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T. Walser
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MINI 02 - Immunotherapy (ID 92)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:P. Forde, S.J. Antonia
- Coordinates: 9/07/2015, 10:45 - 12:15, Four Seasons Ballroom F3+F4
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MINI02.09 - ERK Activation Mediates Increased PD-L1 Expression in KRAS Mutated Premalignant Human Bronchial Epithelial Cells (ID 1620)
11:30 - 11:35 | Author(s): T. Walser
- Abstract
- Presentation
Background:
Immune checkpoint pathways including the PD-1/PD-L1 pathway are involved in tumor evasion from the immune system. Elevated PD-L1 expression in tumor cells inhibits tumor-infiltrating T cell function and may be associated with poor prognosis in lung cancer patients. There is increasing interest in developing immunotherapies that block the immunosuppressive effects of checkpoint pathways such as PD-L1, and identifying patients who may benefit from PD-L1 blockade. Activating KRAS mutations are common driver mutations in non-small cell lung carcinoma. Patients with mutated KRAS demonstrate less benefit from adjuvant chemotherapy and resistance to tyrosine kinase inhibitors. The effect of cancer cell driver mutations on immune checkpoint immune regulation is poorly understood. While recent clinical trials have suggested better response to PD-1 blockade in KRAS mutation subjects, it is unclear if this clinical finding is directly driven by KRAS regulating the PD-1/PD-L1 pathway with resultant improved efficacy to anti-PD-L1 immunotherapy or if the presence of a KRAS mutation is merely a surrogate marker of the overall mutational load and tumor immunogenicity. KRAS mutations are known to activate the RAF-MEK-ERK pathway. We hypothesize that KRAS mutation directly regulates the PD-1/PD-L1 pathway through ERK activation.
Methods:
Immortalized human bronchial epithelial cells (HBEC-vector control), KRAS–mutated (KRAS[v12]) HBEC cells (HBEC-KRAS), p53 knockdown HBEC cells (HBEC-p53), and p53 knockdown/KRAS mutated cells (HBEC-p53/KRAS) were used to assess mRNA and/or surface protein expression levels of immune checkpoints including Lag-3, Tim-3, PD-L1 and PD-L2 by real time-qPCR (RT-qPCR) and flow cytometry, respectively. HBEC-vector and HBEC-KRAS cells were treated with MEK (ERK kinase) inhibitor (PD0325901) at 1µM for 24hrs and evaluated for mRNA and surface protein expression of PD-L1. The premalignant HBEC cell lines were used instead of human lung cancer cell lines in order to assess the role of KRAS mutation in isolation without other mutations.
Results:
PD-L1 and PD-L2 mRNA levels increased 2.4 fold (p<0.001) and 3.6 (p<0.001) fold in comparing HBEC-KRAS to HBEC-vector (wild-type) cells, while Lag-3 and Tim-3 mRNA expression levels were unchanged. Based on mean fluorescence intensity on flow cytometry, cell surface PD-L1 protein expression level was 2.2 and 1.6 fold higher in HBEC-KRAS and HBEC-p53/KRAS, respectively, compared to HBEC-vector cells. There was no increase in surface PD-L1 expression in HBEC-p53 cells compared to HBEC-vector control, suggesting that p53 mutation did not alter PD-L1 expression in HBEC-p53/KRAS cells. With MEK inhibition, PD-L1 mRNA levels decreased 10 and 11 fold in HBEC-vector and HBEC-KRAS cells, respectively. Analogously, PD-L1 surface protein levels were reduced 2.7 fold in HBEC-vector and HBEC-KRAS cells, respectively. These findings suggest that ERK activation mediates intrinsic expression and KRAS mutation mediates over-expression of PD-L1 mRNA and protein.
Conclusion:
Here, we demonstrate that PD-L1 expression is elevated in premalignant KRAS mutated human bronchial epithelial cells, and ERK activation mediates constitutive and KRAS mutation driven up-regulation of PD-L1 in these cells. Our findings suggest that KRAS mutation may directly regulate the PD-1/PD-L1 immune checkpoint pathway. Further understanding of KRAS driven molecular pathways that modulate immune checkpoints may elucidate therapeutic targets for potential combinational drugs to PD-L1 inhibition.
