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P.S. Adusumilli
Moderator of
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ORAL 28 - T Cell Therapy for Lung Cancer (ID 132)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 6
- Moderators:P.S. Adusumilli, E. Smit
- Coordinates: 9/08/2015, 16:45 - 18:15, Four Seasons Ballroom F1+F2
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ORAL28.01 - Checkpoint Blockade Augments TCR Engineered Adoptive T Cell Therapy for Lung Cancer (ID 344)
16:45 - 16:56 | Author(s): E.K. Moon, R. Ranganathan, X. Liu, A. Lo, S. Kim, Y. Zhao, S. Albelda
- Abstract
Background:
Adoptive T-cell immunotherapy (ACT) has shown great promise in melanoma and hematologic malignancies; however one major limitation of engineered T cells targeting solid tumors is likely to be tumor microenvironment-induced hypofunction of the T cells. To study and limit this problem, we have developed a model in which human T cells engineered to target the antigen NYESO1 using a high-affinity engineered TCR (Ly95) are injected into mice bearing human A549 lung cancer cells. Using this model we demonstrate upregulation of PD1 and TIM3 on Ly95 TILs. We were able to augment T cell anti-tumor activity by combining Ly95 T cell therapy with anti-hPD1 and anti-hTIM3 antibodies.
Methods:
In vitro: Human T cells activated by anti-CD3/CD28 Dynabeads and transduced with lentivirus had 50% expression of Ly95 TCR as measured by flow cytometry. They were cocultured with marked tumor cells to measure IFNg release and antigen-specific killing. In vivo: Immunodeficient mice with 200mm[3] flank A549-A2-ESO (AAE) tumors received 10[7] T cells via tail vein. Three weeks later, tumors were harvested/digested, and human TILs were isolated/assesssed for tumor killing/IFNg secretion. This was repeated after the TILs were rested for 24hrs at 37[0]C/5%CO2. The number of TILs and PD1/TIM3 expression on the isolated TILs were assessed by flow cytometry at fresh harvest and post rest. The in vivo experiment was repeated comparing Ly95 T cells alone vs. Ly95 T cells plus either/both intraperitoneal (IP) anti-hPD1 or/and IP anti-hTIM3 at 10mg/kg every 5 days.
Results:
Ly95 TCR T cells were able to kill AAE tumor cells and secrete high amounts of IFNg in an antigen-specific/dose dependent fashion after 18hr coculture. 10[7 ]IV Ly95 T cells were able to slow AAE flank tumor growth as compared to control tumors (498mm[3 ]vs. 1009mm[3], p<0.05.) Flow cytometric analysis of harvested/digested tumors revealed that 5.2% of the tumor digest was human TILs. Freshly isolated TILs were hypofunctional in their ability to kill tumor cells and release IFNg when compared to cryopreserved Ly95 T cells (p<0.05.) After overnight rest away from tumor, TILs improved in function. Further analysis revealed that Ly95 TILs had upregulated their expression of PD1 and TIM3 (increase from 5 to 40% in PD1 and from 17 to 50% in TIM3.) Combining a single Ly95 T cell IV injection with multiple IP anti-hPD1 and anti-hTIM3 injections resulted in 43% reduction in flank tumor size compared to Ly95 T cell injection alone (189mm[3] vs. 332mm[3], p<0.05.)
Conclusion:
The PD1 and TIM3 pathways are involved in tumor-induced hypofunction of TCR engineered TILs. Combining anti-hPD1 and anti-hTIM3 antibodies with TCR T cells, and likely CAR T cells, will likely enhance the efficacy of these approaches in lung cancer and other solid tumors.
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ORAL28.02 - Mesothelin-Targeted CAR T-Cell Therapy for the Treatment of Heterogeneous Antigen-Expressing Lung Adenocarcinoma (ID 3172)
16:56 - 17:07 | Author(s): A. Morello, J. Villena-Vargas, M. Mayor, A.J. Bograd, D.R. Jones, M. Sadelain, P.S. Adusumilli
- Abstract
Background:
Adoptive T-cell therapy using chimeric antigen receptors (CAR) is an emerging strategy by redirecting T-cell effector functions against a cancer cell-surface antigen. To target lung adenocarcinoma (ADC) by CAR T-cell therapy, our laboratory has identified mesothelin (MSLN), a cell-surface antigen based on our published observation that MSLN is expressed in 60% of primary and metastatic lung ADC and is associated with tumor aggressiveness. Unlike hematological malignancies where CAR T-cell therapy has been successful targeting CD19, a cell-surface antigen that is uniformly expressed on B cells, MSLN expression intensity and distribution among lung ADC tumors is heterogeneous. The efficacy of CAR T-cell therapy in a heterogeneous antigen microenvironment is unknown. We hypothesized that the MSLN-targeted CAR T cells will be effective against high-antigen expressing lung ADC cells and the presence of even a small proportion of high MSLN expressing cells can enhance CAR T-cell cytotoxicity against low-antigen expressing lung ADC cells.
