Cancer Centre (ID 10076)
11:00 - 12:30 | Author(s): Stephen P Finn
- Abstract
- Presentation
Background:
The rising imperative to improve our understanding of cancer heterogeneity and individualised drug response has led to a high demand for biopsy material. With improvements in technologies, there is now a move away from more traditional tissue based sampling to liquid based biopsies. ‘Liquid biopsies’ provide a non-invasive means for molecularly profiling patients with cancer, thus benefiting patients and clinicians in terms of treatment choice and shared decision-making. We assessed the willingness of patients to undergo repeated tissue and/or ‘liquid’ based sampling.
Method:
Detailed questionnaires, assessing patients’ perceptions of, and willingness to undergo serial biopsies were distributed to ambulatory patients at a tertiary cancer referral centre (St. James’s Hospital, Dublin). Multivariate analysis was performed using ordinal logistic regression analysis.
Result:
The questionnaire response rate was 97% (247/255). Respondents were primarily female (73%), aged between 51-70 yrs (51%), with breast (39%), colorectal (16%), oesophagogastric (13%), and lung cancer (12%). Of those that responded, repeat biopsy of an easily accessible lesion was acceptable to 203 (82%) patients if recommended by an oncologist. However this reduced to 102 (41%) patients, if the purpose was solely for clinical trial. Acceptability decreased to 168 (68%) and 81 (33%) patients respectively for more invasive biopsies. Additionally, 79 (32%) patients were willing to undergo additional biopsy for research purposes only, with 54 (21%) patients uncertain of its utility in research. Lower performance status (OR=0.44, p=0.04) and the belief that biopsy was unimportant for research (OR=0.74, p=0.04) negatively impacted on willingness to undergo biopsy, while a prior invasive biopsy increased acceptance (OR=1.02, p=0.02). In terms of blood sampling, 82% of patients would consent to repeated blood sampling over the course of their treatment, with >5 samples considered acceptable by 51.5% of patients. Patients with lung cancer had 3.38 greater odds (OR=3.38, p=0.047) of consenting to a repeated blood sample for purely research purposes (compared to any other type of cancer); however their willingness to undergo repeat biopsy was similar to that of other patients (OR=1.99, p=0.129). Data analysis is currently on-going.
Conclusion:
Patients with cancer are willing to participate in serial sampling of blood and urine but are less likely to consent to repeated tissue biopsies. Patients with lung cancer were particularly amenable to repeated blood sampling compared to patients with other cancer types. This is significant given the recent data supporting the use of ‘liquid’ biopsy for real-time monitoring of resistance mutations and treatment response dynamics in patients with lung cancer.
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MA 06 - Lung Cancer Biology I (ID 660)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Biology/Pathology
- Presentations: 1
- Moderators:N. Motoi, Keith M Kerr
- Coordinates: 10/16/2017, 15:45 - 17:30, Room 501
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MA 06.06 - Assessment of RANK Prevalence and Clinical Significance in the NSCLC European Thoracic Oncology Platform Lungscape Cohort (ID 10006)
15:45 - 17:30 | Author(s): Stephen P Finn
- Abstract
- Presentation
Background:
Receptor Activator of Nuclear Factor κappa-B (RANK) is a pathway involved in bone homeostasis. Recent evidence suggests that RANK signalling may also play a role in bone metastasis, and primary breast and lung cancers. The European Thoracic Oncology Platform (ETOP) Lungscape project allows evaluation of the prevalence of RANK expression and its clinical significance in a cohort of surgically-resected NSCLCs.
Method:
RANK expression was assessed on tissue microarrays (TMAs) using immunohistochemistry. Up to 4 cores per patient were analysed based on sample acceptance criteria. An H-Score (staining intensity + % cells stained) was used to assess RANK expression (positivity), as defined by at least 1 core with any degree of positive staining. Prevalence of RANK positivity and its association with clinicopathological characteristics, other cancer-related biomarkers (IHC ALK/MET/PTEN/PD-L1 expression and EGFR/KRAS/PIK3CA mutations) and patient outcome [Relapse-free Survival (RFS), Time-to-Relapse (TTR), Overall Survival (OS)] was explored in a subset of the ETOP Lungscape cohort. The prevalence of RANK overexpression (proportion of positive cancer cells ≥50%) was also investigated.
