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P. Forde
Moderator of
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MINI 02 - Immunotherapy (ID 92)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 14
- Moderators:P. Forde, S.J. Antonia
- Coordinates: 9/07/2015, 10:45 - 12:15, Four Seasons Ballroom F3+F4
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MINI02.01 - Pulmonary Large Cell Carcinoma and Solid Adenocarcinoma Are Highly Mutated with Frequent Expression of PDL1 (ID 2257)
10:45 - 10:50 | Author(s): D.H. Hwang, A. Perry, F. Kuo, S.J. Rodig, L. Sholl, J.L. Hornick
- Abstract
- Presentation
Background:
Large cell carcinoma (LCC) is an uncommon lung tumor that arises predominantly in smokers and shares many features of solid adenocarcinoma (ADC). 40% of LCC/solid ADC harbor mutations in KRAS; EGFR and ALK alterations are rare in this tumor type. The majority of these tumors, however, lack one of the commonly queried oncogenic driver alterations, thus therapeutic options are limited for patients with this tumor type. Immunomodulatory therapies, including targeting PDL1, have shown promise in a variety of tumor types. Tumor neo-antigens, including those induced by smoking, are associated with mutational burden and may predict susceptibility to cytolytic immune response; in addition, high PDL1 expression in non small cell lung carcinoma has been associated with response to anti-PDL1 drugs. Given the high prevalence of smoking in patients with LCC and solid ADC, we hypothesize that these tumors may be amenable to immunomodulatory therapy and sought to define the frequency of PDL1 expression in tumors lacking an oncogenic driver mutation.
Methods:
This study was restricted to 27 LCC and solid ADC known to be negative for KRAS, EGFR, ALK and ROS1 alterations. Hybrid capture targeted next generation sequencing (NGS) on an Illumina HiSeq 2500 was performed using a cancer genomic assay to detect mutations, copy number variations (CNVs) and structural variants. The assay captures exonic sequences of 275 cancer genes and 91 introns across 30 genes for rearrangement detection. Findings were compared to an institutional cohort of 732 consecutive lung tumors sequenced on the same platform. Immunohistochemistry for PDL1 was performed using a rabbit monoclonal antibody (Cell Signaling Technologies) at 1:100 dilution following pretreatment with citrate buffer/pressure cooker and detected using the Envision + polymer system (DAKO). Immunostaining was considered positive in the tumor component or the inflammatory component if ≥5% of the cells showed membranous staining.
Results:
Of the 27 tumors tested, 26 were resected from smokers. NGS revealed an average of 14.9 mutations per case for LCC/solid ADC cohort versus 8.1 mutations in the overall cohort of lung tumors (p<0.0001). 11 cases (41%) were positive for PD-L1. 7 cases (26%) showed strong, diffuse staining (≥70% of cells) for PD-L1. The inflammatory component was positive for PD-L1 in 25 cases (93%). Two cases with strong expression of PD-L1 by immunohistochemistry (>90% of cells) showed focal amplification of CD274 by NGS.
Conclusion:
LCC and solid ADC are strongly associated with a smoking history and harbor a significantly higher average mutational burden than other lung tumors. 41% of LCC/solid ADC are positive for PDL1 by immunohistochemistry with 26% showing very strong PDL1 expression and nearly all cases showing some degree of positivity in the associated inflammatory infiltrate. In some cases, high PDL1 expression is associated with focal amplification of CD274, the gene encoding PDL1. These findings suggest that LCC/ solid ADC is likely to have smoking-associated neo-antigen expression and that PDL1-directed immunotherapies may be a promising therapeutic approach in this otherwise poorly-characterized lung tumor.
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MINI02.02 - Programmed Cell Death Ligand (PD-L1) Expression in Stage II and III Lung Adenocarcinomas and Nodal Metastases (ID 1519)
10:50 - 10:55 | Author(s): H. Uruga, E. Bozkurtlar, T. Huynh, A. Muzikansky, A. Hata, J.F. Gainor, E. Mark, J.A. Engelman, M. Lanuti, M. Mino-Kenudson
- Abstract
Background:
Inhibition of PD-L1 can lead to reactivation of tumor immunity and assist in cancer therapy. PD-L1 expression in tumor cells has been reported to correlate with clinicopathological parameters and prognosis in a variety of cancers including lung adenocarcinomas (ADC). However, it has not been well studied whether PD-L1 expression is altered along with tumor progression. In addition, little is known about the role of PD-L1 expression in predicting response to chemotherapy in ADC. Thus, we sought to compare PD-L1 expression in the main tumor and lymph node metastases of stage II and III ADC, and correlate PD-L1 expression with survival in patients who underwent adjuvant chemotherapy.
Methods:
The study cohort consisted of 109 ADC who underwent curative resection without neoadjuvant therapy and were diagnosed to have stage II or III disease. Of those, 60 cases received platinum-based adjuvant therapy and were followed at our institution. Immunohistochemistry for PD-L1 (E1L3N, 1:200, CST) was performed on sections of the primary tumor and/or metastatic lymph nodes and the primary tumor sections were also stained with CD8 (4B11, RTU, Leica Bond). Membranous staining of any intensity present in 5% or more of the tumor cells was deemed positive for PD-L1 expression. CD8+ tumor infiltrating lymphocytes (TILs) were evaluated using a 4-tier grading system (0-3). The PD-L1 expression in the primary tumor was correlated with that in lymph node metastases as well as clinicopathological parameters, including CD8+ TILs, and recurrence free survival (RFS).
Results:
Of the 109 cases, 53 (48.6 %) exhibited PD-L1 expression in the primary tumor, which was significantly associated with smaller tumor size, lower pT stage, nodal disease, solid-predominant pattern, the presence of tumor islands, necrosis and lymphovascular invasion, and increased CD8+ TILs (grade 2-3). Upon multivariate analysis, only increased CD8+ TILs remained significant (p=0.039). As for the primary – lymph node correlation, PD-L1 expression was seen in 57.6% of 59 N1 nodes, 53.1% of 32 N2 nodes, and 100% of one N3 node available for evaluation. The PD-L1 expression status was the same between the primary tumor and nodal metastases in the majority (76.3 % of N1 nodes, and 75.0% of N2; p<0.001 and p=0.005, respectively), and the upregulation of PD-L1 expression (positive expression was present in nodal metastasis with negative primary) was seen in only small fractions of the cohort (6.8% of N1 nodes and 9.3% of L2 nodes). Interestingly, PD-L1 expression in the primary tumor was associated with longer RFS in patients who underwent platinum-based adjuvant therapy (mean 84 months vs. 41 months in PD-L1 negative patients, p=0.016), but not in those without adjuvant therapy.
