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J. Rodriguez-Canales
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MA 05 - Immuno-Oncology: Novel Biomarker Candidates (ID 658)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Immunology and Immunotherapy
- Presentations: 1
- Moderators:Yoichi Nakanishi, P. Mitchell
- Coordinates: 10/16/2017, 15:45 - 17:30, Room 303 + 304
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MA 05.02 - STK11/LKB1 Loss of Function Genomic Alterations Predict Primary Resistance to PD-1/PD-L1 Axis Blockade in KRAS-Mutant NSCLC (ID 10367)
15:50 - 15:55 | Author(s): J. Rodriguez-Canales
- Abstract
- Presentation
Background:
The genomic landscape of primary resistance to PD-1 blockade in lung adenocarcinoma (LUAD) is largely unknown. We previously reported that co-mutations in STK11/LKB1 (KL) or TP53 (KP) define subgroups of KRAS-mutant LUAD with distinct therapeutic vulnerabilities and immune profiles. Here, we present updated data on the clinical efficacy of PD-1/PD-L1 inhibitors in co-mutation defined KRAS mutant and wild-type LUAD patients and examine the relationship between genetic alterations in individual genes, tumor cell PD-L1 expression and tumor mutational burden (TMB) using cohorts form the SU2C/ACS Lung Cancer Dream Team and Foundation Medicine (FM).
Method:
The cohorts included 924 LUAD with NGS (FM cohort) and 188 patients with KRAS non-squamous NSCLC (SU2C cohort) who received at least one cycle of PD-1/PD-L1 inhibitor therapy and had available molecular profiling. Tumor cell PD-L1 expression was tested using E1L3N IHC (SU2C) and the VENTANA PD-L1 (SP142) assay (FM). TMB was defined as previously described and was classified as high (TMB-H), intermediate (TMB-I) or low (TMB-L).
Result:
188 immunotherapy-treated (83.5% nivolumab, 11.7% pembrolizumab, 4.8% anti-PD1/PD-L1 plus anti-CTLA-4) pts with KRAS-mutant NSCLC were included in the efficacy analysis. The ORR differed significantly between the KL (8.8%), KP (35.9%) and K-only sub-groups (27.3%) (P=0.0011, Fisher’s exact test). KL LUAC exhibited significantly shorter PFS (mPFS 1.8m vs 2.7m, HR=0.53, 95% CI 0.34-0.84, P<0.001, log-rank test) and OS (mOS 6.8m vs 15.6m, HR 0.53, 95% CI 0.34 to 0.84, P=0.0072, log rank test) compared to KRAS-mutant NSCLC with wild-type STK11. Loss-of function (LOF) genetic alterations in STK11 were the only significantly enriched event in PD-L1 negative, TMB-I/H compared to PD-L1 high positive (TPS≥50%), TMB-I/H tumors in the overall FMI cohort (Bonferroni adjusted P=2.38x10[-4], Fisher’s exact test) and among KRAS-mutant tumors (adjusted P=0.05, Fisher’s exact test) . Notably, PD-1 blockade demonstrated activity among 10 PD-L1-negative KP tumors, with 3 PRs and 4SDs recorded. In syngeneic isogenic murine models PD-1 blockade significantly inhibited the growth of Kras mutant tumors with wild-type LKB1 (K), but not those with LKB1 loss (KL), providing evidence that LKB1 loss can play a causative role in promoting PD-1 inhibitor resistance.
Conclusion:
Loss of function genomic alterations in STK11 represent a dominant driver of de novo resistance to PD-1/PD-L1 blockade in KRAS-mutant NSCLC. In addition to tumor PD-L1 status and tumor mutational burden precision immunotherapy approaches should take into consideration the STK11 status of individual tumors.