Result:
RANK expression was assessed in patients from 3 centers, a total of 402 from the 2709 patients of the Lungscape cohort, with median follow-up 44 months; 32.6% female, 40.8/54.2/5.0% adenocarcinomas (AC)/squamous cell carcinomas (SCC)/other, 44.8/28.4/26.9% with stage I/II/III, 20.6/57.7/18.9% current/former/never smokers (and 2.7% with unknown smoking status). Median was 74 months for both RFS and OS, while median TTR was not reached. Prevalence of RANK positivity was 26.6% (107 of the 402 cases), with 95% confidence interval (95%CI):22.4%-31.2%; significantly higher in AC: 48.2% (79 of 164 cases), 95%CI:40.3%-56.1%; vs SCC: 9.2% (20 of 218 cases), 95%CI:5.7%-13.8%; (p<0.001). RANK positivity was more frequent in females (38.9% vs 20.7% in males, p<0.001) and tumors≤4cm (30.7% vs 21.1% in tumors>4cm, p=0.031). Significant associations were also detected between RANK and PTEN expression in AC (RANK positivity 57.4% in PTEN expression vs 30.5% in PTEN loss; p=0.0011) and with MET status in SCC (RANK positivity 27.8% in MET+ vs 7.6% in MET-; p=0.016). No association with outcome was found. RANK overexpression was identified in 43 (10.7%; 95%CI: 7.9%-14.1%) cases.
Conclusion:
In this early-stage NSCLC cohort, RANK positivity (26.6% overall) is found to be significantly more common in adenocarcinomas (48.2%), females, patients with tumors of smaller size, with PTEN expression (in SCC) and MET positivity (in AC). No prognostic significance of RANK expression was found. Analysis of additional cases is ongoing and results will be presented.
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P1.02 - Biology/Pathology (ID 614)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.02-011 - XRCC6BP1: A Key Player in the DNA Repair of Cisplatin Resistant NSCLC Cells (ID 10225)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating resistance to chemotherapeutic agents.
Method:
DNA Repair Pathway RT[2 ]Profiler Arrays were used to elucidate key DNA repair genes implicated in chemoresistant NSCLC cells using cisplatin resistant (CisR) and corresponding parental (PT) H460 cells previously established in our laboratory. DNA repair genes significantly altered in CisR cells were validated at the mRNA and protein level, using RT-PCR and Western blot analysis, respectively. The translational relevance of differentially expressed genes was examined in a cohort of chemo-naïve matched normal and tumour lung tissues from NSCLC patients. Loss of function studies were carried out using siRNA technology. The effect of XRCC6BP1 gene knockdown on apoptosis was assessed by FACS using Annexin-V/PI staining. Cellular expression and localisation of XRCC6BP1 protein and H2AX foci in response to cisplatin were examined by immunofluorescence using the Cytell Imaging System. To investigate a role for XRCC6BP1 in lung cancer stem cells, Side Population (SP) studies were used to characterise stem-like cells in a panel of chemoresistant cell lines. XRCC6BP1 mRNA analysis was also examined in ALDH1[+] and ALDH1[- ]subpopulations. Immunohistochemistry analysis was carried out on a cohort of resected lung tumour tissues (n=20) and XRCC6BP1 expression was correlated with clinicopathological parameters.
Result:
We identified a number of critical DNA repair genes that were differentially regulated between H460 PT and CisR NSCLC cells, where XRCC6BP1 mRNA and protein expression was significantly increased (mRNA; 19.4-fold) in H460 CisR cells relative to their PT counterparts. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in lung adenocarcinoma patients. Gene silencing of XRCC6BP1 induced significant apoptosis of CisR cells and reduced the DNA repair capacity of these cells relative to scrambled (negative) controls. Immunofluorescence studies showed an increase in XRCC6BP1 protein expression and H2AX foci in CisR cells relative to their PT counterparts. While SP analysis revealed a significantly higher stem cell population in CisR cells, XRCC6BP1 mRNA expression was considerably increased in SKMES-1, H460 and H1299 ALDH1[+] CisR cells compared to ALDH1[-] cells. Data analysis of XRCC6BP1 immunohistochemistry is currently ongoing.
Conclusion:
We identified XRCC6BP1 as key DNA repair gene implicated in cisplatin resistant NSCLC. Our data highlight the potential of targeting components of the DNA repair pathway in chemoresistant lung cancer, in particular XRCC6BP1, either alone or in combination with conventional cytotoxic therapies.