Conclusion:
PD-L1 expression in the primary tumor was associated with prominent CD8+ TILs as well as several adverse clinicopathological parameters including nodal disease, but PD-L1 expression in the nodal metastasis was similar to that in the primary tumor in the majority of cases. Although the evaluation was limited due to a small size of the cohort, PD-L1 expression in the primary tumor appears to be predictive of response to platinum-based adjuvant therapy.
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MINI02.03 - Overexpression of CD47, Decrease of Apoptosis and Phagocytosis of Neutrophils in Advanced Non-Small Cell Lung Cancer Patients (ID 2265)
10:55 - 11:00 | Author(s): L. Barrera, O. Arrieta Rodriguez, R. Morales-Flores, A. Garcia-Vicente, E. Montes-Servín, F. Salinas-Parra, A. Ramirez-Tirado
- Abstract
- Presentation
Background:
Lung cancer is the leading cause of cancer death worldwide and most of the patients are diagnosed with advanced disease. Inflammatory components play a key role in tumor progression and survival. Neutrophils are increased in blood of patients with lung cancer and they are associated with poor clinical outcomes. CD47 is a protein which control cell communication, apoptosis, adhesion and proliferation and it has been found increased in cancer and related with phagocytosis evasion mechanism.The aim of this study was to evaluate CD47 expression levels on peripheral neutrophils, also assess the phenotype, apoptosis, activation state, reactive oxygen species production of neutrophils between patients with Non-Small Cell Lung Cancer (NSCLC) and healthy subjects.
Methods:
Fifty NSCLC patients (stage IIIB and IV) naive to treatment and 25 healthy subjects were analized for: CD47 peripheral blood expression, neutrophils phenotype and activation state, evaluation of apoptosis and phagocytosis by flow cytometry. Reactive oxygen species (ROS) production by circulating neutrophils upon stimulation with PMA was assessed by flow cytometry. For the phagocytosis assay, PMNC were labeled with CMFDA and were cultured in RPMI for 24 hrs. To obtain apoptotic target cells, 24h PMNC were labeled with Annexin-V. For the evaluation of phagocytosis, the neutrophils from NSCLC patients were co-cultured with THP-1 cells. The percentage of phagocytosis was assessed by flow cytometry.
Results:
Our results showed a lower percentage of total CD47 in peripheral blood cells in NSCLC patients compared to controls [P=0.042]. Mean Fluorescence Intensity (MFI) of CD47 was higher in patients [P<0.001]. The percentage of CD66b+ cells characterized as neutrophils was higher in patients as well as their MFI of CD47 [P< 0.001]. MFI of CD66b was higher in patients [P< 0.0178]. This would be related with a more activated state. We found that a higher disease stage (IIIB vs. IV) associated with a higher MFI of CD47 [P=0.020]. Plasma pro-inflammatory cytokines, was increased in patients compare to controls IL-6 (P<0.002), IL-8 (P<0.001), IL-12p70 (P<0.008), TNF (0.010) and IFN-g (P<0.001). MFI of CD47 >1635.5 was associated with a higher median Overall survival (P= 0.007). We found a decrease of AnnexinV+/7AAD+ in neutrophils of patients [P=0.0317]. Caspases 3 and 7 were found decreased in neutrophils of patients [P= 0.049]. Oxygen species (ROS) production of neutrophils upon PMA stimulation was increased in patients [P=0.029], suggesting it might play a role in immune effector function. Phagocytosis of apoptotic neutrophils by differentiated THP-1 cells was decrease in cancer patients cells (P=0.0445). Mean fluorescence Intensity of CD47 was increased after 24 hrs in patients [P=0.0408]. This result suggests that neutrophils from patients avoid being engulfed and this may be associated with overexpression of CD47.
Conclusion:
Taken together, these findings suggest that these are altered mechanisms by which neutrophils evade anti-tumor immune response and their increased expression of CD47 is a potential therapeutic target for NSCLC.
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MINI02.04 - Sequential Assessment of DNA Damage Response and PD-L1 Expression in Circulating Tumor Cells of Lung Cancer Patients during Radiotherapy (ID 2511)
11:00 - 11:05 | Author(s): S.H. Lin, J. He, M. Edelman, T. Xu, H. Gao, J. Reuben, Y. Qiao, H. Liu, P. Amstutz, S. Hahn, R.U. Komaki, Z. Liao, C. Tang, D. Adams
- Abstract
- Presentation
Background:
Recent evidence suggests that PD-L1 expression can be induced with radiotherapy and may be a mechanism for resistance to radiotherapy and immunotherapy. Sequentially assessing PD-L1 expression on cancer associated cells in circulation during treatment regimens may be a way to assess the efficacy of radiotherapy and immunotherapy in clinical trials. For this feasibility study, we evaluated the association of RAD50 induction, and PD-L1 expression, on CTCs and Cancer Associated Macrophage-Like Cells (CAMLs) in lung cancer patients (pts) before and during radiotherapy to determine expression changes of these markers.
Methods:
Eleven pts with stage I-IV lung cancer were included in this pilot study. Three pts received Stereotactic Body Radiation Therapy (SBRT) for stage I disease and 8 other pts received chemoradiation for stage II-IV disease. Baseline blood samples (7.5 ml) were drawn prior to the start of radiotherapy (T0) and a second blood sample was drawn at a follow up visit during radiotherapy; or for three pts, after completing SBRT (T1); for a total of 22 samples. Blood was processed using CellSieve™ microfiltration (Creatv Microtech), stained for cytokeratin 8, 18 & 19 and CD45, and imaged. Using the QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique to remove fluoresce signal, all cells were restained for RAD50-AlexaFluor550 and PD-L1-AlexaFluor 488, along with DAPI nuclear stain. The RAD50 foci numbers within nuclear regions were quantified. PD-L1 pixel intensity was measured by the ZenBlue software and grouped into 4 IHC groups: 0-negative (pixel average 0-215), 1-low (pixel average 216-300), 2-medium (pixel average 301-750), and 3-high (pixel average 751+).
Results:
There was at least one cytokeratin positive cell (i.e. CTC or CAMLs) found in each of the samples. Specifically CTCs were found in 82% of T0 and 64% of T1 samples, and CAMLs were found in 91% of T0 and 100% of T1 samples. RAD50 foci ranged from 0-16 per cell, with an average of 0.69 at T0 that increased to 3.46 at T1 (p=0.002) during radiotherapy. Distinctively, there were 6 pts with greater than 2 fold RAD50 foci increase at T1 and 5 pts with ≤ 2 fold induction. PD-L1 expression ranged from 34-2004 pixel intensity, with an average of 170 at T0 and 336 at T1 (p=0.08). Interestingly, 4 pts had no PD-L1 expression at T0 but an increase to 2 to 3+ at T1, 4 pts with low/no PD-L1 expression remained low at T1, and 3 pts had high PD-L1 expression that remained high or decreased at T1. There was no correlation between RAD50 induction and PD-L1 expression.
