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E. Brambilla
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ORAL 07 - Lung Cancer Pathogenesis (ID 91)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 8
- Moderators:J. Sage, E. Brambilla
- Coordinates: 9/07/2015, 10:45 - 12:15, 102+104+106
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ORAL07.01 - Evaluation of Epigenetic Mechanisms of Pluripotency in Human Respiratory Epithelia (ID 3041)
10:45 - 10:56 | Author(s): V. Shukla, M. Rao, J. Beers, H. Zhang, D. Wangsa, D. Wangsa, E. Reardon, J.A. Hong, M. Zhang, S. Davis, G. Chen, T. Ried, M.M. Miettinen, D.S. Schrump
- Abstract
- Presentation
Background:
Smoking is the number one risk factor for lung cancer worldwide. Recent data indicate that stem cells situated throughout the small airway epithelium may initiate cancer formation following direct exposure to inhaled carcinogens. In the present study we sought to generate induced pluripotent stem cells (iPSCs) from normal human small airway epithelial cells (SAECs) in order to investigate epigenetic mechanisms contributing to the cancer stem cell initiation process, and possibly identify novel targets for lung cancer therapy.
Methods:
Several different stocks of SAEC were transduced with Stemcca virus containing OKSM (Yamanaka factors); multiple randomly selected clones were expanded for further analysis. Spectral karyotyping was performed to confirm the purity of pluripotent cells. iPSC cells were injected in SCID mice to study teratoma formation. RNA and DNA were extracted from iPSC and parental SAEC for qRT-PCR and RNA-Seq analyses, as well as pyrosequencing of LINE-1, NBL2 and D4Z4 DNA repetitive elements, and promoter regions of several differentially regulated genes.
Results:
SAEC were reprogrammed to a pluripotent state. Generated iPSCs demonstrated hallmarks of pluripotency including morphology, proliferation, expression of surface antigens, stemness gene expression, and in vivo teratoma formation. Interestingly, no chromosomal aberrations were observed in iPSCs. Pyrosequencing did not demonstrate any significant changes in LINE-1, NBL2 and D4Z4 DNA methylation levels in iPSC compared to parental SAEC, suggesting relatively limited global hypomethylation following reprogramming. Consistent with these observations, cancer-testis genes such as NY-ESO-1, MAGE-A1 and MAGE-A3, which are frequently upregulated by DNA demethylation in lung cancer cells, remained transcriptionally repressed in the iPSC. On the other hand, NANOG and POU5F1 genes were hypomethylated in iPSCs relative to SAEC, correlating with their over-expression in iPSCs. RNA-Seq analysis revealed up-regulation of genes encoding components of Polycomb-Repressive Complex 2 (PRC2), and down-regulation of several tumor suppressor genes such as DKK1, p16 and p21 in iPSC relative to parental SAEC. Several novel pluripotency associated genes were also noted to be up-regulated in pulmonary iPSC, which are the focus of ongoing mechanistic studies.
Conclusion:
This is the first report demonstrating successful reprogramming of human respiratory epithelia to pluripotency. This model may prove useful for elucidating fundamental epigenomic mechanisms of pulmonary carcinogenesis and identification of novel targets for lung cancer therapy.
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ORAL07.02 - Metabolic Reprogramming in the Airway Epithelium of Individuals at High Risk for Lung Cancer (ID 2493)
10:56 - 11:07 | Author(s): S.M.J. Rahman, X. Ji, L.Z. Zimmerman, M. Li, B.K. Harris, M.D. Hoeksema, Y. Zou, J. Qian, R. Slebos, Y. Shyr, A. Spira, J.D. Young, D.C. Liebler, P.P. Massion
- Abstract
- Presentation
Background:
What defines the high risk airway epithelium for lung cancer remains a major challenge. Airway epithelium is prone to assault by the risk factors and considered to be the primary cell type involved in the field cancerization. Transcriptomic aberrations in the airway epithelium of individuals at risk for lung cancer have been reported earlier. However, very limited information exists about proteomic alterations in the airway epithelium. We investigated the molecular underpinnings of risk from proteomic alterations in the cytologically normal airway epithelium from individuals at risk for developing lung cancer.
Methods:
Bronchial brushings specimens were collected from individuals categorized as low, medium and high risk groups based on Bach risk model. Shotgun proteomic profiling data were acquired by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins were identified using a combination of database search tools and candidate proteins were selected based on Jonckheere-Terpstra trend analysis. Pathway analysis was performed using WebGestalt. In vitro model of human bronchial epithelial cell line treated with cigarette smoke condensate (CSC) was used for metabolic flux experiments by gas chromatography mass spectrometry (GC MS) analyses.