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P1.03 - Chemotherapy/Targeted Therapy (ID 689)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Chemotherapy/Targeted Therapy
- Presentations: 4
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.03-039 - Therapeutic Inhibition of the Cancer Stem Cell Marker, ALDH1, a Promising Mechanism by Which Cisplatin Sensitivity Can Be Restored in NSCLC (ID 9909)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
Cisplatin remains the cornerstone of current chemotherapeutic combination startegies in the treatment of NSCLC. Despite initial cisplatin sensitivity tumours develop resistance, which in turn undermines the efficacy of cisplatin as a therapuetic agent. Numerous mechanisms, signalling pathways and theories have been suggested and elucidated in terms of cisplatin resistance and in the development of it, however, to date the clinical issue of resistance has not been overcome. A current avenue of interest is the cancer stem cell (CSC) hypothesis, in which the survival and expansion of highly resistant CSCs during chemotherapeutic treatment are thought to be a contributing factor of resistance and recurrence. Specific inhibition of key CSC markers in combination with chemotherapy may undermine the inherent resistance of the CSC population and sensitise these cells to the cytotoxic effects of therapy. One such CSC marker observed across numerous tumours is aldehyde dehydrogenase 1 (ALDH1), our hypothesis suggests that inhibition of the ALDH1-positive CSC population within cisplatin resistant NSCLC will resensitise the cellls to the cytotoxic effects of cisplatin.
Method:
Using an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines ALDH1 was identified as a CSC marker present within the CisR sublines of each NSCLC histology and characterised as CSCs. ALDH1 was inhibited using two pharmacological ALDH1 inhibitors, diethlylaminobenzaldehyde (DEAB) and disulfiram (commercially known as Antabuse used in the treatment of alcoholism). ALDH1 inhibition was confirmed by flow cytometry. PT and CisR cell lines were treated with inhibitor alone and in combination with cisplatin and assessed in terms of proliferation, clonogenic survival and apoptosis relative to cisplatin-only treatment.
Result:
Both DEAB and the FDA-approved disulfiram significantly decreased the presence of the ALDH1-positive CSC subpopulation across all CisR cell lines. DEAB and disulfiram in combination with cisplatin induced a significant decrease in proliferation and clonogenic survival as well as significant increases in cisplatin-induced apoptosis across CisR sublines when compared to cisplatin alone.
Conclusion:
DEAB and disulfiram significantly reduced the presence of the highly resistant ALDH1-positive CSC subpopulation. This pharmacological CSC depletion in conjunction with cisplatin was associated with the resensitisation of cisplatin resistant cells to the cytotoxic effects of cisplatin, thus restoring cytotoxic efficacy. The resensitisation effect of the disulfiram-based combination strategy, as well as its FDA-approval and extentsive safety profile highlights this strategy as one of great promise. In summary, these data suggest a role for ALDH1 inhibition in the resensitisation and possible circumvention of cisplatin resistance.
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P1.03-041 - Exploitation of the Cancer Stem Cell Marker ALDH1 Within the Vitamin a/Retinoic Acid Axis Promotes Re-Sensitisation of Cisplatin Resistant NSCLC (ID 9938)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
Despite significant advances in personalised medicine in recent decades, cisplatin remains the mainstay chemotherapy in the treatment of NSCLC. The major clinical challenge facing NSCLC today is the development of pan-resistance to platinum agents. Novel drug design, preclinical and clinical trials working toward the approval of new drugs is a lengthy and costly process and in the interim of the drug indentifcation and commercialisation research has turned its focus to two avenues of interest; overcoming cisplatin resistance and the repurposing of approved therapeutics with new indications. Cancer stem cells (CSCs) have been hypothesised to be the initiating cells of therapeutic resistance and tumour recurrence. An ALDH1-positive cell subset has been identified as a key CSC subpopulation present within cisplatin resistant NSCLC sublines. ALDH1 is involved in the metabolism of retinol (vitamin A) and the catalytic conversion of retinal to retinoic acid, where retinoic acid induces cell differentiation. All-trans retinoic acid (ATRA) is a well-established chemotherapeutic agent in the treatment of acute promyelocytic leukaemia; it induces the terminal differentiation of immature cells. We hypothesise that treatment of the cisplatin resistant NSCLC sublines with retinol or ATRA will deplete the ALDH1-positive population and subsequently increase or restore cisplatin sensitivity.
Method:
Flow cytometry on a panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines revealed the greater presence of ALDH1-positive CSC subpopulations within the CisR sublines of each NSCLC histology relative to PT lines. Cells were treated with retinol (substrate of the retinoic acid pathway) or ATRA (product of the retinoic acid pathway) and the presence of the ALDH1-positive CSC subset reanalysed by flow cytometry. Following treatment of the PT and CisR cells with retinol or ATRA alone and in combination with cisplatin the functional parameters of proliferation, clonogenic survival and apoptosis were reassessed relative to cisplatin alone.