Methods:
Human peripheral blood T cells were retrovirally transduced with a 2[nd] generation of CAR targeting MSLN and bearing CD28 and CD3zeta activation domains. In vitro, we analyzed CAR T-cell cytotoxicity ([51]Cr release assay), effector cytokine secretion (Luminex assay), and proliferation (cell-counting assay) against lung ADC cell lines expressing variable levels of MSLN. In vivo, antitumor efficacy was evaluated by median survival and tumor bioluminescence (BLI) in mice bearing established homogeneous or heterogeneous lung ADC tumors.
Results:
In in vitro assays utilizing lung ADC cells with variable level of MSLN expression [low-antigen expression (EKVX or A549) or high-antigen expression (A549M and H1299M), control lung fibroblast (MRC5) or mesothelial cells (MET5A)], CAR T cells exhibit antigen-specific cytolytic activity, effector cytokine secretion and proliferation in proportion to the MSLN expression on cancer cells. In vivo, a single low dose of CAR T cells eradicates primary and metastatic established tumor expressing high-level of MSLN and prolongs tumor free survival (41 days vs not reached, p<0.0001). We next evaluated CAR T-cell efficacy in heterogeneous antigen microenvironment by mixing low and high antigen-expressing cells (A549 expressing firefly luciferase/A549M) and assessed the A549 tumor burden only by bioluminescence imaging. In the presence of A549M cells, CAR T cells are able to prolong progression-free survival of A549 tumor burden (22 days vs 0 days in absence of A549M cells). Further mechanistic studies demonstrated that CAR T cells lysed an additional 5%-15% A549 or EKVX cells in the presence of H1299M or A549M cells (p<0.05) without off-target cytotoxicity. Antigen-activated CAR T cells were effective against low-antigen expressing lung ADC cells without the need for high-antigen expressing cells in the coculture.
Conclusion:
Our results provide scientific rationale to translate MSLN-targeted CAR T-cell therapy for the treatment of the primary and metastatic lung ADC. A phase I clinical trial (NCT02414269) that includes lung ADC patients is initiated at our center.
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ORAL28.03 - Genetic-Engineering Strategies to Enhance CAR T-Cell Therapy Efficacy against PD-L1 Expressing Lung Adenocarcinoma and Mesothelioma (ID 3139)
17:07 - 17:18 | Author(s): L. Cherkassky, A. Morello, J. Villena-Vargas, M. Mayor, D.R. Jones, M. Sadelain, P.S. Adusumilli
- Abstract
Background:
This abstract is under embargo until September 8, 2015 and will be distributed onsite on September 8 in a Late Breaking Abstract Supplement.
Methods:
Results:
Conclusion:
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ORAL28.04 - Tumor-Targeted Radiation Therapy Helps Overcome the Solid Tumor T-Cell Infiltration Barrier and Promotes Mesothelin CAR T-Cell Therapy (ID 3142)
17:18 - 17:29 | Author(s): M. Mayor, J. Villena-Vargas, A. De Biasi, A. Morello, D.R. Jones, M. Sadelain, P.S. Adusumilli
- Abstract
Background:
Translating recent chimeric antigen receptor (CAR) T-cell therapy successes in hematologic malignancies to solid cancers requires overcoming barriers unique to solid tumors such as inadequate tumor infiltration, proliferation, and persistence. Our laboratory has published the rationale to target mesothelin (MSLN), a cell-surface antigen expressed in the majority of thoracic malignancies. We hypothesized that the immune modulating effects of low-dose radiation therapy (RT) would enhance the infiltration and proliferation of mesothelin-targeted CAR T-cell therapy for thoracic cancers, thereby achieving long-term tumor eradication.
Methods:
Using human T cells retrovirally transduced to express mesothelin-targeted CARs, we evaluated T-cell cytotoxicity by chromium release assay, proliferation by cell count assay, cytokine-release by multiplex ELISA, phenotype by flow cytometry, and chemokine receptor profiles by PCR against MSLN-expressing mesothelioma and lung cancer cell lines with and without localized RT. In clinically relevant mouse models (NOD/SCID gamma mice) with established MSLN-expressing tumors, we monitored therapy response, T-cell kinetics and anti-tumor efficacy by utilizing bioluminescent imaging (BLI), and conducted flow cytometric analysis of splenic/peripheral blood T cells for characterization of CAR T-cell effector phenotype.
Results:
RT did not enhance CAR T-cell cytotoxicity. In vitro, RT enhanced CAR T-cell migration in chemotactic assays, and correlatively induced the secretion of chemokines by tumor cells (Fig.1A). In vivo, RT resulted in dose dependent chemokine secretion with robust early intratumoral CAR T-cell accumulation (p<0.05, Fig.1B) as demonstrated by T-cell BLI. Ex vivo tumor analysis by flow cytometry on day 7 post T-cell administration confirmed that RT increased early infiltration and proliferation (p<0.05). Also, single low-dose RT potentiated the efficacy of systemically administered CAR T cells (median survival 30d vs. 79d, p= 0.02) with at least 50% tumor eradication up to 100 days even with a 30-fold decreased dose (Fig.1C&D). Furthermore, in mice with tumor eradication, harvested spleen T-cell analysis at day 56 demonstrated a greater number of persisting CAR T cells in mice treated with RT (p=0.02, Fig.1E).Figure 1
Conclusion:
Our data provides the rationale to use localized RT as a preconditioning regimen prior to CAR T-cell administration in a clinical trial for thoracic malignancies. Furthermore, our mechanistic observation of RT-induced, chemokine-mediated, enhanced T-cell infiltration may also assist the trafficking of endogenous anti-tumor T cells, thereby shifting the balance towards a cohesive anti-tumor immune microenvironment.