Results:
We identified 2901 proteins in bronchial epithelial cells from risk stratified individuals. Jonckheere-Terpstra trend test resulted significantly altered expression of 315 proteins (trend p <0.05) with 238 up and 77 down trends. KEGG pathway analysis with the 315 proteins revealed very early events of possible metabolic reprogramming in the cytologically normal bronchial epithelium of individuals at high risk for lung cancer development. Fourteen enzymes of the glycolytic pathway, TCA cycle, pentose phosphate pathway, and glycogenolysis were over expressed. Six of these fourteen enzymes, PYGB, PFKP, PFKL, PKM2, IDH1, and IDH2 were rate limiting enzymes. In in vitro culture of human bronchial epithelial cells treated with CSC, lactate production and glucose consumption were increased suggesting Warburg effect and metabolic reprogramming. Evidence of glutamine metabolism through reductive carboxylation in CSC treated cells was obtained from the metabolic flux analyses of cells from this in vitro model. Contribution of labeled carbon from [U-[13]C5]-glutamine to TCA cycle in CSC treated cells were more than untreated control cells and there was strong M+5 citrate labeling in CSC-treated cells.
Conclusion:
Shotgun proteomic analysis of cytologically normal bronchial epithelial cells in individuals at increasing risk for lung cancer revealed over expression of carbohydrate metabolic enzymes in high risk individuals suggesting possible metabolic reprogramming. The altered profile of metabolic enzymes may provide a signature of lung cancer risk assessment and serve as the basis of patient selection for surveillance programs and chemoprevention.
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ORAL07.03 - MMP12 and LMO7 Are Key Genes Involved in the Early Pathogenesis of Squamous Cell Carcinoma of the Lung (ID 1173)
11:07 - 11:18 | Author(s): V.H. Teixeira, S. Lourenco, B. Carrol, M. Falzon, A. Capitanio, J. Brown, J.P. George, S.M. Janes
- Abstract
Background:
Lung cancer is the most lethal cancer type worldwide. In order to increase patient survival it is important to improve our understanding of the early changes associated with lung cancer progression. The progression of lung squamous cell carcinoma (SqCC) from pre-invasive lesions involves a series of histological changes which includes squamous metaplasia, mild, moderate and severe dysplasia, and carcinoma in situ (CIS). In these pre-invasive lesions the basement membrane is intact and there is no possibility of metastatic spread, which is in contrast to SqCC where there is the potential for metastasis as soon as invasion occurs. Our laboratory has a unique cohort of patients with pre-invasive lung SqCC lesions. Within this cohort there is a discrepancy between the prevalence of pre-invasive lesions and the incidence of invasive lung cancer, which suggests that not all pre-invasive lesions progress to invasive carcinomas. This tissue collection forms an internationally unique resource of lesions and will shed light on the molecular characteristics of lesions that progress compared to those that either regress or remain stable. The aim of this study was to identify and characterize key genes involved in the early pathogenesis of lung SqCC.
Methods:
Following histological review by two histopathologists to confirm that pre-malignant tissue is present in the biopsy specimens, the epithelial component of interest was laser-capture micro-dissected. This is vital in order to eliminate any cross-contamination from unwanted cells and to ensure that pre-invasive CIS specific gene expression profiles are generated. We have performed genome-wide gene expression Illumina’s Whole-Genome DASL® arrays in 20 progressive and 19 regressive pre-invasive lung SqCC lesions. The protein expression of Matrix metallopeptidase 12 (MMP12) and LIM domain 7 (LMO7) was also determined in the 39 pre-invasive lung cancer lesions by immunostaining analysis. The functional role of MMP12 and LMO7 in cell migration and invasion was demonstrated by MMP12 and LMO7-shRNA knockdown in different squamous cell carcinoma cell lines and human bronchial epithelial cells (HBECs), respectively.
Results:
We found 939 genes significantly differently expressed between the progressive and the regressive pre-invasive lung SqCC lesions. We identified a remarkably elevated expression of a spectrum of genes in the progressive lung SqCC lesions involved in different related cancer pathways including chromosome instability, p53 signalling and Wnt/β-catenin signalling. MMP12 and LMO7 were found within the highest significantly differently expressed genes and were therefore chosen to pursue studies focused on understanding the potential mechanisms leading to the development of lung SqCC. In agreement with the gene expression data the expression of MMP12 and LMO7 proteins were up-regulated and down-regulated, respectively, in progressive when compared with regressive lesions. Inhibiting MMP12 by MMP12 knockdown significantly reduced the migration and invasion of different squamous cell carcinoma cell lines (A431, H357 and H376). We also established HBECs knockdown targeting LMO7. We observed a significant increase in the migration and invasion of HBECs cells in the LMO7 shRNA knockdown compared to control.