Result:
Treatment of the CisR sublines with retinol (1μM) or ATRA (5μM) significantly reduced the presence of the ALDH1-positive CSC subset across CisR sublines. Both retinol and ATRA when used in combination with cisplatin significantly reduced the proliferative and survival capacity of each CisR subline while significantly increasing apoptotic cell death compared to cisplatin alone.
Conclusion:
Exploitation of the vitamin A/retinoic acid pathway in combination with cisplatin re-sensitised resistant cells to the cytotoxic effects of cisplatin. These data suggest vitamin A supplementation or the addition of FDA-approved ATRA to the cisplatin-based chemotherapeutic regimen may be of clinical benefit in overcoming tumour recurrence and cisplatin resistance.
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P1.03-042 - BBI608, a Small Molecule Stemness Inhibitor, Circumvents Cisplatin Resistance in NSCLC (ID 9947)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
The cancer stem cell (CSC) hypothesis is now a well-established and widely investigated field within oncology. It hypothesises that there is a robustly resistant stem-like population of cells that survive and thrive initial chemotherapeutic treatment. These surviving CSCs contribute to the recapitulation of a heterogeneous tumour via a combination of asymmetric and symmetric cell division, subsequently resulting in relapse and therapeutic resistance. BBI608 is a small molecule inhibitor of cancer stemness; it targets STAT3, leading to the inhibition of critical genes required for the maintenance of cancer stemness. Following initial in vitro and in vivo preclinical promise of BBI608 reported in the literature, phase II and III clinical trials are underway and are at various stages of recruitment, progress and completion to investigate BBI608 across a number of advanced malignancies and in combination with numerous chemotherapeutic agents.
Method:
Aldefluor (Stemcell Technologies) staining and flow cytometry analysis of a panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified the ALDH1-positive (ALDH1+ve) subpopulation of cells as an omnipresent CSC subset across cisplatin resistant NSCLC sublines. PT and CisR cell lines were treated with BBI608 (1μM) and the presence of the ALDH1+ve CSC population was reassessed by flow cytometry and expression of stemness factors (Nanog, Oct-4, Sox-2, Klf4 and cMyc) were examined by reverse transcriptase PCR. The functional parameters of proliferation, clonogenic survival and apoptosis were investigated with increasing concentrations of cisplatin in the presence and absence of 1μM BBI608.
Result:
The NSCLC CisR sublines showed a significantly greater ALDH1+ve CSC population relative to their PT counterparts. Treatment of the CisR sublines with 1μM BBI608 significantly depleted the ALDH1+ve CSC population and decreased gene expression of stemness markers. BBI608 significantly decreased the proliferative capacity and clonogenic survival of the CisR sublines when in combination with cisplatin relative to cisplatin alone. Cisplatin in combination with BBI608 significantly increased cisplatin-induced apoptosis in the CisR sublines indicating restoration of cisplatin sensitivity.
Conclusion:
To date, BBI608 has not been investigated in terms of a cisplatin resistant ALDH1+ve CSC population in lung cancer. BBI608, via the inhibition of STAT3, pharmacologically depleted the CSC subpopulation and stemness expression while simultaneously restoring cisplatin sensitivity. There are currently a number of clinical trials in various stages of completion to further investigate BBI608. These data suggest a promising role for BBI608 in the treatment of non-responsive or recurrent NSCLC.
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P1.03-048 - miR-34a and the Micromanagement of Cancer Stemness and Resistance in NSCLC. Does It Hold Therapeutic Benefit? (ID 9968)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
The capacity of microRNAs to post-transcriptionally regulate a myriad of genes has extended their remit into the realm of stemness and furthermore cancer stemness regulation. Disruption of Dicer-1, a crucial component of microRNA biogenesis, has been shown to completely deplete the stem cell pool in early development, indicating a potential role for microRNAs in the maintenance of stem cells. Such logic has led microRNAs to be investigated in the context of cancer stem cells (CSCs). Studies have revealed that microRNAs play a role in CSC self-renewal, differentiation, drug resistance and metastasis. With this, our hypothesis suggests that microRNAs associated with cisplatin resistance and CSC maintenance may be a key target by which the CSC root of cisplatin resistance could be overcome.