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ORAL28.05 - Mesothelin and MUC16 (CA125) Are Antigen-Targets for CAR T-Cell Therapy in Primary and Metastatic Lung Adenocarcinoma (ID 3159)
17:29 - 17:40 | Author(s): T. Eguchi, H. Ujiie, A. Morello, K. Kadota, D.H. Buitrago, K. Woo, D.R. Jones, W.D. Travis, M. Sadelain, P.S. Adusumilli
- Abstract
Background:
Chimeric antigen receptor (CAR) T-cell therapy has shown durable remissions in hematological malignancies targeting cancer-antigen CD19. Ideal cancer-antigen targets for CAR T-cell therapy are antigens overexpressed on cancer cell-surface with limited expression in normal tissues, associated with tumor aggressiveness and expressed in a large cohort of patients. In our search for such candidate antigens in lung adenocarcinoma (ADC), we investigated the overexpression of Mesothelin (MSLN), MUC16 (CA125), and the combination of MSLN-MUC16 as the interaction of both antigens has been shown to play a role in tumor metastasis.
Methods:
In patients with stage I lung ADC (n = 912, 1995 - 2009), a tissue microarray consisting of 4 cores from each tumor and normal lung tissue was used to examine the antigen-expression characteristics, and their association with cumulative incidence of recurrence (CIR). Autologous metastatic tumor tissue was available from 36 patients. Differences in CIR between groups were tested using the Gray method (for univariate nonparametric analyses) and Fine and Gray model (for multivariate analyses).
Results:
MSLN and MUC16 were not expressed in normal lung tissue. In primary and metastatic lung ADC tumors, MSLN was expressed in 69% and 64%, MUC16 was expressed in 46% and 69%, both antigens were present in 50% and 33%, and either antigen were present in 33% and 49% respectively. On univariate analysis, patients with high MSLN expression had high risk of recurrence than low expression [5-year CIR, High: 25.1% vs Low: 17.6%, P = 0.017]. Patients with high MUC16 expression had high risk of recurrence than low expression [5-year CIR, High: 24.2% vs Low: 14.0%, P < 0.001]. Patients with high MUC16 and high MSLN had higher risk of recurrence than low expression [5-year CIR, High risk (High MUC16 and High MSLN): 27.6%, Intermediate risk (High MUC16 and Low MSLN): 24.2%, Low risk (Low MUC16): 13.6%, P < 0.001]. On multivariate analysis, increased MUC16-MSLN expression was associated with recurrence [Hazard ratio, 2.57 95% Confidence interval 1.41 – 4.68 P = 0.002], even after adjustment for currently known markers of lung ADC aggressiveness (gender, surgical procedure, stage, architectural grade and lymphatic invasion). High expression of MUC16 in the primary tumor was associated with high expression at recurrence sites.
Conclusion:
MSLN, MUC16 or a combination of expression of both antigens in patients with primary lung ADC is associated with increased risk of recurrence, a retained overexpression at metastatic sites in advanced lung ADC indicating that MUC16-MSLN expression is a marker of tumor aggressiveness. Expression in the majority of lung ADC patients imparting aggressiveness with no expression in normal lung provides the rationale to target MSLN and MUC16 for lung ADC CAR T-cell therapy.
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ORAL28.06 - Discussant for ORAL28.01, ORAL28.02, ORAL28.03, ORAL28.04, ORAL28.05 (ID 3463)
17:40 - 17:50 | Author(s): E. Smit
- Abstract
- Presentation
Abstract not provided
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Author of
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MINI 01 - Pathology (ID 93)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:W.A. Franklin, A.G. Nicholson
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI01.07 - Comparison of Grading Systems Based on Histologic Patterns and Mitotic Activity to Predict Recurrence in Stage I Lung Adenocarcinoma (ADC) (ID 3030)
11:20 - 11:25 | Author(s): P.S. Adusumilli
- Abstract
- Presentation
Background:
An established grading system for lung adenocarcinoma does not exist but is greatly needed. The histologic classification proposed by the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS) and the European Respiratory Society (ERS) has been shown to define prognostically significant subgroups of lung adenocarcinoma (ADC). Since then, various grading systems based on histologic patterns have emerged as promising methods to further discriminate patient risk of clinical outcomes. The aim of this work is to quantitatively assess the discrimination properties of a set of grading systems proposed in recent years to identify the best grading scale(s) independent of other clinical factors to predict recurrence.
Methods:
We considered five grading systems: (1) single predominant pattern as six subtypes; (2) as three grades of low (lepidic), intermediate (acinar, papillary) and high (micropapillary, solid); (3) two most predominant grades; (4) predominant grade with mitotic grade; and (5) predominant grade with cribriform pattern and mitotic activity criteria. We evaluated the performance of each grading system with the concordance predictive estimate (CPE). The CPE represents the probability that for any pair of patients, the patient with the better predicted outcome from the Cox model had the longer survival time. CPE > 0.80 demonstrates strong performance. To compare the performance of the grading systems, we determined the significance of the differences between the CPEs. Five-year recurrence-free probability (RFP) was derived using the Kaplan-Meier method.