Conclusion:
Our results suggest that MMP12 and LMO7 may be potential therapeutic markers for lung cancer at early stage.
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ORAL07.04 - Discussant for ORAL07.01, ORAL07.02, ORAL07.03 (ID 3304)
11:18 - 11:28 | Author(s): M.B. Beasley
- Abstract
- Presentation
Abstract not provided
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ORAL07.05 - Differential Tumorigenic Properties of Mesenchymal Cells From Neoplastic and Non-Neoplastic Human Lung in NSCLC (ID 1006)
11:28 - 11:39 | Author(s): D. Madeddu, A. Falco, L. Ampollini, C. Lagrasta, C. Frati, A. Gervasi, B. Lorusso, F. Saccani, G. Graiani, R. Alfieri, P. Petronini, P. Carbognani, L. Gnetti, P. Rossetti, G. Bocchialini, F. Ricci, E. Quaini, K. Urbanek, F. Quaini
- Abstract
- Presentation
Background:
Cancer Initiating Cells (CICs) and their niches may open new avenues in the pathogenesis and management of lung cancer. A relevant component of the niche is represented by supportive stromal cells that control the fate of CICs by a reciprocal cross-talk. The understanding of these cellular events could represent a significant advancement in cancer biology and treatment. Recent observations by our and other laboratories have suggested that mesenchymal stromal cells (MSC) regulate lung cancer growth and resistance, thus generating large expectations in novel anti-cancer strategies. The aim of our study was to determine whether MSC isolated from NSCLC and from non-neoplastic human lung samples possess different biologic properties and tumorigenic potential.
Methods:
Fresh samples of neoplastic and spared lungs from 58 male patients (80% smokers) affected by primary pulmonary adenocarcinoma undergoing surgical resection were processed. Stromal cells were separated from epithelial cells by negative selection using EpCAM (CD326)-based immunomagnetic sorting. After further enrichment, we could expand for at least 14 passages a population of CD90, CD105, CD73 and CD44 positive MSC from lung cancer (Lc-MSC) and non-neoplastic (Nn-MSC) lung tissue. The oncogenic potential of these cells from the same patient was tested on a Calu-3-based in vitro model of NSCLC by co-culture and conditioned media (CM) and in vivo by xenotransplantation in Balb/c Nude mice. In vivo cell tracking was achieved by pre-labeling MSC with Quantum dots 585 (Qdots). Morphometric assessment of tissue composition and immunofluorescence combined with FISH analysis of human X and Y chromosomes was performed on xenografted tumors.
Results:
Nearly 30x10[6] cells could be typically obtained after 3 passages in each case, however, compared to Nn-MSC, cultures of Lc-MSC displayed lower growth kinetic and mitotic index while higher survival and HIF-1-alpha (Hypoxia-inducible-factor-1) upregulation in response to hypoxia was observed. A larger fraction of Lc-MSC expressed transcription factors involved in stemness (Oct3/4, SOX2) and in bronchioalveolar (TTF1, ETS-1, CCL10) commitment. Co-cultures demonstrated that Lc-MSC significantly increased Calu-3 growth as compared to Nn-MSC in transwell assay and by contact. CM from Lc-MSC similarly promoted Calu-3 expansion as compared to Nn-MSC. When 2.5x10[6] Lc-MSC or Nn-MSC from the same patient were subcutaneously co-injected with Calu-3, a 38% and 17% increase in tumor volume was respectively observed, compared to the injection of an equal number of Calu-3 alone (CTRL). Lc-MSC or Nn-MSC injected alone did not generate tumors. Quantitative estimation of the in vivo expansion of neoplastic cells indicated that the addition of Lc-MSC increased by 6-fold and 29-fold Calu-3 replication compared to Nn-MSC and CTRL, respectively. Cell tracking documented that Qdots labelled MSC were located at the boundary of neoplastic epithelial glands generated by X-chromosome polysomic Calu-3 cells. A comparative molecular analysis of Lc-MSC and Nn-MSC is ongoing for the identification of distinctive signalling pathways implicated in the microenvironemental control of CIC on NSCLC development.
Conclusion:
Profound differences exist in the biology and oncogenic potential of intratumoral and normal lung MSC strongly supporting the notion that the tumor microenvironment may represent a potential target of new customized therapeutic strategies.
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ORAL07.06 - Sox2 Cooperates with Lkb1 Loss to Promote Mouse Model of Squamous Cell Lung Cancer (ID 2910)
11:39 - 11:50 | Author(s): A. Mukhopadhyay, G. Mollaoglu, B. Witt, T.G. Oliver
- Abstract
- Presentation
Background:
Squamous cell carcinoma (SCC) of the lung is the second most common subtype of lung cancer with limited treatment options and a poor survival rate. Until recently, mouse models of SCC have been limited.