Conclusion:
Both RAD50 foci and PD-L1 expression were quantifiable in both CTCs and CAMLs, and had variable responses to radiotherapy +/- chemotherapy. These data suggest that sequential tracking of CTCs or immune-related cells from the primary lung tumor is feasible using microfiltration and potentially can serve as predictive biomarkers for cancer therapy.
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MINI02.05 - Discussant for MINI02.01, MINI02.02, MINI02.03, MINI02.04 (ID 3299)
11:05 - 11:15 | Author(s): D.P. Carbone
- Abstract
- Presentation
Abstract not provided
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MINI02.06 - Humanized Immuno-Mouse for Study of Anti-PD-1 Therapy in KRAS-Mutated Lung Cancer Patient Derived Xenotransplant (PDX) (ID 3104)
11:15 - 11:20 | Author(s): M. Wang, J.W. Riess, J. Keck, K. Palucka, L. Shultz, M. Cheng, D. Cai, C. Bult, D.R. Gandara, P.C. Mack
- Abstract
- Presentation
Background:
Preclinical modeling of immunotherapeutics in PDX-bearing mice has been limited by the absence of a relevant immune microenvironment, as a highly immunosuppressive environment is often required for the implanted tumor to grow. Checkpoint inhibitors including anti-PD-1 and anti-PD-L1 antibodies (mAbs) are promising new treatments in non-small cell lung cancer (NSCLC). The creation of a PDX model system that supports human tumor growth and recapitulates the relevant genomics in NSCLC while providing the immune microenvironment necessary for anti-PD-1 and anti-PD-L1 mAb activity is critical for validation of combination checkpoint inhibitor strategies in NSCLC.
Methods:
Hematopoietic CD34+ progenitor stem cells (CD34+ HPC) were engrafted into the tail vein of sublethally irradiated NSG mice. A KRAS G12D PDX was assayed for PD-L1 expression by FACS (Biolegend; clone 29E. 2A3, San Diego CA) and implanted into Hu-CD34 NSG mice with > 25% Hu-CD45+ cells 12 weeks post CD34+ HPC injection. Multilineage engraftment of immune cell subsets was assayed in peripheral blood, spleen and tumor by FACS (CD45, CD3, CD4, CD8, CD19). PDX were treated with vehicle Q5D x 6, pembrolizumab (Merck, Whitehorse Station PA) 5 mg/kg Q5D x 6, and combination pembrolizumab and docetaxel (Hospira, Lake Forest) 10 mg/kg Q7D x4 at the same single agent dosages. Body weight and tumor growth were assessed twice weekly.
Results:
Hu-CD45+ cells were detected in peripheral blood, spleen and tumor by flow cytometry on single cell suspension. The majority of Hu-CD45+ cells were T-cells: CD3CD4+ (mean blood 50%, spleen 53%, tumor 52%) and CD3CD8+ (mean blood 14%, spleen 15%, tumor 39%). KRAS G12D tumor had 89% surface expression of PD-L1. No significant change in Hu-CD45+ cell composition was noted between the different treatment groups. Pembrolizumab both alone and in combination with docetaxel showed activity in KRAS G12D PDX with substantial tumor growth inhibition and decreased mean tumor volume at day 24 post-treatment.
Conclusion:
Multilineage engraftment of relevant immune cell subsets for PD-1 inhibition is present in the humanized immune-mouse (Hu-CD34 NSG). PD-1 inhibition in a KRAS G12D Hu-CD34 NSG with high PD-L1 expression demonstrated substantial tumor growth inhibition both alone and in combination with chemotherapy. Additional studies are underway exploiting the Hu-CD34 NSG mouse model for study of anti-PD-1/PD-L1 therapies in KRAS mutant and other important molecular subsets of NSCLC.
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MINI02.07 - Preclinical Rationale for a Phase I/II Study of Pembrolizumab (P) and Vorinostat (V) in Immune Therapy Naïve and Pretreated Stage IV NSCLC (ID 734)
11:20 - 11:25 | Author(s): N. Tchekmedyian, H. Zheng, A.A. Beg, E. Haura, D. Chen, S.J. Antonia, J. Gray
- Abstract
- Presentation
Background:
The WHO estimated that 1.6 million people died of lung cancer in 2012. Nivolumab, an anti-PD-1 immune checkpoint inhibitor, was FDA approved on March 4, 2015 for platinum-refractory, metastatic, squamous-cell NSCLC, based upon a RR to single agent nivolumab of ~15% and improved OS. Combinatorial strategies may enhance these outcomes. Increased tumor expression of T cell chemokines, such as CCL5 and CXCL10, is associated with a better response to immunotherapy. Furthermore, expression of T cell chemokines is strongly and positively associated with increased T cell infiltration and improved patient survival. Therefore, enhancement of expression of T cell chemokines may augment response to PD-1 blockade immunotherapy.
Methods:
FDA-approved oncology agents were utilized from the Approved Oncology Drugs Set (97 agents) from the Developmental Therapeutics program of NCI. LKR cells were plated in 96-well plates, and a viability assay was performed 48 hours after drug administration (Cell Counting Kit-8, Dojindo Laboratories). Mice were bred and housed in the animal facility at Moffitt Cancer Center. Cells were harvested in logarithmic growth phase after being cultured for less than 2 weeks. 1x10[6] LKR or 344SQ cells were injected s.c. and tumors were monitored for growth by measurements 2-3 times per week. Romidepsin was injected i.p. (2mg/kg) on days 14,16, and 18 after tumor inoculation. Anti-PD-1 was injected i.p. (300μg/mouse) on days 15, 17, and 19 after tumor inoculation. Relative tumor size between treatment groups was analyzed using the t test with Welch’s correction.
Results:
Histone deacetylase inhibitors (HDACi), including vorinostat, emerged as the only class of agents in a 97-drug screen capable of inducing expression of multiple T cell chemokines, including CCL5, CXCL9, and CXCL10, in mouse and human lung cancer cell lines and primary tumors. HDACi’s ability to induce T cell chemokine expression was dependent on both JAK-STAT and NF-kB pathways. HDACi (romidepsin) treatment of mice bearing LKR tumors did not substantially cause tumor shrinkage but significantly reduced growth (p<0.0001; final tumor volume). This effect of HDACi was completely T cell dependent. LKR tumor cells had low cell surface expression of PD-L1 but which was substantially increased by IFN-g. PD-1 blockade with mAb reduced tumor growth but rarely induced rejection. However, when PD-1 blockade was combined with HDACi, 9 out of 11 mice demonstrated complete tumor rejection. HDACi anti-tumor response correlated with T cell chemokine induction in tumors and greater presence of tumor-infiltrating lymphocytes (TILs). We next used a mouse tumor model (344SQ) that was relatively resistant to anti-PD-1 treatment. PD-1 blockade combined with HDACi significantly reduced growth of these tumors compared to untreated (p=0.0003), anti-PD-1 alone (p=0.01), or HDACi (p=0.004) alone treated mice.