Results:
We applied the grading systems to a uniform large cohort of stage I lung ADC (N=909). The scale based on the single predominant pattern as five subtypes yielded a CPE of 0.63 (95% CI, 0.59-0.67), indicating moderate discrimination. Our analysis showed that grading systems (1), (2), and (3) were not significantly different from each other, suggesting that identifying finer subtypes and second predominant pattern may not improve discrimination. Grading system (4) [CPE, 0.67; 95% CI, 0.63-0.71] yielded a significantly higher CPE than (1), (2) and (3) [p<0.01]. Grading system (5) [CPE, 0.67; 95% CI, 0.63-0.71] was significantly better than (1), (2) and (3) but not (4) [p=0.776]. The lack of improvement in discrimination with the inclusion of cribriform between (4) and (5) can be attributed to the significant relationship between cribriform pattern and mitoses. As the proportion of cribriform pattern increased, the amount of mitotic activity also increased (p<0.001). Under (2), the 5-year RFP of the intermediate grade was 0.81. The addition of cribriform and mitotic counts further classified the intermediate (acinar, papillary) grade such that those with <10% cribriform and low mitotic count had 5-year RFP of 0.89, while the 5-year RFP for the other combinations are between 0.73-0.75.
Conclusion:
Grading systems based on histologic patterns and mitotic activity out-perform those with only histologic pattern. This comparison study suggests that proposed grading systems (4) or (5) provide valuable information in discriminating patients with different risks of disease-recurrence in patients with lung ADC.
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MINI 06 - Quality/Prognosis/Survival (ID 111)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Treatment of Localized Disease - NSCLC
- Presentations: 1
- Moderators:R. Meguid, J. Yoshida
- Coordinates: 9/07/2015, 16:45 - 18:15, 605+607
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MINI06.02 - T1a Lung Adenocarcinomas: Presence of Spread of Tumor through Alveolar Spaces (STAS), Micropapillary and Solid Patterns Determines Outcomes (ID 3068)
16:50 - 16:55 | Author(s): P.S. Adusumilli
- Abstract
- Presentation
Background:
Our previous reports highlighting the significance of presence of micropapillary (MIP) (JNCI 2013), STAS- spread of tumor through alveolar spaces (JTO 2015), and predominant solid (SOL) (Modern Pathol 2011) histological subtype as poor prognostic markers in stage I lung adenocarcinomas (ADC) are reproduced by others. In this study, we hypothesized that presence of STAS, MIP or SOL patterns (≥5%) in small stage I lung ADC (≤2 cm) is a marker of invasion and poor prognosis, and can influence the recurrence patterns based on the type of surgical resection – lobectomy (LO) versus limited resection (LR).
Methods:
All available tumor slides from patients with therapy-naive, surgically resected small (≤ 2cm), solitary stage I lung ADC were reviewed (1995-2011; n = 909). STAS was defined as isolated tumor cells within alveolar spaces separate from the main tumor. MIP and SOL patterns were considered present in the tumor when it comprised ≥5% of the overall tumor. Cumulative incidence of recurrence (CIR; any types, locoregional or distant) was estimated using a cumulative incidence function. Differences in CIR between groups were assessed using Gray’s method.
Results:
Figure 1 The association of outcomes with the presence of STAS, MIP, or SOL patterns is shown in the table. The risk of developing any types of recurrence was significantly higher in patients with both STAS and MIP positive tumors than others (P < 0.001); and the risk of developing any types of recurrence was significantly lower in patients with both STAS and SOL negative tumors than others (P < 0.001). In the LR group, STAS, MIP and SOL patterns were independent prognostic factors for any types of recurrence (HR: 4.5, 1.4, and 1.3, respectively), locoregional recurrence (HR: 5.2, 1.3, and 1.3, respectively), and distant recurrence (HR: 3.1, 1.4, and 1.2, respectively).
Conclusion:
Tumor STAS, presence of MIP and SOL patterns are independent risk factors of recurrence especially in the LR group of small stage I lung ADC patients. Importantly, of these factors, tumor STAS was the strongest predictor of locoregional recurrence in this group. These results suggest that the identification of STAS in small lung ADC may identify LR patients who need further management, one of which may be completion lobectomy.
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MINI 38 - Biology and Prognosis (ID 167)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:R. Tsuchiya, M. Wynes
- Coordinates: 9/09/2015, 18:30 - 20:00, 702+704+706
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MINI38.02 - BAP1 Inactivation in Mesothelioma Is Highly Prevalent (ID 657)
18:35 - 18:40 | Author(s): P.S. Adusumilli
- Abstract
- Presentation
Background:
Efforts to elucidate tumorigenic mutations in mesothelioma are essential to advance therapy. Prior efforts to characterize the molecular heterogeneity of this disease have been limited by sample condition and testing platforms. Herein, we describe efforts to prospectively test patients using next-generation sequencing with matched patient germline controls.