Methods:
Using lentiviral delivery of Sox2 and Cre recombinase to the mouse lung, we tested the ability of Sox2 to promote tumorigenesis in multiple tumor suppressor backgrounds. Mouse lungs were imaged for tumor formation using micro-CT imaging. Resulting mouse tumors were evaluated for histological markers including Nkx2.1, Sox2, p63, cytokeratin-5, cytokeratin-14 and compared to human squamous tumors. Phospho-signaling proteins including pAkt, pErk, pStat3, pAMPK, p4EBP1 were also evaluated in mouse and human tumors by immunohistochemistry.
Results:
Expression of Sox2 specifically cooperates with loss of Lkb1 to promote squamous lung tumors. Importantly, Sox2 expression and mTOR pathway activation frequently co-occur in human squamous tumors. Mouse squamous tumors exhibit characteristic histopathology and biomarker expression similar to human SCC. They also mimic human SCCs by activation of therapeutically relevant pathways including STAT and mTOR. Sox2 expression is sufficient to induce phosphorylated Stat3 in vitro (Mukhopadhyay et al, Cell Reports, 2014). Sox2-driven tumors also exhibit immune cell infiltration consistent with other squamous lung cancer models.
Conclusion:
This mouse model of Sox2-driven squamous lung cancer may be a useful model to study immunotherapies and their mechanism of action. This model may also be used to test the contribution of additional driver alterations in SCC, as well as for preclinical drug discovery. Our data suggest mTOR, Jak-Stat and immunotherapies may be relevant targets for squamous lung cancer.
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- Abstract
Background:
Non-small-cell lung cancer (NSCLC) is featured with genetic and histopathological heterogeneity. LKB1-mutant NSCLC represents a unique and prevalent molecular subtype with limited treatment options. Originally characterized as a tumor suppressor, LKB1 phosphorylates and activates several downstream targets to inhibit cell growth; on the other hand, LKB1 also regulates cellular energy sensing and metabolic homeostasis. This raises an interesting question about how LKB1 inactivation coordinates in vivo lung tumor progression with metabolic adaptation. We have shown recently that the Kras/Lkb1 lung tumor heterogeneity results from p63-mediated ADC to SCC transdifferentiation (AST) through mixed Ad-SCC at late stage, suggesting an unexpected plasticity upon LKB1 inactivation in NSCLC. However, it remains unclear how LKB1 inactivation coordinates tumor progression with metabolic adaptation in orchestrating this tumor plasticity.
Methods:
We integratively analyze the transdifferention process of mouse lung adenocarcinoma to squamous cell carcinoma in Kras/Lkb1 Adeno-Cre nasal inhalation model as well as the lineage-defined Kras/Lkb1 model. Moreover, we have also systematically analyzed the clinical lung adenosquamous cell carcinoma to prove the findings from our animal models.
Results:
Here in Kras[G12D];Lkb1[lox/lox ](KL) mouse model, we reveal differential reactive oxygen species (ROS) levels in lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). ROS can functionally modulate the ADC-to-SCC transdifferentiation (AST). Furthermore, pentose phosphate pathway deregulation and impaired fatty acid oxidation collectively contribute to the redox imbalance and functionally affect AST. Similar tumor and redox heterogeneity also exist in human NSCLC with LKB1 inactivation. In preclinical trials towards metabolic stress, certain KL ADC can develop drug resistance through squamous transdifferentiation. This study uncovers critical redox control of tumor plasticity that may affect therapeutic response in NSCLC.
Conclusion:
LKB1-mutant tumor represents a unique and prevalent molecular subtype of NSCLC with limited treatment options. Through integrative human lung cancer sample analysis and modeling tumor development in mouse model, we have uncovered the accumulation of ROS during ADC progression, which modulates the phenotypic transition as squamous transdifferentiation and metabolic adaptation. This metabolic adaptation reflects the dynamic function of LKB1: a tumor suppressor at early lung ADC progression and an essential metabolic regulator at late phenotypic transition. The redox-controlled tumor plasticity for squamous transdifferentiation enables ADC to progress under stress, and more importantly to escape certain treatment towards cancer metabolism. The plasticity represents as a potentially important mechanism for lung cancer metabolic adaptation and drug resistance, and holds important therapeutic implications.