Method:
MicroRNA expression within a panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC sublines was profiled using the 7[th] generation miRCURY LNA arrays (Exiqon) and validated by qPCR. Cell lines were stained for the presence of the CSC marker, aldehyde dehydrogenase 1 (ALDH1) and FACS was used to isolate the ALDH1-positive CSC population from the ALDH1-negative bulk cell population. Expression of the panel of cisplatin resistance-associated microRNAs was investigated within the ALDH1-positive CSC population relative to their negative counterparts by qPCR. Significantly altered miRNAs were inhibited in the CisR subline using antagomirs (Exiqon) and the presence of the ALDH1-positive subset reassessed by flow cytometry and expression of stemness genes (Nanog, Oct-4, Sox-2, Klf4, cMyc) determined. The presence of the cisplatin-associated miRNAs was investigated in FFPE murine tumours within a xenograft model of CSCs, in which 1x10[3] ALDH1-positive and negative subsets were injected into NOD/SCID mice.
Result:
Upon validation, a 5-miR signature was identified across NSCLC histologies to be associated with cisplatin resistance. When this panel was further investigated within the ALDH1-positive CSC subpopulation, it was observed that there was a significant up-regulation of miR-34a-5p relative to corresponding ALDH1-negative populations. Interestingly, the ALDH1-positive subpopulations showed significantly greater miR-34a-5p expression when compared to the CisR sublines from which they were isolated. This up-regulation was also observed within the FFPE xenograft tumours. However, inhibition of miR-34a-5p with antagomiRs did not significantly alter the presence of the ALDH1-positive CSC population, or the expression of stemness-associated genes.
Conclusion:
These data suggest that miR-34a-5p while significantly up-regulated in cisplatin resistance and CSCs may not play a functional role in CSC maintenance and further investigation is required to fully elucidate the role of miR-34a-5p in cancer stemness.
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P1.09 - Mesothelioma (ID 695)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Mesothelioma
- Presentations: 2
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.09-006 - JMJ and BRD Domain Family Members in Malignant Pleural Mesothelioma: Potential Therapeutic Targets or Not? (ID 9919)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive rare cancer affecting the pleura and is predominantly associated with prior exposure to asbestos. Treatment options are limited, and most patients die within 24 months of diagnosis. There is an urgent unmet need to identify new therapeutic options for the treatment of MPM. Asbestos fibres contain transition metals such as iron, and may cause an alteration of iron homeostasis in the tissue. In addition, asbestos fibres have also been shown to have high affinity for histones, and therefore may result in high accumulation of iron around chromatin. Lysine Demethylases (KDMs) containing a JmjC domain require both Fe2+ and 2-oxoglutarate as co-factors to regulate gene expression. Bromodomain containing proteins a family of chromatin reader proteins, have potential therapeutic efficacy against various malignancies. Long non-coding RNAs (lncRNAs) have also been shown to play a role as oncogenic molecules in different cancers. Several such lncRNAs have now been shown to locate to the same chromosomal region as various KDMs. We therefore examined the expression of various JmjC and Brd members (along with any associated lncRNAs) in MPM and assessed some for their clinical potential using existing small molecule inhibitors.
Method:
A panel of MPM cell lines and a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies were screened for expression of various BRD and JmjC members and associated lncRNAs by RT-PCR. IHC for KDM4A was performed on a cohort of FFPE specimens. The effects of treatments with small molecule inhibitors targeting these proteins on both cellular health and gene expression were assessed.
Result:
The expression of the various KDMs was detectable across our panel of cell lines. In primary tumours the expression of many of these genes were significantly elevated in malignant MPM compared to benign pleura (p<0.05), and significant differences were also observed when samples were analysed across different histological subtypes. Treatment of mesothelioma cell lines with various small molecule inhibitors caused significant effects on cellular health and on the expression of a panel of genes.
Conclusion:
The expression of various KDMs, BRD genes and associated lncRNAs are significantly altered in MPM. Small molecule inhibitors directed against these show potential therapeutic efficacy with significant anti-proliferative effects. We continue to assess the effects of these compounds on gene expression and cellular health to confirm their potential utility as novel therapies for the treatment of MPM.
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P1.09-007 - Targeting MET/TAM Receptors in Mesothelioma: Are Multi-TKIs Superior to Specific TKI? (ID 9959)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos, and most patients die within 24 months of diagnosis. There is an urgent need to identify new therapies for treating MPM patients. Targeting “addicted” receptor tyrosine kinase (RTK) signalling networks has become a critical therapy option in cancer therapy. RTK hetero-dimerization may however, be a key element in the development of resistance to such therapy. As such Tyrosine kinase inhibitors (TKIs) with the ability to target multiple receptors may have superior efficacy to those targeting individual receptors. We and others have identified c-MET, MST1R (also known as RON), Axl and Tyro3 as RTKs frequently overexpressed and activated in MPM, making these attractive candidate targets. Several agents have been developed which target these. LCRF0004 specifically targets MST1R, whereas BMS-777607, RXDX-106 or Merestinib (LY2801653) are orally bioavailable small molecule inhibitors which inhibit c-MET, MST1R, Axl and Tyro3 at nM concentrations. These drugs may therefore have clinical utility in the treatment/management of MPM.