Methods:
Sequential mesothelioma patients were approached for consent to our IRB protocol NCT01775072 to perform MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), a comprehensive molecular profiling platform based on solution-phase exon capture and next generation sequencing to detect somatic genetic alterations in FFPE tumor specimens. MSK-IMPACT involves hybridization capture and deep sequencing of all protein-coding exons of 341 key cancer-associated genes, including all genes that are druggable by approved therapies or are targets of experimental therapies being investigated in clinical trials at MSKCC.
Results:
51 patients with mesothelioma underwent MSK-IMPACT testing (see Table 1). 12 samples had low tumor content. Among 39 samples with reliable results, BAP1 was the most common alteration (46%). Another 3 samples had changes also thought to inactivate BAP1 (2 samples had gene copy number changes just below the cutoff for whole gene deletions and 1 had an inversion of LIMD-BAP1 thought to inactivate BAP1), making the incidence of BAP1 alterations possibly as high as 56%. In 4 samples with sufficient tumor content, no alterations were identified. Table 1N=39 (%) Gender M/F 26/13 (67/33) Primary site of disease * Pleural * Peritoneal * Testicular 32 (82) 6 (15) 1 (3) # identified alteration, average 3 Alterations present in >6% * BAP1 * NF2 * CDKN2Ap16INK4A * SETD2 * CDKN2Ap14ARF * LATS1 * CREBBP * WT1 * CDKN2B * PI3KCA * PBRM1 * TP53 18 (46) 8 (21) 5 (13) 5 (13) 4 (10) 4 (10) 4 (10) 4 (10) 3 (8) 3 (8) 3 (8) 3 (8)
Conclusion:
Using MSK-IMPACT, BAP1 inactivation is the most common alteration. Other aberrations previously reported at high frequency were identified but albeit at lower frequencies (NF2 and p16, previously reported as 40% and 75% respectively). For multiple samples with deep coverage, no alterations were identified. The high incidence of BAP1 mutations in this systematic testing makes this pathway ideal for developing and testing targeted therapies.
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ORAL 28 - T Cell Therapy for Lung Cancer (ID 132)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 4
- Moderators:P.S. Adusumilli, E. Smit
- Coordinates: 9/08/2015, 16:45 - 18:15, Four Seasons Ballroom F1+F2
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ORAL28.02 - Mesothelin-Targeted CAR T-Cell Therapy for the Treatment of Heterogeneous Antigen-Expressing Lung Adenocarcinoma (ID 3172)
16:56 - 17:07 | Author(s): P.S. Adusumilli
- Abstract
Background:
Adoptive T-cell therapy using chimeric antigen receptors (CAR) is an emerging strategy by redirecting T-cell effector functions against a cancer cell-surface antigen. To target lung adenocarcinoma (ADC) by CAR T-cell therapy, our laboratory has identified mesothelin (MSLN), a cell-surface antigen based on our published observation that MSLN is expressed in 60% of primary and metastatic lung ADC and is associated with tumor aggressiveness. Unlike hematological malignancies where CAR T-cell therapy has been successful targeting CD19, a cell-surface antigen that is uniformly expressed on B cells, MSLN expression intensity and distribution among lung ADC tumors is heterogeneous. The efficacy of CAR T-cell therapy in a heterogeneous antigen microenvironment is unknown. We hypothesized that the MSLN-targeted CAR T cells will be effective against high-antigen expressing lung ADC cells and the presence of even a small proportion of high MSLN expressing cells can enhance CAR T-cell cytotoxicity against low-antigen expressing lung ADC cells.
Methods:
Human peripheral blood T cells were retrovirally transduced with a 2[nd] generation of CAR targeting MSLN and bearing CD28 and CD3zeta activation domains. In vitro, we analyzed CAR T-cell cytotoxicity ([51]Cr release assay), effector cytokine secretion (Luminex assay), and proliferation (cell-counting assay) against lung ADC cell lines expressing variable levels of MSLN. In vivo, antitumor efficacy was evaluated by median survival and tumor bioluminescence (BLI) in mice bearing established homogeneous or heterogeneous lung ADC tumors.
Results:
In in vitro assays utilizing lung ADC cells with variable level of MSLN expression [low-antigen expression (EKVX or A549) or high-antigen expression (A549M and H1299M), control lung fibroblast (MRC5) or mesothelial cells (MET5A)], CAR T cells exhibit antigen-specific cytolytic activity, effector cytokine secretion and proliferation in proportion to the MSLN expression on cancer cells. In vivo, a single low dose of CAR T cells eradicates primary and metastatic established tumor expressing high-level of MSLN and prolongs tumor free survival (41 days vs not reached, p<0.0001). We next evaluated CAR T-cell efficacy in heterogeneous antigen microenvironment by mixing low and high antigen-expressing cells (A549 expressing firefly luciferase/A549M) and assessed the A549 tumor burden only by bioluminescence imaging. In the presence of A549M cells, CAR T cells are able to prolong progression-free survival of A549 tumor burden (22 days vs 0 days in absence of A549M cells). Further mechanistic studies demonstrated that CAR T cells lysed an additional 5%-15% A549 or EKVX cells in the presence of H1299M or A549M cells (p<0.05) without off-target cytotoxicity. Antigen-activated CAR T cells were effective against low-antigen expressing lung ADC cells without the need for high-antigen expressing cells in the coculture.