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ORAL07.08 - Discussant for ORAL07.05, ORAL07.06, ORAL07.07 (ID 3470)
12:01 - 12:11 | Author(s): Y. Yatabe
- Abstract
- Presentation
Abstract not provided
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Author of
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MS 21 - Immunotherapy Predictive Biomarkers (ID 39)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:K. Fong, P. Dennis
- Coordinates: 9/09/2015, 14:15 - 15:45, Four Seasons Ballroom F1+F2
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MS21.02 - PD1/PDL1 Biomarker Strategies (ID 1942)
14:40 - 15:00 | Author(s): E. Brambilla
- Abstract
- Presentation
Abstract:
Introduction: Cancer cells express antigens that potentially differentiate them from normal cells. These are known to be numerous in lung cancer and characterized by a high mutational rate (7-11 mutations / MegaBase), especially in relation with smoking derived genetic instability, P53 mutations, and/or the presence of targetable mutations in adenocarcinoma. These tumor antigens should confer immunogenicity to lung cancer transformed cells. However, immune-editing occurs in most lung cancer along a three phases sequence: 1) Elimination, where transformed cells are destroyed by the immune system; 2) Equilibrium, equivalent to a functional state of dormancy in which tumor cells growth is controlled by adaptive immunity, a state characterized by typically dense lymphocytic infiltration rich in CD8 cytotoxic cells (E. Brambilla et al. JCO, under review); 3) Escape from immune surveillance. PD-L1 in NSCLC is expressed on the membrane of tumor cells, and/or on immune infiltrating cells dendritic cells (DC), other antigen presenting cells (APC) and T lymphocytes. PD-1, the PDL1 receptor, is expressed on tumor infiltrating lymphocytes (TILS), mainly CD4 T cells, T regulatory (T-reg) and B, NK, monocytes and DC. Upon PD-L1 binding, PD-1 inhibits kinases involved in T cell activation. There are two mechanisms of expression of immune checkpoints on tumor cells and their immune stromal counterparts: oncogenic signaling, and response to inflammatory signals, both of which occur potentially in lung cancer. Tumor cells express multiple ligands and receptors and antitumor immune response can be enhanced by multi-level blockade of immune checkpoints. PD-1/PD-L1 engagement leads to HSP-2 phosphatase activity which dephosphorylates Pi3K and thus downregulate AKT. The necessary patient selection for immunotherapy has stressed the search for predictive biomarker of PD-1/PD-L1 pathway inhibition. The cutoff for positivity on tumor cells[1–3]: The cutoff for positivity in and out of trials on tumor cells has never been assessed nor optimized or standardized. The percentage of PD-L1 membrane staining considered as the cutoff for positivity was from ≥1%, ≥5%, ≥10%, ≥50% and the intensity was or not defined and taking into account (any intensity, 1+, 2+, 3+, or a scale from 1 to 3+/H Score , or 2+3+only). At least, most if not all reports considered only membrane staining on tumor cells, although cytoplasmic staining was also considered with AQUA techniques. Stromal expression of PD-L1 on immune infiltrate (T cells, macrophages, DC) is also needed for scoring. Whereas DC and macrophages display a clear cytoplasmic membrane stain, this is not appreciated on lymphocytes. We have set up a study to assess a cutoff of positivity for prognosis analysis (1500 randomized early stage operable NSCLC patients with or without adjuvant cisplatin therapy after surgery) using E1L3N Cell Signaling antibody commercially available. We found that 20% of lung tumors cell expressed PD-L1 (≥20% intensity 2+3+), and 29% the immune stromal cells (T, macrophages, DC ) ≥10% intensity 2+3+. PD-L1 positivity in both tumor and immune cells were seen in only 9% of NSCLC, 20,7% were both negative . We double-check the scoring cells with Ming Tsao. The best concordance was for intensity 2+ /3+ (83%) although the intensity 1 was not reproducible ( 40%) . There was no prognostic relevance of PD-L1 (tumor cells or stroma) in the control arm and pooled analysis, whatever cutoff by 10% increment or linear scoring was used. There was no statistical correlation between PDL1 expression (Tumor or Immune cells ) with clinicopathological criteria or histology . Only immune PD-L1 expression was correlated with a highly intense immune infiltrations (TILs ) ( P = 002 ). Not surprisingly, previous published evaluations of prognostic value were discordant likely because immune checkpoints modulators play both positive and negative roles in the immune inhibitory pathways with some redundancy, and patients series and assays were not comparable .The two meta-analyses with their numerous biases ( different antibodies, cutoffs, patient series composition in early and advanced stage, ethnicities and contribution of oncogene driven cancers, time of use of the