Conclusion:
HDACi not only enhanced anti-tumor response against PD-1 blockade sensitive tumors (LKR), but also induced response against PD-1 blockade resistant tumors (344SQ). HDACi induces JAK-STAT and NF-kB dependent chemokine expression and may induce tumor-infiltrating lymphocytes. Thus, a Phase I/randomized Phase II clinical trial of vorinostat, an orally active, small molecule HDACi, plus pembrolizumab, an anti-PD-1 humanized monoclonal IgG4-kappa antibody, is planned in patients with immune therapy naïve and pre-treated metastatic NSCLC.
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MINI02.08 - Bidirectional Cross-Talk between CD14+ Monocytes and Human Lung Cancer Cell Lines Results in Different Phenotypic and Functional Outcomes (ID 2497)
11:25 - 11:30 | Author(s): E. Schenk, A. Mansfield, A. Dietz
- Abstract
- Presentation
Background:
Myeloid cell infiltration of the tumor microenvironment is associated with decreased overall survival in multiple tumor types, including lung cancer. This myeloid cell infiltration represents a tissue component of the heterogeneous group of cells termed myeloid derived suppressor cells (MDSC), which can inhibit the endogenous anti-tumor response, direct angiogenesis, and promote tumor progression. In several studies of patients with non-small cell lung cancer (NSCLC), there is wide variation in the presence of myeloid cells in the tumor with increasing levels peripheral blood MDSC associated with poor survival. We have previously shown that CD14+ monocytes can be converted by the tumor microenvironment to an immune suppressive phenotype in non-Hodgkin lymphoma and glioblastoma. In this work, we expand on our earlier observations to include recruitment of CD14+ cells by lung cancer cell lines and their conversion to an immune suppressive phenotype. While most models of myeloid cells in the microenvironment describe the effects of these cells on non-malignant systems, we show that myeloid cells may have profound direct effects on the tumor.
Methods:
Human lung cancer cell lines were cultured and supernatants collected for ELISA. CD14+ cells were isolated from the peripheral blood of healthy volunteers using anti-CD14 immunomagnetic beads. Lung cancer cell lines and CD14+ cells were cocultured under a variety of low serum conditions with or without cisplatin. Changes in CD14+ cell HLA-DR expression and tumor cell survival were measured by flow cytometry. CD14+ cell migration through a permeable transwell membrane was measured in real time with live cell imaging.
Results:
Under normal culture conditions, 7 of 8 human lung cancer cell lines secreted detectable levels of CCL2, a major chemoattractant for monocytes, ranging from 30 to 10,000 pg/ml of CCL2 found in culture supernatants. CD14+ cells more robustly migrated towards cell lines with higher production of CCL2. The coculture system showed a differential impact on monocytes by the tested lung cancer cell lines which either reliably upregulated or downregulated CD14+ cell expression of HLA-DR. In 3 of 8 lung cancer cell lines, CD14+ cell HLA-DR was downregulated in a manner expected to promote local immune suppression. Under serum starvation conditions, one lung cancer cell line showed improved survival when cocultured with CD14+ cells. Similarly, coculture with CD14+ cells enhanced tumor survival of two cell lines after exposure to cisplatin.
Conclusion:
The studied lung cancer cell lines differ in the degree of CD14+ cell recruitment, CD14+ cell HLA-DR expression after coculture, and level of conferred survival benefit under stressful conditions. Taken together these results suggest that the variable myeloid involvement in lung cancer patients can be modeled using lung cancer lines. In addition, we have identified that for some tumors, monocytes confer a significant survival advantage that is not associated with immune or angiogenic responses. Future work is needed to explore the impact of CD14+ cells on lung tumor invasiveness, angiogenesis, and the mechanisms underlying these pro-tumor effects.
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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): M. Lee, J. Yanagawa, S. Wu, T. Walser, G. Wang, J.W. Goldman, E.B. Garon, G. Zeng, S. Sharma, J. Minna, D.P. Carbone, S.M. Dubinett, J.M. Lee
- 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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MINI02.10 - Discussant for MINI02.06, MINI02.07, MINI02.08, MINI02.09 (ID 3300)
11:35 - 11:45 | Author(s): I.I. Wistuba
- Abstract
- Presentation
Abstract not provided
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- Abstract
- Presentation
Background:
As one of the novel therapy strategies, PD-L1 has been shown the function of down-regulating T-cell activation through receptor PD-1. Moreover, prognosis of cancer patients are based not only on tumor-related factors but also on host-related factors, particularly systemic inflammatory response. As significant indicators of patients’ inflammation status, circulating monocyte count, neutrophil ratio and lymphocyte ratio were proved as predictors of prognosis in various cancers. Squamous non-small cell lung cancer (NSCLC) revealed to be divergent clinical and molecular phenotypes compared with non-squamous NSCLC. Significantly, combining application of appropriate biomarkers in prognosis prediction is emerging its high importance in cancer research.
Methods:
Chart review was performed on 1286 consecutive patients, 156 of these patients were enrolled in the final analysis. Patients with squamous NCSLC were randomly assigned (2:1) centrally by computer into training group and validation group. Monocyte ratio, Neutrophils to Lymphocytes Ratio, PD-L1 immunostaining score and PD-1-positive stained tumor-infiltrating lymphocytes counts were assessed by Fisher’s linear discriminant analysis to discriminate if OS would exceeding 5 years. The final model was used to calculate the discriminant score in each study participant. And this prediction model was validated in a second set of squamous NCSLC patients. We internally validated the model using a cross-validation procedure.
Results:
4 independent predictors of OS were identified by using FLDA with stepwise variant-selection. The clinical classifying model was described by the following equation: Y = −1.212 + 0.211 × NLR ratio + 0.437 × monocyte ratio - 0.390 × PD-L1 + 0.035 × PD-1 (eigenvalue 0.673, canonical correlation 0.634, P < 0.001). In this equation, PD-L1 represented PD-L1 immunostaining score; and PD-1 represented PD-1 positive TILs counts. For the training set of 104 leave-one-out-cross-validated cases, 27 of 29 OS > 5 years (93.1% sensitivity) and 61 of 75 OS <= 5 years (81.3% specificity) were correctly classified with an overall accuracy of 84.6% (88 of 104) and an AUC of 0.938 [P < 0.001, 95% confidence interval (CI) 0.864–1] Next, the predicting model consisting of the 4 predictors (NLR ratio, monocyte ratio, PD-L1 and PD-1) were applied to the validation set of 52 patients (14 OS > 5 years and 38 OS <= 5 years). A survival prediction for 38 of the 52 patients (73.1%) with an AUC of 0.908 (P < 0.001, 95% CI 0.806–1) was achieved. Also, 12 of 14 OS > 5 years (85.7% sensitivity) and 26 of 38 OS <= 5 years (68.4% specificity) were correctly identified.