Conclusion:
Our results provide scientific rationale to translate MSLN-targeted CAR T-cell therapy for the treatment of the primary and metastatic lung ADC. A phase I clinical trial (NCT02414269) that includes lung ADC patients is initiated at our center.
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ORAL28.03 - Genetic-Engineering Strategies to Enhance CAR T-Cell Therapy Efficacy against PD-L1 Expressing Lung Adenocarcinoma and Mesothelioma (ID 3139)
17:07 - 17:18 | Author(s): P.S. Adusumilli
- Abstract
Background:
This abstract is under embargo until September 8, 2015 and will be distributed onsite on September 8 in a Late Breaking Abstract Supplement.
Methods:
Results:
Conclusion:
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ORAL28.04 - Tumor-Targeted Radiation Therapy Helps Overcome the Solid Tumor T-Cell Infiltration Barrier and Promotes Mesothelin CAR T-Cell Therapy (ID 3142)
17:18 - 17:29 | Author(s): P.S. Adusumilli
- Abstract
Background:
Translating recent chimeric antigen receptor (CAR) T-cell therapy successes in hematologic malignancies to solid cancers requires overcoming barriers unique to solid tumors such as inadequate tumor infiltration, proliferation, and persistence. Our laboratory has published the rationale to target mesothelin (MSLN), a cell-surface antigen expressed in the majority of thoracic malignancies. We hypothesized that the immune modulating effects of low-dose radiation therapy (RT) would enhance the infiltration and proliferation of mesothelin-targeted CAR T-cell therapy for thoracic cancers, thereby achieving long-term tumor eradication.
Methods:
Using human T cells retrovirally transduced to express mesothelin-targeted CARs, we evaluated T-cell cytotoxicity by chromium release assay, proliferation by cell count assay, cytokine-release by multiplex ELISA, phenotype by flow cytometry, and chemokine receptor profiles by PCR against MSLN-expressing mesothelioma and lung cancer cell lines with and without localized RT. In clinically relevant mouse models (NOD/SCID gamma mice) with established MSLN-expressing tumors, we monitored therapy response, T-cell kinetics and anti-tumor efficacy by utilizing bioluminescent imaging (BLI), and conducted flow cytometric analysis of splenic/peripheral blood T cells for characterization of CAR T-cell effector phenotype.
Results:
RT did not enhance CAR T-cell cytotoxicity. In vitro, RT enhanced CAR T-cell migration in chemotactic assays, and correlatively induced the secretion of chemokines by tumor cells (Fig.1A). In vivo, RT resulted in dose dependent chemokine secretion with robust early intratumoral CAR T-cell accumulation (p<0.05, Fig.1B) as demonstrated by T-cell BLI. Ex vivo tumor analysis by flow cytometry on day 7 post T-cell administration confirmed that RT increased early infiltration and proliferation (p<0.05). Also, single low-dose RT potentiated the efficacy of systemically administered CAR T cells (median survival 30d vs. 79d, p= 0.02) with at least 50% tumor eradication up to 100 days even with a 30-fold decreased dose (Fig.1C&D). Furthermore, in mice with tumor eradication, harvested spleen T-cell analysis at day 56 demonstrated a greater number of persisting CAR T cells in mice treated with RT (p=0.02, Fig.1E).Figure 1
Conclusion:
Our data provides the rationale to use localized RT as a preconditioning regimen prior to CAR T-cell administration in a clinical trial for thoracic malignancies. Furthermore, our mechanistic observation of RT-induced, chemokine-mediated, enhanced T-cell infiltration may also assist the trafficking of endogenous anti-tumor T cells, thereby shifting the balance towards a cohesive anti-tumor immune microenvironment.
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ORAL28.05 - Mesothelin and MUC16 (CA125) Are Antigen-Targets for CAR T-Cell Therapy in Primary and Metastatic Lung Adenocarcinoma (ID 3159)
17:29 - 17:40 | Author(s): P.S. Adusumilli
- Abstract
Background:
Chimeric antigen receptor (CAR) T-cell therapy has shown durable remissions in hematological malignancies targeting cancer-antigen CD19. Ideal cancer-antigen targets for CAR T-cell therapy are antigens overexpressed on cancer cell-surface with limited expression in normal tissues, associated with tumor aggressiveness and expressed in a large cohort of patients. In our search for such candidate antigens in lung adenocarcinoma (ADC), we investigated the overexpression of Mesothelin (MSLN), MUC16 (CA125), and the combination of MSLN-MUC16 as the interaction of both antigens has been shown to play a role in tumor metastasis.
Methods:
In patients with stage I lung ADC (n = 912, 1995 - 2009), a tissue microarray consisting of 4 cores from each tumor and normal lung tissue was used to examine the antigen-expression characteristics, and their association with cumulative incidence of recurrence (CIR). Autologous metastatic tumor tissue was available from 36 patients. Differences in CIR between groups were tested using the Gray method (for univariate nonparametric analyses) and Fine and Gray model (for multivariate analyses).