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OA 13 - Immuno-Biology (ID 677)
- Event: WCLC 2017
- Type: Oral
- Track: Immunology and Immunotherapy
- Presentations: 1
- Moderators:Hiroyuki Suzuki, Scott N. Gettinger
- Coordinates: 10/18/2017, 11:00 - 12:30, Room 301 + 302
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OA 13.01 - CD38-Mediated Immunometabolic Suppression as a Mechanism of Resistance to PD-1/PD-L1 Axis Blockade (ID 10157)
11:00 - 11:10 | Author(s): J. Rodriguez-Canales
- Abstract
- Presentation
Background:
Although immune checkpoint inhibitors of the PD-1/PD-L1 axis provide significant clinical benefit for patients with lung cancer, effective use of these agents is encumbered by a high rate of primary or acquired resistance. Strategies for optimal therapeutic application of immunotherapy require a thorough understanding of resistance mechanisms. To date, there have been only a few studies reporting potential mechanisms of resistance to PD-1/PD-L1 blockade.
Method:
In multiple immunocompetent syngeneic and spontaneous animal models of K-ras/p53 mutant lung cancer, we explored the resistance mechanisms to PD-1/PD-L1 blockade using both pharmacologic and genetic approaches (therapeutic antibody treatment and CRISPR/Cas9-mediated editing). The molecular and immune profiles of the tumor microenvironment were evaluated. Additionally, to determine the applicability to patients with lung cancer, we analyzed 259 tumor specimens with IHC staining and mRNA expression, and further confirmed the analyses in publically-available TCGA datasets.
Result:
In multiple models of antibody blockade and genetic knockout of PD-L1, we identified the up-regulation of CD38 on tumor cells as a marker of treatment resistance. Furthermore, by manipulating CD38 on a panel of lung cancer cell lines we demonstrated in vitro and in vivo that CD38 expression inhibits CD8[+] T cell proliferation, anti-tumor cytokine secretion, and tumor cell killing capability. The T cell suppressive effect is dependent upon the ectoenzyme activity of CD38 that regulates the extracellular levels of adenosine. To test whether CD38 blockade might be therapeutically efficacious to prevent anti-PD-L1/PD-1 resistance, we applied combination therapy with anti-CD38 and anti-PD-L1 and demonstrated dramatic therapeutic benefit on primary tumor growth and metastasis. Additionally, in a set of 259 resected lung cancer specimens, ~15% exhibited positive staining for CD38 on tumor cells, and the expression correlated with cytolytic T cell score and an immune/inflammatory signature across multiple large datasets.
Conclusion:
CD38 was found to be a novel mechanism for tumor escape from immune checkpoint PD-1/PD-L1 inhibitor therapy. Targeting this resistance pathway may broaden the benefit of PD-L1/PD-1 axis blockade for lung cancer treatment.
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P2.02 - Biology/Pathology (ID 616)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.02-013 - Investigation of Genomic and TCR Repertoire Evolution of AAH, AIS, MIA to Invasive Lung Adenocarcinoma by Multiregion Exome and TCR Sequencing (ID 9192)
09:30 - 09:30 | Author(s): J. Rodriguez-Canales
- Abstract
Background:
Carcinogenesis may result from accumulation of molecular aberrations (molecular evolution) and escaping from host immune surveillance (immunoediting). It has been postulated that atypical adenomatous hyperplasia (AAH) represents preneoplastic lesion that may progress to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and further to frankly invasive adenocarcinoma (ADC). However, due to lack of appropriate study materials, the molecular and immune landscape of AAH, AIS or MIA have not been well studied and the definition and management of these lesions remain controversial.
Method:
With the intent to delineate the pivotal molecular and immune events during early carcinogenesis of lung adenocarcinoma, we have collected 119 resected pre- and early neoplastic lung lesions including AAH (N=24), AIS (N=27), MIA (N=54) and ADC (N=14) from 53 patients including 41 patients presenting with multifocal lesions and 25 patients carrying more than one type of pathology. Two to five spatially separated regions from each lesion were subjected to whole exome sequencing and T cell receptor sequencing.