Conclusion:
The analysis of a set of immunological markers could effectively and reproducibly classify patients with squamous NCSLC according to their overall survival. Further prospective validation in larger independent cohorts of patients with similar or different regimens is warranted to fully assess its predictive power. The 4-immunological-marker model offers a novel tool for survival prediction and could have important clinical implications for the consideration of differential treatment strategies in patients with squamous NCSLC, thus providing a framework for future individualized therapy.
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MINI02.12 - Distribution of Immune Markers and Their Association with Overall Survival and Time to Progression in Non-Small-Cell Lung Cancer (NSCLC) (ID 3108)
11:50 - 11:55 | Author(s): A.T. Kuykendall, F. Khalil, E. Haura, S.J. Antonia, M. Schabath, D. Gabrilovich, B. Creelan
- Abstract
- Presentation
Background:
Inducible nitric oxide synthase (iNOS) and reactive nitrosylation are important mediators of tumor immunosuppression by myeloid-derived suppressor cells (MDSCs). However, the role of CD33+ peritumoral PMN-MDSCs in these pathways remains unclear. We conducted a retrospective cohort study of NSCLC subjects treated with surgery, with the primary objective to determine the association of MDSC biomarkers with time to progression (TTP) and overall survival (OS).
Methods:
Inclusion criteria: Surgically treated NSCLC of all stages at a single institution between 1996 and 2010. Somatic mutations tested by PCR. Anti-human antibodies optimized for immunohistochemistry. Samples scored by blinded pathologist based on intensity and percentage of peritumoral cells. Peritumoral nitrotyrosine (NT) and iNOS used Allred scoring. Time to progression (TTP) defined as time from resection to progression event or censored at last evaluation. Overall survival (OS) defined as time from resection to death.
Results:
Of 458 tumor samples, 366 lung primaries, 38 soft tissue metastases, and 39 brain metastases. Demographics: median age 67 yrs, 54% female, 96% Caucasian. Of 151 tested for somatic mutations, 36% KRASm, 8.6% EGFRm, 25% p53m, respectively. Histology: adenocarcinoma 76%, squamous 10%. Higher % CD3+ tumor infiltrating lymphocytes (TILs) and CD33+ myeloid cells were observed in tumors than normal tissue (p < .0001 and p = .002, respectively). More CD3+ TILs observed in soft tissue metastases than primary lung tumors (p < .0001). No difference in iNOS expression between tumor and normal lung tissue. More CD3+ TIL was observed in p53 mutant tumors (p=.03). iNOS was positively correlated with CD3+ TIL (p < .001) and CD73+ epithelial cells (p <.001), but not CD33+ myeloid cells. NT expression correlated with the absence of CD3+ TIL (p = .02), consistent with its putative immunosuppressive activity. Median TTP: 10.4 months; 320 (69.7%) events. Median OS: 35.4 months; 353 (77.1%) events. Expectedly, presence of CD3+ TIL was associated with favorable OS; HR 0.5 [0.4 – 0.7], p < .0001, and TTP; HR 0.7 [.5 – .9], p =.009. CD33+ myeloid cells were associated with favorable OS; HR 0.6 [0.5-0.8], p = .0002. Presence of peritumoral iNOS trended toward favorable OS; HR 0.81 [0.6-1.0], p = .07. Peritumoral iNOS was not associated with TTP. Figure 1
Conclusion:
Increased presence of TILs in p53 mutant tumors has been reported in other cancers, and may be related to somatic mutational load. An inflamed tumor phenotype was associated with improved overall survival. Unexpectedly, iNOS was positively correlated with both CD3+ infiltration and overall survival.
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- Abstract
- Presentation
Background:
Cancer development and biology is influenced by the host immune system. Emerging data indicate that the context of immune cell infiltrates within the tumor is associated with cancer prognosis. Both the activation state and type of immune cells present can provide mediators that either promote or inhibit tumor growth. While the presence of activated cytotoxic T lymphocytes (CTL) may correlate with better patient survival, the presence of tumor associated macrophages and effector lymphocytes that lack cytotoxic properties may promote tumor growth. Thus, in established tumors, a balance between pro and anti-tumor mediators are present, but as advanced tumors rarely regress without therapeutic intervention, this balance is likely skewed towards tumor-promoting inflammation. In attempts to gain insight into the immune networks that regulate tumorigenesis, we used genome wide gene expression datasets of primary lung cancer tissues to identify and functionally validate immune related genes that are associated with patient survival.
Methods:
Gene expression analysis was conducted on microarray datasets from 128 early-stage NSCLC resected tumor samples. Limiting analysis to immune-related gene sets curated by NIAID ImmPortal, we identified a minimum gene set using MAximizing R Square Algorithm (MARSA) that selected for the greatest separation between good and poor prognostic patient subgroups. The prognostic value of this gene signature was validated in nine additional independently published microarray datasets of NSCLC. From the gene signature, we functionally characterized the potential role of the soluble protein LAIR2 in immune regulation of lung cancer.
Results:
We identify a 9-gene signature that separate patients into high and low-risk subgroups for 5-year cancer-free survival (hazard ratio 10.26, 95% confidence interval 4.32-24.34, p<0.0001). The prognostic accuracy of this signature was validated in additional NSCLC datasets (total 1095 patients without adjuvant treatment). Amongst the 9-genes, the gene encoding the soluble protein LAIR2 was highly expressed within the high-risk patient subgroup. Functionally, we found that addition of recombinant LAIR2 resulted in increased NK cell expression of cytotoxicity receptors and secretion of pro-inflammatory cytokines in the presence of lung cancer cell lines.
Conclusion:
By limiting gene expression analysis to immune related genes, we identify a 9-gene prognostic immune signature in resected early stage NSCLC patients. The signature identifies a role for the soluble protein LAIR2 in the modulation of immune cell activation during lung cancer development and may suggest that LAIR2 induce a pro-inflammatory microenvironment which promote tumorigenesis and poor patient outcome.