Results:
MSLN and MUC16 were not expressed in normal lung tissue. In primary and metastatic lung ADC tumors, MSLN was expressed in 69% and 64%, MUC16 was expressed in 46% and 69%, both antigens were present in 50% and 33%, and either antigen were present in 33% and 49% respectively. On univariate analysis, patients with high MSLN expression had high risk of recurrence than low expression [5-year CIR, High: 25.1% vs Low: 17.6%, P = 0.017]. Patients with high MUC16 expression had high risk of recurrence than low expression [5-year CIR, High: 24.2% vs Low: 14.0%, P < 0.001]. Patients with high MUC16 and high MSLN had higher risk of recurrence than low expression [5-year CIR, High risk (High MUC16 and High MSLN): 27.6%, Intermediate risk (High MUC16 and Low MSLN): 24.2%, Low risk (Low MUC16): 13.6%, P < 0.001]. On multivariate analysis, increased MUC16-MSLN expression was associated with recurrence [Hazard ratio, 2.57 95% Confidence interval 1.41 – 4.68 P = 0.002], even after adjustment for currently known markers of lung ADC aggressiveness (gender, surgical procedure, stage, architectural grade and lymphatic invasion). High expression of MUC16 in the primary tumor was associated with high expression at recurrence sites.
Conclusion:
MSLN, MUC16 or a combination of expression of both antigens in patients with primary lung ADC is associated with increased risk of recurrence, a retained overexpression at metastatic sites in advanced lung ADC indicating that MUC16-MSLN expression is a marker of tumor aggressiveness. Expression in the majority of lung ADC patients imparting aggressiveness with no expression in normal lung provides the rationale to target MSLN and MUC16 for lung ADC CAR T-cell therapy.
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P1.02 - Poster Session/ Treatment of Localized Disease – NSCLC (ID 209)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Localized Disease - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.02-029 - Long and Short-Term Predictors of Outcome in Elderly Patients (≥ 75 Years) Undergoing Lobectomy for Stage I Non-Small Cell Lung Cancer (ID 3126)
09:30 - 09:30 | Author(s): P.S. Adusumilli
- Abstract
Background:
More than 65% of patients diagnosed with non-small cell lung cancer (NSCLC) are above the age of 65 years. Half of this cohort are ≥75 years who are at higher risk following surgical resection, which is the mainstay of treatment for early-stage NSCLC. The purpose of this study is to determine the factors influencing the outcomes in patients ≥75 years who underwent lobectomy for stage I NSCLC: postoperative complications, short-term (30- and 90-day mortality) and long-term (overall survival (OS) and cancer-specific survival (CSS)). In addition to the routinely used clinical factors, we investigated the utility of lung age, the tool commonly used for smoking cessation.
Methods:
Patients with pathological stage I NSCLC who underwent lobectomy between 2000 and 2011, age ≥75 years at surgery with no induction therapy, and no previous lung resection were included in the study (n =435). We investigated the influence of smoking history, preoperative history of cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD), Carlson comorbidity index (CCI), serum creatinine level, lung age (calculated by height and forced expiratory volume in one second), percent predicted diffusing capacity of the lung for carbon monoxide (%DLCO), and p-stage. Outcomes studied were postoperative in-hospital complication (CTCAE grade ≥3), 30- and 90-day mortality, OS, and CSS. Complications and mortality were analyzed by chi-square tests for univariate analysis. OS and CSS were analyzed by Kaplan-Meier methods with log-rank tests for univariate analysis, and Cox proportional analysis for multivariate analysis.
Results:
Median chronological age was 79 years, whereas median lung age was 89 years (female gender n = 334, positive smoking history n = 391, p-stage IA/IB were 282/153). In univariate analysis, low %DLCO and CVD history were significantly associated with postoperative complications (p = 0.032 and 0.018, respectively), and only high serum creatinine level was significantly associated with 30- and 90-day mortality (p = 0.02 and 0.027, respectively). P-stage, lung age, %DLCO, and COPD history were significantly associated with poor OS (p <0.001, p <0.001, p = 0.009 and 0.008, respectively). P-stage, lung age, and COPD history were significantly associated with poor CSS (p =0.003, 0.004, and 0.046, respectively). In multivariate analysis, both p-stage and lung age were independently associated with poor OS (p <0.001 and <0.001, respectively) and poor CSS (p = 0.006 and 0.01, respectively).
Conclusion:
In elderly patients with stage I NSCLC undergoing lobectomy, p-stage and lung age were independent risk predictor for long-term prognosis (OS and CSS); serum creatinine level was associated with short-term mortality; and %DLCO and CVD history were associated with postoperative complications. Our observations from this large cohort are useful for treatment decision making in elderly patients with stage I NSCLC.