Method:
Expression of RON/MET/TAM and associated ligands were assessed in a cohort of patient samples and MPM cell lines comprising benign, epithelial, biphasic, and sarcomatoid histologies. In vitro and in vivo experiments were undertaken to determine the efficacy of single and multi RTK targeting agents (LCRF0004, RXDX-106, BMS-777607). The effects of LCRF0004 and BMS-777607 were subsequently examined in an in vivo SQ xenograft tumour model.
Result:
mRNA expression of the RON/MET/TAM family and associated ligands (MSP, GAS6) was detected in a large panel of normal pleural and MPM cell lines. In a cohort of patient samples, mRNA levels of c-MET, Axl, Tyro3 and various isoforms of MST1R (flRON, sfRON, t-ΔRON) and MSP but not Gas6 or MERTK were increased in tumours compared with benign pleural samples (p<0.05). No MET Exon 14 skipping mutations were detected. RTK targeting agents displayed in vitro efficacy in terms of reduced proliferation. In vivo, the multi-target TKI (BMS-777607) demonstrated superior anti-tumour activity compared with LCRF0004 (MST1R specific compound). IHC analysis of the xenograft tumours showed high cytoplasmic expression of Vimentin, Cytokeratin and Calretinin, with significant necrosis in many.
Conclusion:
Our data suggests that a multi-TKI, targeting the RON/MET/TAM signalling network, is superior to selective RTK inhibition as an interventional strategy in MPM.
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P2.02 - Biology/Pathology (ID 616)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 4
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.02-024 - False Positivity Due to Polysomy in Fluorescence in Situ Hybridization (ID 10523)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
Pathologists may recognize the phenomenon of polyploidy in FISH, which may be misleading in interpretation of break apart fluorescence in-situ hybridization (FISH). The chance for single or split probe signals is likely to increase with the degree of polysomy. The aim of this study was to explore whether false positivity due to polyploidy occurs in practice.
Method:
A cohort of cases referred for study or patient care was collected from the archives. From the cases where the ALK and/or ROS1 in-situ hybridization test was repeated in our hospital the outcome of testing was compared. Additionally tumor DNA of an occasional case was tested by an orthogonal method (Ion Torrent Oncomine Focus Assay) for translocations.
Result:
Three cases with ALK FISH rearrangement elsewhere were diagnosed with polyploidy in the referral center. One case was reported with rearrangements in both the ALK and the ROS1 gene detected by FISH analysis. In the repeated FISH analysis the average number of co-localization signals in the tumor cell nuclei was 7.6 for ALK and 9.5 for ROS1 respectively (range 1 - 30). Moreover, the morphology of this case was a giant cell carcinoma, variant of pleomorphic carcinoma of the lung. Examination with an orthogonal method (Ion Torrent Oncomine Assay) revealed no translocations and the tumor cells were negative for ALK and ROS1 by immunohistochemistry proving the original report as false positive, supported by absence of response on crizotinib. In break apart FISH the 15% threshold for positivity was obtained in cells emphasizing that in cross sections of normal nuclei occasionally split signals or 3’ probe signals may be present even in diploid nuclei. In the range of 15-20% the chance of false positive FISH is >1%.[1] However, in polyploid tumors the higher number of probe signals within one nucleus comes with an increased chance of split or 3’ signals and a higher rate of false-positive results when maintaining a uniform threshold 15% irrespective of ploidy. Moreover, this may in case of ALK be an additional reason for discordancy with ALK immunohistochemistry, explaining the lack of response on targeted therapy in these patients.[2] 1. vLaffert Lung cancer. 2015;90:465 2. vdWekken. Clin Cancer Res.epub.
Conclusion:
In case of polysomy there is a increased chance of false positive in break apart FISH results. An addition technique should be used to confirm a positive FISH status in tumors with highly increased gene copy number due to polysomy.