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MINI02.14 - Discussant for MINI02.11, MINI02.12, MINI02.13 (ID 3469)
12:00 - 12:10 | Author(s): V. Papadimitrakopoulou
- Abstract
- Presentation
Abstract not provided
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MS 04 - Harnessing the Full Potential of the Immune System (ID 22)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 4
- Moderators:C. Butts, P. Forde
- Coordinates: 9/07/2015, 14:15 - 15:45, Four Seasons Ballroom F3+F4
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MS04.01 - PD1/PDL1 Studies (ID 1860)
14:20 - 14:40 | Author(s): R. Herbst
- Abstract
- Presentation
Abstract not provided
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MS04.02 - Other Immune Inhibitors (ID 1861)
14:40 - 15:00 | Author(s): N.A. Rizvi
- Abstract
- Presentation
Abstract not provided
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MS04.03 - Vaccines (ID 1862)
15:00 - 15:20 | Author(s): J. Vansteenkiste
- Abstract
- Presentation
Abstract:
Cancer immunotherapy in a broad sense is any interaction with the immune system to treat cancer. One approach is non-antigen-specific modulation of the immune system. Historical examples with e.g. BCG, interferon, interleukins, were disappointing in lung cancer. More recently, specific antibodies against the Programmed Death 1 (PD-1) receptor or its ligands (PD-L1) have delivered exciting results, with major patient benefits in randomised controlled trials (RCTs) in relapsing NSCLC {Brahmer, 2015 19949 /id}. Antigen-specific immunotherapy aims at specific priming of immune system to recognize the tumour as foreign, thereby generating specific antibodies and/or cytotoxic T cells. This is “therapeutic cancer vaccination (TCV)”. Conditions for optimal TCV are: 1/ specificity (well-defined target antigen(s) in the tumour, not in other tissues); 2/ selectivity (use in the population expressing the target); 3/ immunogenicity (interaction with antigen leads to effective humoral and/or cellular response); 4/ tumour sensitive to immune kill in order to obtain improvement in patients’ outcome. Better knowledge of tumour immunity has led to encouraging data in phase II RCTs with several TCVs, which then have entered large phase III trials. Examples are the MAGE-A3 vaccine studied in resected early stage NSCLC, the BLP-25 vaccine in locally advanced NSCLC after chemoradiotherapy, and e.g. belagenpumatucel-L and the TG4010 vaccine in advanced stage NSCLC. The MAGE-A3 protein is totally tumour-specific and present in about 35% of early stage NSCLC. In the hypothesis generating double-blind, randomized, placebo-controlled phase II study, 182 patients with completely resected MAGE-A3-positive stage IB-II NSCLC received recombinant MAGE-A3 protein combined with an immunostimulant (13 doses over 27 months) or placebo (2). No significant toxicity was observed. There was a 24% – non-significant – improvement in disease-free survival (DFS, HR 0.76; 95% CI 0.48 to 1.21). The ensuing large phase III study MAGRIT (MAGE-A3 as Adjuvant Non-Small Cell LunG cancer ImmunoTherapy) was reported at the ESMO 2014 meeting (3). MAGE-A3 positive patients with completely resected stage IB-II-IIIA NSCLC and adjuvant chemotherapy as clinically indicated, were randomly 2:1 assigned to receive MAGE-A3 vaccine or placebo. Almost 14,000 surgical patients were screened, 4210 patients were MAGE-A3 positive (33%), and 2312 patients were randomised. The median DFS (primary endpoint) was slightly better with MAGE-A3 (60.5 versus 57.9 months), but the difference was unfortunately not significant (Hazard Ratio, HR, 1.02, 95%CI: 0.89, 1.18, P=0.74). No subgroups with potential benefit could be identified. Based on this disappointing result, further development of the MAGE-A3 vaccine in NSCLC has been abandoned. Mucins like the MUC1 protein are present in many epithelia, but MUC1 expression is altered (mainly by aberrant glycosylation) in many cancer types, including NSCLC. The tandem repeat MUC1-peptide liposomal vaccine BLP-25 has been studied in patients with stage IIIB-IV NSCLC (4). While overall survival (OS) was not significantly different in the total group, a challenging effect was observed in stage IIIB patients (HR 0.524; 95%CI 0.261-1.052). This led to START (Stimulating Targeted Antigenic Responses to NSCLC Trial), a phase III, double blind, RCT comparing maintenance therapy with Tecemotide (n=829) or placebo (n=410) in patients with unresectable stage III NSCLC who did not progress after sequential or concurrent chemo-radiotherapy (5). The primary endpoint – OS – was not significantly different between the vaccine and placebo group (25.6 and 22.3 months). However, pre-planned subgroup analysis showed that the patients treated with concurrent chemoradiotherapy (N=829) had a 10.2-month improvement in OS (30.8 versus 20.6 months, adjusted HR 0.78, P=0.016). The consequential trial was START 2, a similar large RCT in patients who completed concurrent chemoradiotherapy for unresectable stage III NSCLC (NCT02049151). However, this RCT and further development of Tecemotide was abandoned after disappointing results of a smaller trial in Japanese patients with stage III NSCLC and concurrent chemoradiotherapy. Belagenpumatucel-L is a vaccine based on a mixture of allogeneic tumour cells with TGF-β2 antisense blockade as adjuvant. A phase III trial in patients with stage III-IV NSCLC in disease control after first-line therapy was reported at the 2013 ESMO meeting (STOP, NCT00676507) (6). Patients without progression after 1[st] line chemotherapy, were randomly assigned to intradermal belagenpumatucel-L (N=270) versus placebo (N=262)for 24 months. Median OS was 20.3 months with belagenpumatucel-L versus 17.8 months with placebo (HR 0.94, p=0.594). In subgroup analysis of patients randomized <12 weeks after the last chemotherapy, the HR of the median OS was 0.77 (P=0.092). For patients enrolled within 12 weeks and treated with previous radiotherapy, the HR was HR 0.45 (P=0.014). The vaccine was well tolerated with mainly mild local administration side-effects. TG4010 is a vaccine based on a recombinant viral vector (attenuated strain of vaccinia virus) expressing both the tumour-associated antigen MUC1 and interleukin-2. This vaccine is explored in the phase IIB/III RCT TIME trial (NCT01383148). This double-blind, placebo-controlled trial evaluates standard first-line chemotherapy with or without TG4010 in MUC1-positive stage IV NSCLC patients. In the phase IIB part, the predictive value of activated NKs (TrPAL: triple positive activated lymphocytes) was evaluated based on a PFS endpoint, and reported in an interim report at the 2014 ESMO meeting (7). Based on a Bayesian analysis, the predefined endpoint of a HR <1 in the patients with low level of NK cells was met. The PFS was not significantly different between vaccine and placebo (HR 0.78, 95%CI 0.55-1.10]. In subgroup analyses, the effect was more pronounced in patients with non-squamous NSCLC (HR 0.71, 95CI 0.51-0.97) than in squamous histology. Therefore, a decision was made to continue the phase III part of the trial in non-squamous NSCLC only, with OS a the primary endpoint. References 1. Brahmer J, Reckamp KL, Baas P et al. Nivolumab versus docetaxel in advanced squamous cell non-small cell lung cancer. N Engl J Med 2015; on-line May 31. 2. Vansteenkiste J, Zielinski M, Linder A et al. Adjuvant MAGE-A3 immunotherapy in resected non-small cell lung cancer: Phase II randomized study results. J Clin Oncol 2013;31:2396-2403. 3. Vansteenkiste JF, Cho BC, Vanakesa T et al. MAGRIT, a double-blind, randomized, placebo-controlled phase III study to assess the efficacy of the recMAGE-A3 + AS15 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small cell lung cancer (NSCLC). Ann Oncol 2014; 25 Suppl 4: abstract 1173O. 4. Butts C, Murray N, Maksymiuk A et al. Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small cell lung cancer. J Clin Oncol 2005;23:6674-6681. 5. Butts C, Socinski MA, Mitchell PL et al. Tecemotide (L-BLP25) versus placebo after chemoradiotherapy for stage III non-small-cell lung cancer (START): A randomised, double-blind, phase 3 trial. Lancet Oncol 2014;15:59-68. 6. Giaccone G, Bazhenova L, Nemunaitis J et al. A phase III study of belagenpumatucel-L therapeutic tumor cell vaccine for non-small cell lung cancer (NSCLC). Eur J Cancer 2013; 47 Suppl 2: abstract LBA 7081. 7. Quoix E, Losonczy G, Forget F et al. TIME, a phase 2B/3 study evaluating TG4010 in combination with first-line therapy in advanced non-small lung cancer (NSCLC). Phase 2B results. Ann Oncol 2014; 25 Suppl 4: abstract 1055PD.