initial resection sample or contemporary biopsy…) rendered their interpretation extremely problematic . Global result was favoring a poor prognosis of “PD-L1 positivity” on tumor cells. PD-L1 expression as a predictive biomarker in cancer immunotherapy[1,4–7]: In the majority of phase I trials with four antibodies targeting the co-inhibitory receptor PD-1 or its primary ligand PD-L1 (Table 1), response rates appear higher in patients with increased tumor PD-L1 membrane expression by immunohistochemistry (IHC). However, different antibody assays, lack of standardization, different cutoff point to determine PD-L1 positivity, the usual various pharmaceutic companies to recommend their companion test, and the small number of specimens available for testing, in addition with the variability of the intervals between biopsy and test, has surely hampered the conclusion and prevent consensus to be reached[7,8]. The most pertinent threshold was provided by Garon et al, with ≥50% of tumor cells PD-L1 positive to allow the highest response rate of 45% in pembrolizumab treated patients in the validation group[1]. In most trial series, biopsies or resected specimen were used restropectively although considerable difference between these samples occurs due to tumor heterogeneity. The reliability of small biopsy samples is questionned[9]. Indeed lung tumor heterogeneity is exemplary , and PD-L1 is typically heterogeneous in its distribution in the tumor bulk as is PD-L1 positive immune cells . Multiple issues are yet addressed before PD-L1 is considered as a robust and definitive molecular predictor of efficacy. Various clones are currently being evaluated in and out of clinical trials (Ventana SP263, SP6242, Dako 28-8 and 22C3, Cell Signaling E1L3N). As for prognostic evaluations, thresholds of ≥1%, ≥5%, ≥10%, ≥50% or continuous H score have been used. In addition in a few trials, PD-L1 expression in TILs was predictive more than PD-L1 on tumor cells but the cutoff was not disclosed. IASLC pathology panel is leading a large multicentric reproducibility study ( Fred Hirsch )with lung pathologists of the IASLC Pathology Committee to address these questions. Alternative regulations of PD-1/PD-L1 pathway The ability of cancer cells to evade immunosurveillance results from the production of immunosuppressive chemokines by the tumor cells, loss of MHC antigen expression, a higher number of T-reg cells in the tumor microenvironment and inhibitory pathways referred to as immune checkpoints, which result in a link of inhibitory ligands to their receptors (CTLA~4~-PD-1, PD-L1/PD-L2-PD-1) are unfrequently upregulated in lung cancer. Moreover immune-editing was associated with the illegitimate expression of tumor germ cell (testis /placenta) antigens[10], normally absent in normal tissue but testis and placenta, inducing a state of immune escape when aberrantly expressed in lung cancer correlating with highly and metastatic aggressive behavior. While patients with PD-L1 overexpression based on different assays, cutoff, tumor material, have more robust response to PD-L1 (67-100% ORR), PD-L1 negative NSCLC ranges from 0 to 15%, suggesting that PD-L1 IHC is not a clear and exclusive predictive biomarker. This is not surprising due to multiple regulations at the two clinically relevant immunologic synapses: the tumor-T cell interface, and the APC-T cell interface, both playing role in tumor control. In all cohorts, PD-L1 in tumor cells was observed with or without immune infiltration. TILs intense infiltration occurred in 10% of NSCLC across histology and was a statistically significant good prognosis factor although the oncogene driven adenocarcinomas lack immune infiltrate. EGFR pathway upregulates PD-L1 as well as PTEN loss[11–14]. In addition the 2 synapses are functionally affected by HLA loss (>50% of NSCLC), EGFR signaling, PTEN loss, the density of CD8 in infiltrate available for cytotoxicity and even more CD8 +/PD1+ exhausted cytotoxic T cells among TILs . The best predictive biomarker might not be simply binary . Biopsies may underevaluate the pertinent tumor-stroma interface , PD-L1 biologically relevant ( more than 1-10% of tumor cell ! ) has already taken place and destroyed the potentially reactive CD8 T cells. Indeed secondary biomarkers may drive the tumor in association or independently of PD-1/PD-L1 pathway. Table 1: Prevalence of PD-L1 in NSCLC:
References: 1. Garon EB, et al. Pembrolizumab for the treatment of NSCLC. N Engl J Med. 2015;372(21):2018-2028. 2. Sorensen S, et al. PD-L1 expression and survival among advances NSCLC patients treated with chemotherapy. Ann Oncol. (25 (Supplement 4)). 3. Soria J-C, et al. Clinical activity, safety and biomarkers of PD-L1 blockade in NSCLC: Additional analyses from a clinical study of the engineered antibody MPDL3280A (anti-PDL1). 4. Patel SP, Kurzrock R. PD-L1 Expression as a Predictive Biomarker in Cancer Immunotherapy. Mol Cancer Ther. 2015;14(4):847-856. 5. Taube JM, et al. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. CCR. 2014;20(19):5064-5074. 6. Herbst RS, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014;515(7528):563-567. 7. Soria J-C, et al. Immune checkpoint modulation for non-small cell lung cancer. CCR. 2015;21(10):2256-2262. 8. Brahmer JR, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. NEJM. 2012;366(26):2455-2465. 9. Kitazono S, et al. Reliability of Small Biopsy Samples Compared With Resected Specimens for the Determination of PD-L1 Expression in NSCLC. Clin Lung Cancer. 2015. 10. Rousseaux S, et al. Ectopic activation of germline and placental genes identifies aggressive metastasis-prone lung cancers. Sci Transl Med. 2013;5(186):186ra66. 11. Akbay EA, et al. Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov. 2013;3(12):1355-1363. 12. D’Incecco A, Andreozzi M, Ludovini V, et al. PD-1 and PD-L1 expression in molecularly selected NSCLC patients. Br J Cancer. 2015;112(1):95-102. 13. Chen N, et al. Upregulation of PD-L1 by EGFR Activation Mediates the Immune Escape in EGFR-Driven NSCLC: Implication for Optional Immune Targeted Therapy for NSCLC Patients with EGFR Mutation. J Thorac Oncol. 2015 14. Lin C, et al. Programmed Death-Ligand 1 Expression Predicts TKI Response and Better Prognosis in a Cohort of Patients With EGFR Mutation-Positive Lung Adenocarcinoma. Clin Lung Cancer. 2015.Percent tumor samples expressing PD-L1 Tumor surface expression cutoff for positivity PD-L1 detection antibody Reference 49% 5% 28-8 Grosso et al. JCO, 2013 52% NR R&D B7-H1 Gatalica et al. Cancer Epidemiology biomarkers prevention, 2014 95% >10% 5H1 Dong et al. Nature Medicine, 2002 50% 11% MIH1 Konishi et al. CCR, 2004 21% (squamous only) >1% vs >5% vs H-score 5H1 Marti et al. JCO, 2014 60% 5% DAKO IHC Gettinger et al. JCO, 2014 50% 1% NR Sun et al. JCO, 2014 25% ≥50% NR Garon et al. NEJM, 2015
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ORAL 06 - Next Generation Sequencing and Testing Implications (ID 90)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:G. De Lima Lopes, V. Miller
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL06.01 - Genomic Characterization of Large-Cell Neuroendocrine Lung Tumors (ID 1667)
11:05 - 11:16 | Author(s): E. Brambilla
- Abstract
Background:
Neuroendocrine lung tumours account for 25% of all lung cancer cases, and they range from low-aggressive pulmonary carcinoids (PCA) to highly malignant small-cell lung cancer (SCLC) and large-cell neuroendocrine lung carcinoma (LCNEC). The last two are strongly associated with heavy smoking and are typically detected at a clinically advanced stage, having a poor survival. Comprehensive genomic analyses in lung neuroendocrine tumours are difficult because of limited availability of tissue. While more effort has been done in the context of SCLC, the detailed molecular features of LCNEC remain largely unknown.
Methods:
We conducted 6.0 SNP array analyses of 60 LCNEC tumours, exome sequencing of 55 tumor-normal pairs, genome sequencing of 11 tumour-normal pairs, transcriptome sequencing of 69 tumours, and expression arrays on 60 tumors. Data analyses were performed using in house developed and published pipelines.
Results:
Analyses of chromosomal gene copy number revealed amplifications of MYCL1, FGFR1, MYC, IRS2 and TTF1. We also observed deletions of CDKN2A and PTPRD. TTF1 amplifications are characteristic of lung adenocarcinoma (AD); CDKN2A deletions are frequent alterations in both AD and squamous-cell lung carcinoma (SQ); FGFR1 amplifications are found in SQ and, less frequently, in SCLC; and MYCL1 and IRS2 amplifications are frequent events in SCLC. Similar to the copy number data, we found patterns of mutations characteristic of other lung cancer subtypes: TP53 was the most frequently mutated gene (75%) followed by RB1 (27%), and inactivation of both TP53 and RB1, which is the hallmark of SCLC, occurred in 20% of the cases. Mutations in STK11 and KEAP1-NFE2L2 (frequently seen in AD and SQ) were found in 23% and 22% of the specimens, respectively. Interestingly, mutations in RB1 and STK11/KEAP1 occurred in a mutually exclusive fashion (p-value=0.016). Despite the heterogeneity observed at the mutation level, analysis of the pattern of expression of LCNEC in comparison with the other lung cancer subtypes (AD, SQ, SCLC, and PCA) points to LCNEC as being an independent entity. An average mutation rate of 10.7 mutations per megabase was detected in LCNEC, which is in line with the rate observed in other lung tumours associated with smoking. We found that, similar to SCLC, the mutation signatures associated with APOBEC family of cytidine deaminases, smoking, and age (based on Alexandrov et al 2013) were the predominant ones in LCNEC. However, the contribution of the individual SCLC and LCNEC samples to these three signatures was quite different, and we are currently exploring it.