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P2.02-048 - Survival Correlation Between TP53 Gene and PD-L1 Tumour Expression in Resected Non-Small Cell Lung Carcinoma (ID 10272)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
Tumour suppressor gene TP53 mutation is common in human cancers, especially playing an important role in lung cancer tumourgenesis. Some clinical studies have shown that TP53 alterations in non-small cell lung carcinoma (NSCLC) carry a worse prognosis and may relatively more resistant to chemotherapy and radiation. We conducted this study to evaluate the impact of TP53 assessed by limited targeted profiling, correlating with PD-L1 tumour expression and clinicopathological variables in resected NSCLC.
Method:
NSCLC patients who underwent curative resection between 1998 and 2006 at our institution were included. PD-L1 status was assessed using Ventana SP142 antibody on archival FFPE surgical tumour specimens, arrayed on tissue microarrays (TMAs) with triplicate 0.6 mm cores. PD-L1 was scored as positive if membranous staining was present in >1% of tumour cells aggregated across the replicate cores to address heterogeneity. In collaboration with the Lung Cancer Genomics Ireland Study, a targeted panel of 49 genes was assessed by Sequenom MassArray including TP53 and genes in MAPK and PI3K pathways. Clinicopathological data was obtained from hospital electronic database.
Result:
Seventy-two patients were included, of which 40 (58.0%) were males, with a median age of 66.0 years (range: 51.0 – 82.6). 54.2%, n=39 with adenocarcinoma histological subtypes, 45.8%, n=33 were ex-smoker and 42.9%, n=30 had Stage IB disease. Most patients had T2 stage (71.4%, n=50), N0 nodal disease (55.2%, n=37) and grade 2 differentiation (65.7%, n=46). Presence of TP53 mutation was identified in 22 patients (30.5%). Five patients had co-presence of TP53 mutation and PD-L1 positivity. There was no correlation between PD-L1 positivity with TP53 status, KRAS, PTPN11, PHLPP2, PIK3CA, MET and PIK3R1. The median disease-free survival in TP53 mutation with PD-L1 positivity was not reached. In univariate/unadjusted analysis, co-presence of TP53 mutation and PD-L1 positivity appear to have superior disease-free survival over TP53 wild-type and PD-L1 negativity, HR 0.17 (95%CI 0.01-0.78, p=0.018). A trend was seen with overall survival but not statistically significant (TP53 mutant, PD-L1 positive vs TP53 wild-type, PD-L1 negative: NR vs 23.1 months, HR 0.34 (95% CI: 0.0.5-1.11, p=0.079). Independent PD-L1 positivity appears to be associated with better prognosis: DFS HR 0.36 (95% CI 0.11-0.90, p=0.0272) and OS HR 0.47 (95% CI 0.19-0.98, p=0.0427).
Conclusion:
In our cohort, co-presence of TP53 mutation and PD-L1 expression was not associated with poorer survival among resected NSCLC patients. Independently, PD-L1 expression was associated with better survival, a finding which warrants further investigations as potential biomarker.
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P2.02-064 - A Novel 5-miR Signature Shows Potential as a Diagnostic Tool and as a Predictive Biomarker of Cisplatin Response in NSCLC (ID 9957)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumour biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumours, including lung cancer. Their role in cisplatin resistance has yet to be determined.
Method:
MicroRNA expression within a panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7[th] generation miRCURY LNA arrays (Exiqon) and subsequently validated by qPCR. Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumour tissue and serum from NSCLC patients of different histologies. A xenograft model of cisplatin resistance was carried out in which 1x10[3] H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumour volume was measured over time and harvested once the tumour mass measured 500mm[3] and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumours and compared between PT and CisR tumours.
Result:
Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT.
Conclusion:
A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature showed both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological media.
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P2.02-069 - Targeting Neuropilin-1 in NSCLC (ID 10205)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
Neuropilin-1 (NP1) is expressed by a wide variety of human tumour cell lines and diverse human neoplasms, and is implicated in mediating the effects of VEGF on the proliferation, survival and migration of cancer cells. It is extensively expressed in tumour vasculature, where NP1 over-expression is associated with tumour progression and poor clinical outcome. In this study, we examined the effects of targeting NP1 in NSCLC both in vitro and in vivo.
Method:
A panel of NSCLC cell lines (H460, H647, A549 and SKMES) were screened for NP1 at the mRNA and protein levels by RT-PCR and Western blotting, respectively. Cellular expression and localisation of NP1 was further examined by immunocytochemistry, while a panel of retrospective resected lung tumours and matched normal tissues were stained by immunohistochemistry. The effects of targeting NP1 on cell proliferation (BrdU ELISA), apoptosis (FACS, HCS) and downstream survival signalling pathways (Western Blot) were examined under normoxic and hypoxic (0.1% O~2~) cell growth using anti-NP1 neutralising antibodies. Cell survival was assessed in response to treatment of NSCLC cells with a range of chemotherapeutic agents in combination with NP1 neutralising antibodies. Using a human xenograft model, tumour growth studies were carried out in nude mice following subcutaneous injection of NP1 over-expressing cells relative to empty vector controls.