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MS04.04 - Microenvironment as a Target (ID 1863)
15:20 - 15:40 | Author(s): S. Albelda
- Abstract
- Presentation
Abstract:
The traditional approaches to lung cancer therapy have focused on treating the malignant epithelial cancer cells within the tumor. However, it is now realized that in most cases, most of the tumor consists of “supporting cells” that include endothelium, pericytes, fibroblasts, and a variety of innate and acquired (B cells and T cells) immune cells. Thus, targeting these non-tumor cells could be an alternative therapeutic strategy. This concept is already being used in clinical practice. One example is targeting the endothelial cells within the tumor using an anti-VEGF antibody (bevacizumab). Another example are the checkpoint inhibitors (anti-CTLA4 and anti-PD1 antibodies) that target endogenous T cells. However, it may also be possible to attack other targets such as macrophages, Tregs, neutrophils or cancer-associated fibroblasts (CAFs). Tumor-associated macrophages represent one target. These cells take on a tumor-supportive phenotype and produce anti-inflammatory cytokines/chemokines (i.e. TGFbeta, PGE2, IL10, VEGF), arginase (which inactivates T cells), and angiogenic factors. This has led to the hypothesis that changing the state of the macrophage to an anti-tumor phenotype in which immune-activating mediators would be made and antigen-presentation could be enhanced would have direct anti-tumor activities and would allow endogenous T cells to kill tumor cells. Macrophage activation has been attempted for many years using agents such as bacterial endotoxin, TNF, liposomal-encapsulated muramyl tripeptides, lipopeptides or oligonucleotides/agents that activate toll-like receptors. To date, however, this approach has not been very successful, primarily due to lack of specificity for tumor infiltrating macrophages resulting in intolerable systemic toxicity. Our group explored the use of a cell permeable flavonoid compound called DMXAA for this purpose. Administration of DMXAA causes activation of tumor-associated macrophages via multiple pathways with release of cytokines and chemokines resulting in hemorrhagic tumor necrosis, a subsequent inflammatory/immuno-permissive tumor environment, and ultimately attracts CD8 T cells into tumors (Jassar et al. 2005). Although intra-tumoral treatment of both large and small lung cancers in mouse models led to striking tumor regression, there was a major problem in translating this work- DMXAA does not react with human macrophages. Since we did not know how DMXAA was working (i.e. what was the DMXAA receptor that triggered macrophage activation) progress was stalled. This changed recently, when it was discovered that DMXAA worked by binding to a newly described intracellular sensor of cytosolic DNA (working through binding to cyclic dinucleotides) called STING (stimulator of Interferon Genes). STING activates innate immunity by signaling through the TBK/IRF3 axis, NF-kB and STAT6 pathways. Interestingly, it was noted that DMXAA bound well to mouse STING but NOT to human STING (explaining its lack of efficacy in humans). A company (Aduro Biotech) has designed a compound that binds to human STING and thus activates human macrophages like DMXAA activates mouse macrophages. Their lead compound has strong in vivo anti-tumor activity (much like DMXAA) and clinical trials using intra-tumoral injections are about to start (Corrales et al., 2015). Another potential target in the tumor microenvironment is the cancer-associated fibroblasts (CAFs). Fibroblasts and their associated stroma promote tumor growth through multiple mechanisms, including suppression of anti-tumor immunity, supporting angiogenesis, as a depot for growth factors/ cytokines/chemokines, modulating the inflammatory response, and shielding the tumor from infiltrating cells. Our group at Penn has been developing genetically altered T cells that can be targeted to any expressed surface antigen by transducing autologous T cells with a chimeric antigen receptor (CAR). A CAR is composed of a single chain antibody fused to the cytoplasmic sequences from the CD3zeta chain and a co-activating receptor (41BB/CD137). This construct combines antibody specificity with the ability to activate the killing machinery of T cells. Our lead CAR T cell target is CD19 to treat B cell malignancies, however, we are also testing CARs targeted to mesothelin (mesothelioma, lung cancer, pancreas cancer, ovarian cancer) and other solid tumor cell targets. We hypothesized that we could use this approach to eliminate CAFs. To do so, we identified Fibroblast Activation Protein (FAP) as a target antigen for CAFs. In epithelial-derived tumors, FAP is selectively expressed by cancer-associated stromal cells It is highly expressed in the stroma of lung (and many other) cancers, but not in benign tumors or normal adult quiescent tissues (although it is upregulated in wounds and fibrotic tissues). We thus produced T cells expressing anti-mouse FAP CARs. The FAP CAR T cells selectively killed FAP-expressing cells. Immune-competent C57BL/6 mice bearing large established subcutaneous murine lung cancers and human A549 tumors in immune-deficient mice were treated. FAP-CAR T cells reduced the number of FAP+ cells, markedly reduced the amount of tumor matrix and limited tumor growth in all three lung cancer models (Wang et al., 2014; Lo et al., 2015). We hope to move this approach forward to clinical trials in lung cancer and mesothelioma. We will likely combine anti-fibroblast therapy with chemotherapy, vaccines, or other types of immunotherapy. In summary, a new therapeutic paradigm is now emerging based on therapy aimed at the non-malignant host cells, NOT directly targeting the cancer cells. Examples include antibodies targeting endothelial cells and checkpoint inhibitors that target T cells. An advantage of this approach is that stromal cells are more genetically stable compared with tumor cells and they are unlikely to lose their antigen(s) and become invisible to T cells. Another advantage is the same targets could be used in multiple tumors. Future applications will likely include activation or elimination of TAMS, targeting fibroblasts, and deletion of T-regulatory cells. References: Corrales L, et al. Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Reports 2015. 11:1-13. Jassar, A., et al. Activated Tumor-Associated Macrophages and CD8[+] T-cells are the Key Mediators of Anti-tumor Effects of the Vascular Disrupting Agent DMXAA in Murine Models of Lung Cancer and Mesothelioma”. Cancer Research 2005. 65:11752-11761. Lo A, et al. Tumor-promoting desmoplasia is disrupted by depleting FAP-expressing stromal cells. Cancer Res. 2015 May 15. [Epub ahead of print] Wang LC, et al. Targeting Fibroblast Activation Protein in Tumor Stroma with Chimeric Antigen Receptor T Cells Can Inhibit Tumor Growth and Augment Host Immunity Without Severe Toxicity. Cancer Immunology Research 2014. 2:154-166.