Result:
Mutation burden (average SNVs) was found to progressively increase from 1.32/Mb in AAH to 2.55/MB in AIS, 5.42/MB in MIA and 15.38/MB in ADC. Genomic heterogeneity has also become more complex with neoplastic progression with mean Shannon index of 1.53 in AAH, 1.78 in AIS, 1.56 in MIA and 1.79 in ADC. An increase in C>A transversions coincident with a decrease in A>G transitions and progressively increasing APOBEC enrichment scores (4.13 in AAH, 5.63 in AIS, 6.02 in MIA and 6.59 in ADC) were observed with neoplastic disease progression. Furthermore, phylogenetic analysis revealed varying evolutional processes in AAH, AIS, MIA and ADC with canonical cancer gene mutations in KRAS, ATM, TP53 and EGFR etc. as key drivers in a subset of patients. TCR sequencing demonstrated a progressive decrease in T cell density (average percent T cells among all nuclear cells: 12% in AAH, 8% in AIS, 7% in MIA and 4% in ADC) and a progressive decrease in productive TCR clonality (average productive TCR clonality: 0.0434 in AAH, 0.0427 in AIS, 0.0399 in MIA and 0.0395 in ADC) suggesting suppressive T cell repertoire in more advanced diseases.
Conclusion:
Our results provide molecular evidence supporting the model of early lung carcinogenesis from AAH, to AIS, MIA and ADC and demonstrated that with disease progression, genomic landscape of lung neoplastic lesions has become progressively more complex along with progressive immunosuppressive TCR repertoire.
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P2.02-046 - Assessment of PDL1 and Immunoprofiling Using Multiplex Quantitative Immunofluorescence in Lung Cancer: Clinical Implications (ID 10245)
09:30 - 09:30 | Author(s): J. Rodriguez-Canales
- Abstract
Background:
Understanding of the “profile” of PD-L1 expression and its interplay with immune cells will provide important insights into lung cancer pathogenesis, and immunotherapeutic strategies targeting this important immune checkpoint protein. The aim was to investigate the correlation between multiplex immunofluorescence (mIF) expression of PD-L1, density and nature of tumor infiltrating immune cells in non-small cell lung carcinomas (NSCLC), and correlate those profiles with clinical and pathological variables including patient outcome.
Method:
We studied 194 stage II/III patients that underwent pulmonary resection, including 98 adenocarcinoma (ADC), 59 squamous cell carcinoma (SqCC), 15 large cells carcinomas (LCC) and 22 neuroendocrine carcinomas (NEC), primary tumors. Formalin-fixed and paraffin embedded (FFPE) tissue microarrays were constructed with five 1.5 mm cores representative of histologic patterns found in each tumor. mIF was performed using the Opal 7-color fIHC Kit™, scanning in the Vectra™ multispectral microscope and analyzed using the inForm™ software (Perkin Elmer, Waltham, MA). The markers studied were grouped in two 6-antibody panels: Panel 1, AE1/AE3 pancytokeratins, PD-L1 (clone E1L3N), PD-1, CD3, CD8 and CD68; and Panel 2, AE1/AE3, Granzyme B, CD45RO and CD57, FOXP3, and CD20. General linear model was used to evaluate the interaction among primary vs metastatic tumors, histologic type and TAICs and Cox's proportional hazard model for overall survival (OS).