Conclusion:
Taking into account somatic copy number and mutation data, we distinguished two well-defined groups of LCNEC: an SCLC-like group, carrying alterations in MYCL1, ISR2, and in both RB1 and TP53; and a group resembling AD and SQ, with alterations in CDKN2A, TTF1, KEAP1-NFE2L2, and STK11. Although these results suggest that LCNEC might be a mix of different lung cancer subtypes, mutation clonality and expression analyses show that they are likely to be a separate entity, sharing molecular characteristics with the other lung cancer subtypes. Their heterogeneity suggests that LCNEC might represent an evolutionary trunk that can branch to SCLC or AD/SQ.
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ORAL06.03 - Genome-Wide Gene Copy Number Analysis by OncoScan<sup>TM</sup> FFPE Assay in 976 Resected NSCLC From LACE-Bio2 (ID 1561)
11:27 - 11:38 | Author(s): E. Brambilla
- Abstract
- Presentation
Background:
Genome wide SNP array studies have identified systematic gene copy number aberrations (CNA) in non-small cell lung cancer (NSCLC), but their prognostic implication is unknown. This study aimed to investigate associations between CNAs and survival using the LACE-Bio bio-bank. The LACE-Bio consortium includes large clinical trials comparing adjuvant platinum-based chemotherapy to observation after complete resection of stage I-III NSCLC.
Methods:
DNA was extracted from FFPE tumor samples from 3 pivotal adjuvant chemotherapy trials (CALGB 9633, IALT, JBR.10); 1013 samples were profiled using Affymetrix OncoScan[TM] arrays with over 300,000 probes and normalized relative to a pool of normal tissues. Segmentation was performed using the CBS algorithm and minimally recurrent regions (MCR) across the series identified by CGHregions. All analyses were performed on the level of MCRs. CNAs were correlated with clinicopathological factors and adjusted for the False Discovery Rate (FDR). The primary endpoint, disease-free survival (DFS), was assessed via univariate Cox models stratified by trial and adjusted for treatment, age, sex, PS, histology, T, and N stage.
Results:
Among 976 successfully profiled samples, 414 (42%) were adenocarcinoma (ADC), 430 (44%) squamous cell carcinoma (SCC) and 132 (14%) other NSCLC; 710 (73%) were male. Across the 431 MCRs identified, patients had on average 94 (SD 69) CNAs: 51 gains and 43 losses. A gain or loss was observed in at least 10% of patients for 177 and 166 regions respectively. The most common gains (up to 48%) were on chromosomes 1p, 3q, 5p, 6p, and 22q. The most common losses (up to 40%) were on chromosomes 3p, 8p and 9p. The size of 253 of the 431 MCRs (59%) was smaller or equal to 3Mb (and 79% ≤10 Mb). Sensitivity analyses on the subset of samples with optimal quality (n=777, defined by MAPD<0.3) gave consistent results. The CNA frequency of 195 regions was significantly different with FDR≤0.05 between ADC and SCC (of which 49% regions of size ≤3Mb and 71% ≤10Mb); the most significant were more gains in 3q, 22q and 12 in SCC and more losses in 3p, 4, 5q in SCC. With a median follow-up of 5.3 years, 510 DFS events and 451 deaths were recorded. In univariate analyses for DFS, 13 regions in loci 19p11–13, 7p12, 9p21, 15q14 had a raw p-value <0.005 (FDR<0.13, the top 8 corresponded to FDR≤0.05); 9 of those 13 regions were of size ≤3Mb (12 regions ≤10Mb). In adjusted analyses, 10 of the 13 regions retained raw adjusted p-values ≤0.005 (FDR≤0.15). Losses of focal regions including CDKN2A/B and STK11 (≤3Mb) were associated with poorer DFS: the hazard ratio (HR) for a 2-fold copy number decrease in region 9p21.3 (including CDKN2A/B) was 1.50 (95% CI: 1.2–1.9, P<0.001, FDR=0.02), and the HR for a 2-fold copy number decrease in 19p13 (including STK11) was 2.4 (1.3–4.3, P=0.005, FDR=0.15). Similar results were obtained for overall survival and lung-cancer specific survival. Results of histology-specific analyses will be presented.
Conclusion:
These large-scale genome-wide analyses of gene CNA provide new candidate prognostic markers for stage I-III NSCLC.
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