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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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P3.04-092 - HNF4α Is a Marker for Invasive Mucinous Adenocarcinoma (IMA) and a Prognostic Factor in Stage I Lung Adenocarcinoma (LADC) (ID 3066)
09:30 - 09:30 | Author(s): P.S. Adusumilli
- Abstract
Background:
According to the 2015 WHO classification, invasive LADC with prominent apical intra-cytoplasmic mucin and small basally oriented nuclei, formerly referred to as mucinous bronchioloalveolar carcinoma, is classified as IMA. Hepatocyte nuclear factor 4 alpha (HNF4α) is a recently recognized marker for IMA although it is also infrequently positive for other subtypes of LADC. However, the prognostic significance of HNF4α is not known. We investigated the frequency of HNF4α expression in IMA as well as non-IMA subtypes, and the prognostic significance of HNF4α in Stage I LADC.
Methods:
Slides from patients with therapy-naive, surgically resected solitary stage I LADC (1995-2009) were subtyped according to the 2015 WHO classification. Tissue microarrays were constructed from each tumor (n=793), and stained for HNF4α. HNF4α expression intensity (0-3) and distribution (1, 1%-50%; 2, 51%-100%) were summed into a total score (0-5) and dichotomized as negative (score <2) or positive (score ≥2). Comparisons were made with TTF-1 expression. Recurrence-free probability (RFP) was estimated using the Kaplan-Meier method, and multivariate analyses were performed using the Cox proportional hazards model.
Results:
32 cases were identified as IMA. Of all LADC, HNF4α was positive in 68 cases (9%) including72% (n = 23) of IMA, 6% (n = 45) of tumors with non-IMA subtypes (P < 0.001). Among non-IMA subtypes, HNF4α was positive in 6% of lepidic, 4% of papillary, 2% of micropapillary, 7% of solid, and 29% of colloid tumors. HNF4α was positive in 12% of KRAS mutant tumors while it was negative in all EGFR mutant tumors (P < 0.001). HNF4α was more frequently positive in TTF-1 negative tumors (40%) than TTF-1 positive tumors (5%; P < 0.001). The RFP for patients with HNF4α-positive tumors was significantly lower than that for patients with HNF4α-negative tumors (P = 0.002) in the entire cohort. This finding was confirmed in subgroup analysis of patients with non-IMA subtypes (P = 0.009). In multivariate analysis, HNF4α was an independent prognostic factor for recurrence (HR=1.61, 95%CI =1.27-2.02, p<0.001).
Conclusion:
HNF4α expression was significantly associated with IMA histology, negative EGFR mutation status, and TTF-1 negativity. Furthermore HNF4α was also expressed infrequently in non-IMA subtypes, however in these patients it was a significant prognostic factor.
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P3.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 226)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.08-038 - Targetable Cancer-Associated Antigens for Immunotherapy in Malignant Pleural Mesothelioma (MPM) - Mesothelin, CA125 and WT-1 (ID 3165)
09:30 - 09:30 | Author(s): P.S. Adusumilli
- Abstract
Background:
Mesothelin (MSLN), CA125 (also know as mucin-16, MUC16) and WT-1 are cancer-associated antigens currently under investigation as targets for tumor-specific immunotherapy, based on published observations that antigen-specific immune responses to these antigens prolong survival. In solid malignancies, we (Clin Cancer Res 2012, 2013) and others have published the role of MSLN in promoting tumor aggressiveness. Additionally, MSLN has been demonstrated to interact with CA125 in promoting invasion and metastasis, resulting in poor clinical outcomes. In this study, we investigated the individual and correlative expressions of MSLN, CA125 and WT-1 in both epithelioid and non-epithelioid MPMs.
Methods:
All available H&E-stained slides from patients who were diagnosed with MPM (1989-2010) were reviewed; tumors were classified according to the WHO classification. We constructed tissue microarrays (6 tumor cores/tumor) from 273 patients (epithelioid=224; non-epithelioid, including biphasic and sarcomatoid =49). MSLN, CA125, and WT-1 immunohistochemistry were performed, and total scores for each antigen were determined by assessing the combined intensity and distribution of the antigen expression.
Results:
Epithelioid MPMs demonstrated positive MSLN expression in 92% of patient samples (73% high-moderate expression), CA125 expression in 72% (19% high-moderate), and WT-1 in 94% (63% high-moderate) (Fig.1A). Triple positive antigen expression was recognized in 68% of patients; co-expression of two antigens was demonstrated in 23% of epithelioid MPMs (Fig.1B). In non-epithelioid MPMs, MSLN, CA125, and WT-1 were positive in 57% (16% high-moderate), 33% (2% high-moderate), and 98% (43% high-moderate) of patient tumors, respectively. Triple and double antigen co-expression were demonstrated in 29% and 33% of non-epithelioid MPMs, respectively. Only 1% of epithelioid and 2% of non-epithelioid MPMs demonstrated absence of expression of all three antigens: MSLN, CA125, and WT-1.Figure 1
Conclusion:
Our observation from a large cohort of MPM patients inclusive of all histological subtypes demonstrating greater than 98% positive expression of at least one of the three cancer-associated antigens in epithelioid and non-epithelioid MPMs, with strong antigen expression and high frequency of double and triple antigen expression, provides rationale to develop targeted therapies to these cancer-associated antigens for the treatment of MPM patients We are initiaiting a phase I clinical trial (NCT02414269) of mesothelin-targeted T-cell therapy for MPM patients at our center.