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ORAL 31 - PD1 Axis Inhibition (ID 143)
- Event: WCLC 2015
- Type: Oral Session
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:J. Weiss, B. Luey
- Coordinates: 9/09/2015, 16:45 - 18:15, Four Seasons Ballroom F1+F2
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ORAL31.05 - High Intratumoral T Cell Infiltration Correlated with Mutational Load and Response to Pembrolizumab in Non-Small Cell Lung Cancer (ID 2728)
17:28 - 17:39 | Author(s): T. Walser
- Abstract
- Presentation
Background:
Responses to PD-1 blockade have been induced in approximately 20% of advanced non-small cell lung cancer (NSCLC) patients with progressive disease after standard therapy [Garon, NEJM 2015]. One challenge is to understand how the immune response was initiated in responding patients. Tumor mutational burden has been associated with response to PD-1 checkpoint inhibitors in NSCLC [Rizvi, Science, 2015]. In addition, studies in melanoma patient-derived tumor specimens revealed that responses to PD-1/L1 blockade rely on pre-therapy tumor infiltration of activated T effector cells [Tumeh, Nature, 2014]. We hypothesize that clonal T cell infiltration is correlated with tumor mutational load and clinical response with PD-1 blockade.
Methods:
We studied tumor specimens in NSCLC patients treated with pembrolizumab at UCLA on the KEYNOTE -001 clinical trial. All patients signed informed consent for the trial as well as separate specimen acquisition protocols. Responses were classified by the investigators according to irRC. DNA was extracted and whole exome sequencing was performed at the UCLA Immunogenetics Core. DNA from the same patient’s PBMC or other non-cancerous tissue was sequenced for baseline comparison. Immunohistochemistry (IHC) was done for CD8 (Clone C8/144B, Dako), CD4 (Clone SP35, Cell Marque) and PD-L1 (Clone SP142, Spring Bioscience).
Results:
We report results from 27 patients (14 responders, and 13 nonresponders). Significantly higher density of pre-dosing CD8+ cells (percentage of CD8+ nucleated cells) in the tumors of the responding patients was observed (mean of 17.7% in responders vs 5.6% in non-responders, p=0.02 by unpaired t test) suggestive of a pre-existing immune response. Mutational load in 5 patients (3 responders and 2 nonresponders) showed a trend towards correlation with response (mean of 19 nonsynonymous somatic mutations per MB in responders vs 6 in nonresponders, p=0.33). Interestingly, a strikingly significant correlation between mutational load and CD8 expression was observed (R[2]=0.96, p=0.003). In addition, pre-dosing tumor PD-L1 expression demonstrated a trend towards correlation with response (mean of 72.1% in responders vs 51.5% in nonresponders, p=0.07) but not with CD8 tumor infiltration (R[2]=0.05, p=0.28). No significant association of CD4+ T cell tumor infiltration with response (mean of 37.4% CD4 + cells in responders vs 27.0% in nonresponders, p=0.32) was observed.
Conclusion:
We observed strong correlation of pre-dosing intratumoral T cell infiltration with response and mutational load in NSCLC patients treated with pembrolizumab. Our results have direct implications for the design and interpretation of ongoing and planned immunotherapy studies for NSCLC and evaluation of potential predictive biomarkers to select patients most likely to benefit.
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-063 - Exposure to IL-1β Leads to EMT via Distinct Mechanisms in Acute and Chronic Inflammation in NSCLC (ID 3034)
09:30 - 09:30 | Author(s): T. Walser
- Abstract
Background:
Dysregulated inflammation is associated with the development and progression of lung cancer. Pulmonary diseases characterized by increased inflammation, including emphysema and pulmonary fibrosis, are strongly related to heightened risk of lung cancer. Moreover, lung cancer patients with increased levels of inflammatory mediators or inflammatory cells have poor outcomes. It has been shown that dysregulated inflammatory cytokines in the tumor microenvironment can promote cancer metastasis. However, the mechanisms of this effect in lung cancer have not been fully understood. Interleukin 1β (IL-1β), a key pro-inflammatory cytokine, is associated with tumor aggressiveness and poor patient outcomes in NSCLC. Herein, we report that treatment of IL-1β leads to epithelial-to-mesenchymal transition (EMT) in NSCLC cell lines. Delineation of the underlying molecular pathway(s) may potentiate novel therapeutic strategies.