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MINI 27 - Biology and Other Issues in SCLC (ID 152)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:P.A. Bunn, Jr, J. Sage
- Coordinates: 9/09/2015, 16:45 - 18:15, 605+607
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MINI27.03 - PD-L1 Expression in Small Cell Lung Carcinoma: An Immunohistochemical Analysis of 26 Cases Using Two Anti-PD-L1 Antibodies (ID 2936)
16:55 - 17:00 | Author(s): P. Forde
- Abstract
- Presentation
Background:
Small cell lung carcinoma (SCLC) represents 15% of lung cancers and is treated using chemotherapy +/- radiation but despite initial responses most recur within a few months and become resistant to therapy. Novel immune checkpoint inhibition of programmed death-1 (PD1) targeted therapy has shown promise in other solid tumors including non-small-cell lung cancer (NSCLC) and malignant melanoma. In some tumor types correlation with response and significant expression of programmed death- ligand 1 (PD-L1), the lead candidate biomarker of anti-PD-1 therapy, has been described but no data is available regarding expression levels in SCLC. Here we report the rate of PD-L1 expression in SCLC and in associated tumor infiltrating immune cells lymphocytes and macrophages.
Methods:
Immunohistochemistry (IHC) for PD-L1 using two monoclonal antibodies (clone 5H1 and clone SP142) and for CD3 (clone PS1) was performed on standard formalin fixed paraffin embedded tissue sections of 21 resected SCLC specimens (median age: 67) and three additional tumors with pre- and post-therapy biopsies. Since there is no generally accepted scoring system for PD-L1 expression we chose to evaluate staining in tumor cells and immune cells infiltrating the tumor nests and in adjacent stroma using a 4 tier semi quantitative scoring system (score 0 -no or <1%, 1+ 1-<5%, 2+ 5-25% and 3+ >25% of cells staining). Both cytoplasmic and membranous staining was accepted as positive. The number of tumor infiltrating lymphocytes (TIL) were estimated utilizing a CD3 stain while macrophages were identified on corresponding H&E stains.
Results:
PD-L1 staining of tumor cells and Immune Cells (TIL & Macrophage) are shown in the table below. Membranous PD-L1 staining was only seen in two tumors and in variable number of immune cells with 2+ or 3+ PD-L1 scores. The majority of positive staining was cytoplasmic with both antibodies. The staining intensity was stroger with the 5H1 antibody. The paired pre- and post-therapy samples were all negative for PD-L1.
* Tumors with membranous staining; IC: immune cellsClone/score PD-L1 staining in 5H1 Tumor IC in tumor IC in stroma 0 (<1%) 19/21 (90%) 7/21 (33%) 5/21 (24%) 1+ (1-<5%) 1/21 (5%)* 11/21 (53%) 6/21 (29%) 2+ (5-25%) 1/21 (5%)* 3/21 (14%) 7/21 (33%) 3+ (>25%) 3/21 (14%) SP142 Tumor IC in tumor IC in stroma 0 (<1%) 20/21 (95%) 8/21 (38%) 10/21 (48%) 1+ (1-<5%) 1/21 (5%)* 11/21 (52%) 7/21 (33%) 2+ (5-25%) 2/21 (10%) 4/21 (19%) 3+ (>25%)
Conclusion:
Most SCLC are tumor membrane PD-L1 negative by IHC. A subset of SCLC contain PD-L1 positive TILs and/or macrophages in the tumor and the stroma. No up regulation of PD-L1 expression was seen in a small pilot sample of matched pre- and post-therapy biopsies. It is unclear whether PD-L1 expression assessed by IHC will be a predictive marker for PD-1 targeted therapy in SCLC. Preliminary data indicates single agent and combined checkpoint inhibitors (PD1 plus CTLA-4 inhibitors) are active in previously treated SCLC indicating additional research is required to understand their mechanism of action in a tumor type that has seen no therapeutic advances in the last two decades.
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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.04 - Comprehensive Genomic Profiling (CGP) of Advanced Cancers Identifies MET Exon 14 Alterations That Are Sensitive to MET Inhibitors (ID 3156)
17:17 - 17:28 | Author(s): P. Forde
- Abstract
- Presentation
Background:
Amplifications and activating mutations in the c-MET proto-oncogene are known oncogenic drivers that have proven responsive to targeted therapy. Mutations causing skipping of MET exon 14 are also oncogenic, but less well characterized. We undertook comprehensive genomic profiling (CGP) of a large series of advanced cancers to further characterize MET exon 14 alterations.
Methods:
DNA was extracted from 40 microns of FFPE sections from 38,028 advanced cancer cases. CGP was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of >500x using three versions of the FoundationOne test. Hybridization capture baits for the MET gene were identical for all three versions of the test. Base substitution, indel, copy number alteration, and rearrangement variant calls were examined to identify those nearby to the splice junctions of MET exon 14. These genomic alterations were then manually inspected to identify those likely to affect splicing of exon 14, or delete the exon entirely.
Results:
221 cases harboring MET ex14 alterations were identified. These patients had a median age of 70.5 years (range 15-88), with 97 males and 124 females. The cases were lung carcinoma (193), carcinomas of unknown primary (15), brain glioma (6), and one each of adrenal cortical carcinoma, hepatocellular carcinoma, histiocytic sarcoma, renal cell carcinoma, rhabdomyosarcoma, skin merkel cell carcinoma, and synovial sarcoma. The majority were stage IV. Identification of this alteration has lead to treatment with MET inhibitors such as crizotinib, and to durable partial responses or better exceeding 3 months in histiocytic sarcoma (1), sarcomatoid lung carcinoma (1), and nsclc (1+). Multiple patients (5+) have initiated treatment on either crizotinib or MET inhibitors in clinical development, and additional outcome data will be reported. One patient with locally advanced unresectable disease harbored a MET exon 14 skipping alteration. On initiation with treatment with an MET inhibitor, symptomatic relief was observed in 3 days, radiographic response was observed at two weeks, and resection was performed 8 weeks after initiation of the MET inhibitor.
Conclusion:
MET exon 14 alterations define a hereto unrecognized population of advanced cancer cases, particularly in NSCLC. Multiple case reports demonstrate that these alterations confer sensitivity to multiple small molecule MET inhibitors. This finding expands the population of advanced NSCLC patients who can derive benefit from MET-targeted therapies.
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