Result:
All lung cancer cell lines examined expressed NP1 with the exception of the H460 cell line. Immunocytochemistry analysis confirmed cellular expression and localisation of this receptor, particularly in the leading edges of migrating cells, suggesting a possible role in cell migration. In a small cohort of resected NSCLC patients, tumour expression of NP1 was high relative to their matched normal lung tissues in adenocarcinoma, squamous cell and large cell neuroendocrine carcinomas. Cell proliferation and apoptosis were significantly altered in NSCLC cells expressing NP1. While hypoxia induced the expression of NP1, treatment of cells with NP1 neutralising antibodies reduced hypoxia-mediated cell proliferation and decreased expression of PI3K and MAPK signalling pathways. In a preliminary study, treatment with NP1 neutralising antibodies sensitised NSCLC cells to the cytotoxic effects of chemotherapy. In vivo, H460 cells over-expressing NP1 significantly increased tumour growth in NOD/SCID mice relative to empty vector controls.
Conclusion:
These data suggest a role for the Neuropilin-1 receptor in promoting cell survival and tumour growth in NSCLC and may offer potential as a therapeutic biological strategy in lung cancer.
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-053 - Optimization and Characterization of Assays to Identify Met Exon 14 Skipping in FFPE Embedded NSCLC Samples (ID 9881)
09:30 - 16:00 | Author(s): Stephen P Finn
- Abstract
Background:
The hepatocyte growth factor (HGF)receptor (MET), is frequently altered in NSCLC. Despite having a significant number of diverse mutations/alterations, randomized trials with MET inhibitors have proved disappointing, with no clinical benefit (1). More recently MET exon 14 skipping alterations have emerged as potential therapeutic targets as MET exon as they inhibit the degradation of Met, prolonging its oncogenic activity (2). Patients with Met exon 14 skipping have been found to sensitive to MET inhibitors such as crizotinib, and clinical trials of MET TKIs in METex14 mutated NSCLC are ongoing (1).
Method:
A one-step RT-PCR end-point PCR assay to examine for the detection of Met exon14 skipped mRNAs in FFPE was designed, optimized and tested on a cohort of NSCLC patients. Positive samples were confirmed by targeted next-generation sequencing of these samples. Finally RNA in situ hybridization (RISH) was optimised on a cMET exon 14 skipped cell line (NCI-H596) and subsequently performed on full-face sections using a specific BaseScope™ Assay (Techne).
Result:
Initial studies found that standard end-point PCR resulted in significant false-positives. However, a one-step RT-PCR methodology resolved this issue. Met exon 14 skipped samples were then examined in a cohort of pulmonary sarcomatoid carcinomas (PSCs). In agreement with another study of Caucasian patients (3), we identified Met Exon 14 skipped mutations in 2/20 (10%) of patients. Expression of Met exon 14 skipped was confirmed using targeted resequencing by NGS. RISH was also examined in the same samples.
Conclusion:
These results demonstrate the optimization of a methodology to robustly detect Met exon 14 mutated patients in FFPE material by a PCR based assay, with results comparable to those obtained in similar studies. This methodology can be utilised by any standard hospital diagnostic laboratory without the need for any specialized technology such NGS, RISH or FISH. A qPCR based version of this assay is currently being optimized and the results will be presented at the meeting. References Reungwetwattana, T. et al., (2017). Lung Cancer 103: 27–37 Pilotto, S. et al. (2017). Ann Transl Med 5(1):2 Saffroy, R. et al (2017). Oncotarget. 2017 Mar 21. doi: 10.18632/oncotarget.16403. [Epub ahead of print]
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SS 01 - Supporting the Clinical Management of Lung Cancer Patients through Innovation in Diagnostics - Roche (ID 761)
- Event: WCLC 2017
- Type: Workshop
- Track: Radiology/Staging/Screening
- Presentations: 1
- Moderators:
- Coordinates: 10/15/2017, 08:15 - 12:00, Room 315
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SS 01.06 - Integrated Tissue and Molecular Diagnostics for Optimal Lung Cancer Patient Care (ID 10973)
08:15 - 12:00 | Presenting Author(s): Stephen P Finn
- Abstract
- Presentation
Abstract not provided
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