Methods:
We treated NSCLC cell lines with IL-1β acutely (3 days) and chronically (21 days) in vitro and identified EMT mediators using RNA interference and chemical inhibitors. Histone modifications and DNA methylation were analyzed with chemical inhibitors, ChIPassays and methylation-specific PCR. We utilized transwell migration, cell proliferation and anchorage-independent cell growth assays to evaluate the functional phenotypes
Results:
We found that following acute IL-1β exposure (within 7 days), the activator protein 1 (AP-1) transcription factor components, including Fra-1 and c-jun, mediate EMT. AP-1 functions downstream of ERK1/2 and JNK signaling and resides upstream of the transcription factors Slug and Zeb2. Importantly, inhibition of slug, zeb2, fra-1 or ERK1/2 and JNK signaling by RNA interference or chemical inhibitor is sufficient to abolish IL-1β-induced E-cadherin repression. This occurs concomitantly with decreased cell migration and invasion. Surprisingly, following prolonged IL-1β exposure (21 days), cells do not revert back to the epithelial state despite inhibition of these acute EMT mediators. We also found that following withdrawal of IL-1β after twenty one-day exposure, the treated cells are able to maintain their mesenchymal phenotype for more than 30 days before reverting back to an epithelial phenotype. We refer to this prolonged but reversible EMT program that persists in the absence of the original inflammatory stimulus as EMT “memory.” Further studies showed that fra-1 is only required to establish but not to maintain EMT memory. Chemical inhibition of a variety of enzymes involved in histone modifications and DNA methylation indicates the repression of E-cadherin is mediated by different mechanisms depending on the duration of IL-1β exposure. H3K27Me3 and histone acetylation mediate E-cadherin repression during acute EMT but DNA methylation is responsible for the downregulation of E-cadherin in EMT memory. In fact, we have found increased CpG island methylation in the E-cadherin promoter region in EMT memory. In vitro functional studies further showed that EMT memory enables cancer cells to enhance their motility but gradually regain proliferative advantage.
Conclusion:
We conclude that lung cancer cells utilize distinct mechanisms for EMT in response to acute and chronic inflammation. We also demonstrate that dynamic alteration of histone modification and DNA methylation can lead to prolonged but reversible EMT, subsequently creating a time window for cancer cells to migrate to distant organs and eventually undergo mesenchymal-epithelial transition to form macro-metastases.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-063 - The Mutational Landscape of Pulmonary Premalignancy in the Context of Lung Adenocarcinoma (ID 1614)
09:30 - 09:30 | Author(s): T. Walser
- Abstract
Background:
While genomic alterations in lung cancer are being actively investigated, the early mutational events that occur within the pulmonary field of cancerization that subsequently drive early carcinogenesis are poorly understood. As a result, the clinical importance of premalignant lesions remains enigmatic. Epithelial cells in the field of lung injury can give rise to distinct premalignant lesions that may bear unique genetic aberrations. A subset of these lesions may progress to invasive cancer, however the mutational landscape that may predict progression has not been determined. In the present study we performed whole exome DNA sequencing to measure the incidence of somatic DNA alterations in matched sets of primary tumor, premalignant lesions and adjacent normal lung tissues.
Methods:
FFPE tissue blocks from 41 patients were obtained from the UCLA Lung Cancer SPORE Tissue Repository. The following regions were dissected from distal airways utilizing Laser Capture Microdissection: a) normal airway epithelial cells (1-3 regions), b) premalignant atypical adenomatous hyperplasia (AAH, 2-4 regions), c) adenocarcinoma in situ (AIS, 1-3 regions) and, d) adenocarcinoma (ADC, 1-3 regions). DNA was extracted and sequencing libraries were constructed followed by exome capture. Sequencing was performed on an Illumina HiSeq2000 with a mean coverage of ~50x per base.
Results:
Data analysis included analyses for germline and somatic variants, loss of heterozygosity and copy number alternations. Within each case, position-specific missense and nonsense mutations were compared. Different cases were compared for the mutations at a gene-specific level. Mutations found only in AAH lesions were defined as premalignant, in ADC as malignant, and in both AAH and ADC as progression-associated mutations. The analysis demonstrated that AAH lesions from the same patient often have different mutational profiles. We identified novel recurring progression-associated mutations in 33 genes, most of which have not been previously described as key drivers for lung cancer. Interestingly, recurring mutations were found in genes involved in calcium signaling and extracellular matrix/receptor interaction. The data was compared to the TCGA and COSMIC databases. Among affected proteins, only 3% overlapped with the COSMIC and approximately 6% with the TCGA database. Interestingly, all of the mutations overlapping with the COSMIC, were found to be common mutations in AAH. Furthermore, pathways affected by the mutated genes were identified utilizing Gene Ontology and pathways from the KEGG, Biocarta or Reactome databases. The observation that few genes mutated in both AAH and ADC are known as key drivers, indicates that: a) progression-associated mutations might facilitate malignant transformation by mutated key driver(s), or b) a combination of two or more progression-associated mutations that are not oncogenic alone, might drive malignant transformation. These hypotheses will be further tested by mapping progression- and malignant-associated genes in the context of pathways.
Conclusion:
Our data indicate that premalignant lesions from the same patient may have different mutational profiles. This inter-lesion heterogeneity suggests that a progression-associated mutational landscape could be defined in longitudinal studies of pulmonary premalignancy. These results could help identify targets for the development of targeted chemopreventive strategies for lung cancer. Supported by EDRN (U01CA152751-AS).