Result:
Fifty-eight % out of 164 tumors were positive for PDL-1+ expression (5% cut-off) in malignant cells (EA1/EA3+). Significant higher levels of PD-L1+ expression were detected in NEC compared with other histologies (ADC, SqCC and LCC) (P=0.006). In the same way, we observed higher densities of cytotoxic T lymphocytes (CD3+CD8+) in NEC when compared with the lowest expression in SqCC (P=0.02). Large cell carcinomas presented high levels of memory/regulatory T cells (CD3+FOXP3+CD45RO+) compared with other histologic types but the difference didn´t achieve statistical significance. No difference was found for CD3+PD-L1+, CD68+PD-L1+, natural killer T lymphocytes (CD3+CD57+) and B lymphocytes (CD20+) among the histologic types. Difference between primary and metastatic tumors was found only for naive/memory T lymphocytes (CD3+ CD45RO+) (P=0.04). High CD3+FOXP3+CD45RO+ and CD3+PDL1+ expression were independent favorable prognostic factor for DFS and OS adjusted by smoking, primary vs metastatic, and histologic type [HR 2.68, 95% (CI 1.37–5.24), P=0.004; HR 2.11 (CI 1.07-4.18, P=0.03].
Conclusion:
High abundance of CD3+PD-L1+ cells and memory/regulatory T cells CD3+FOXP3+CD54RO are favorable prognostic factors for resected NSCLC, highlighting the importance of comprehensive assessment of both tumor and immune cells. Supported by CNPq P246042/2012-5 e CNPq 301411/2016-6; FAPESP 2013/10113-7.
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P3.03 - Chemotherapy/Targeted Therapy (ID 719)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Chemotherapy/Targeted Therapy
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.03-027 - LKB1 Loss Is Associated with Resistance to Anti-Angiogenic Therapy in Non-Small Cell Lung Cancer Mouse Models (ID 10259)
09:30 - 09:30 | Author(s): J. Rodriguez-Canales
- Abstract
Background:
LKB1 is a protein kinase that is mutated and down-regulated in 20-30% of non-small cell lung cancer (NSCLC). LKB1 mutations co-occur with KRAS alterations in 7%-10% of NSCLC, resulting in an aggressive phenotype with short survival. Because LKB1 activates AMPK, the master sensor of cellular energy, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations in the cells. However LKB1 also plays an important role in regulating angiogenesis, likely as a strategy to overcome energetic depletion of tumor microenvironment. Bevacizumab, the human anti-VEGF antibody, improves the PFS and OS of NSCLC patients combined with chemotherapy, but often the benefit is transient and therapeutic resistance occurs. Our laboratory has previously identified alterations in cell metabolism and in vasculature of LKB1-deficient tumors when compared to LKB1 wild type in NSCLC.
Method:
LKB1 KO murine NSCLC cell lines were generated using CRISPR/Cas9 system in a KRAS[G12D] mutant background (LKR10 & LKR13). Syngeneic NSCLC models were established via s.c. injection of LKB1 intact and KO murine cells in immunocompetent mice. After tumors reached 150 mm[3] mice were randomly assigned to treatment groups consisting of vehicle, mouse anti-VEGF and nintedanib. Tumor volumes were measured and compared using student’s t test and samples were collected for vasculature analysis. Survival curves will be calculated using log rank test. Hypoxia experiments were preformed and apoptosis was measured using annexin V and 7ADD staining.
Result:
Treatment with anti-VEGF or nintedanib significantly inhibited tumor progression in LKB1 wt KRAS[G12D] mutant mouse model (p<0.001) but did not show any therapeutic effect in the LKB1 KO KRAS[G12D] group. Furthermore in the LKB1 wt group, the median survival of anti-VEGF and nintedanib treated mice was 111 days and 84 days respectively and 37 days in the vehicle group. No improvement in survival was detected in the LKB1 KO group after treatment with anti-VEGF. In vitro studies showed that LKB1 loss is associated with a decrease in oxygen consumption and enhanced glycolysis. Furthermore LKB1 KO NSCLC cells showed a decrease in apoptosis under hypoxic and low nutrient conditions compared to LKR13 LKB1 wt cells.
Conclusion:
NSCLC LKB1-deficient tumors showed resistance to anti-angiogenic therapy and this effect is driven by the regulation of metabolic adaptations that allow cells to survive under hypoxic and low nutrient conditions.
