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Edwin Parra
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MA17 - Molecular Mechanisms and Therapies (ID 143)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:Eloisa Jantus-Lewintre, Hongbin Ji
- Coordinates: 9/09/2019, 15:45 - 17:15, Melbourne (1991)
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MA17.10 - Lactate Transporter Blockade as a Strategy to Overcome VEGF Inhibitor-Resistance in LKB1-Deficient NSCLC (Now Available) (ID 2647)
15:45 - 17:15 | Author(s): Edwin Parra
- Abstract
- Presentation
Background
STK11/LKB1 alterations are found in 20-30% of NSCLC and used to co-occur with KRAS mutations. Because LKB1 activates AMPK, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations in cells. Our laboratory have previously reported that loss of LKB1 promotes enhanced glycolysis and elevated lactate production and more recently we demonstrated that STK11/LKB1 mutations are the strongest predictors of de novo resistance to immunotherapy in NSCLC. Prior studies have revealed an association between alterations in the LKB1/AMPK pathway and worse clinical outcomes in NSCLC and in patients treated with chemotherapy and bevacizumab. Given the roles of LKB1 in the regulation of cell metabolism and resistance to immunotherapy, it is feasible that LKB1 also impacts on the response to anti-angiogenic therapies.
Method
Xenograft mouse models were established by subcutaneous injection of H460 cells (LKB1-deficient) and H460 LKB1-expressing in nude mice and LKR10 (KRASG12D) LKB1 wild-type (K) or LKB1- knockout (KL) into 129Svmice. Mice were randomized to vehicle or B20-4.1.1 anti-VEGF antibody. Glycolytic activity of LKB1-intact and -deficient NSCLC cells was measured by Seahorse assay. We analyzed gene expression of SLC16A3 (MCT4) by qPCR and Western blot. Genetic disruption of MCT4 in the K and KL cell lines was done using CRISPR-Cas9 and mouse models were established by subcutaneous injection into mice.
Result
Mice bearing LKB1-expressing H460 xenografts treated with anti-VEGF antibody showed a significant decrease in tumor volume (p<0.05) compared with their vehicle-treated counterparts. However, mice bearing LKB1-deficient H460 xenografts showed markedly reduced efficacy of anti-VEGF therapy compared with that in LKB1-expressing xenografts. Anti-VEGF therapy significantly reduced growth of LKR10 K tumors (p<0.001) but not in LKR10 KL tumors. Microvascular density was not increased in KL tumors following anti-VEGF treatment compared to K. Human isogenic LKB1-deficient cells showed a significantly increased rate of glycolysis and lactate secretion compared with cells expressing LKB1. Human and murine LKB1-deficient cells also had increased MCT4 expression compared to K cells. Immunofluorescence and RPPA analysis of tumor samples from the K and KL mouse models showed that KL tumors upregulated MCT4 protein expression compared with K tumors (p<0.0001). The genetic disruption of MCT4 KL tumors significantly improved tumor volume reduction to anti-VEGF therapies in vivo (p<0.001).
Conclusion
LKB1 loss is associated with increased lactate secretion and resistance to VEGF inhibition in NSCLC. The targeting of the lactate transporter MCT4 enhance the sensitivity of LKB1-deficient NSCLC to anti-VEGF therapy.
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MS17 - Pathology of the Future (ID 80)
- Event: WCLC 2019
- Type: Mini Symposium
- Track: Pathology
- Presentations: 1
- Now Available
- Moderators:Erik Thunnissen, Teh-Ying Chou
- Coordinates: 9/10/2019, 14:30 - 16:00, Interlaken (1988)
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MS17.04 - Multiplex Immunohistochemistry (Now Available) (ID 3540)
14:30 - 16:00 | Author(s): Edwin Parra
- Abstract
- Presentation
Abstract
Multiplexed imaging platforms to simultaneously detect multiple epitopes in the same tissue section emerged in the last years as very powerful tools to study tumor immune contexture. These revolutionary technologies are providing a deep methodology for tumor evaluation in formalin-fixed and paraffin-embedded (FFPE) to improve the understanding of tumor microenvironment, new targets for treatment, prognostic and predictive biomarkers, and translational studies. Multiplexed imaging platforms allow the identification of several antigens simultaneously from a single tissue section, core needle biopsies, and tissue microarrays. In recent years, multiplexed immunohistochemistry, immunofluorescence, mass spectometry and other imaging modalities have improved the abilities to characterize the different types of cell populations in malignant and non-malignant tissues, and their spatial distribution in relationship to clinical outcomes. Multiplexed technologies associated with digital image analysis software offer a high-quality throughput assay to study cancer specimens, inc;luding lung cancer, at multiple timepoints before, during and after treatment. The aim of this resentation is to provide a review of multiplexed tissue imaging applied to lung cancer focusing in the use of multiplex immunofluorescence with tyramine signal amplification staining for lung cancer immune profiling and translational research.
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OA15 - Targeted Agents and Immunotherapy for Small Cell Lung Cancer (ID 152)
- Event: WCLC 2019
- Type: Oral Session
- Track: Small Cell Lung Cancer/NET
- Presentations: 1
- Now Available
- Moderators:Raffaele Califano, Charles M Rudin
- Coordinates: 9/10/2019, 14:30 - 16:00, Hilton Head (1978)
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OA15.04 - Genomic and TCR Intratumor Heterogeneity of Small-Cell Lung Cancer by Multiregion Sequencing: An Association with Survival (Now Available) (ID 1458)
14:30 - 16:00 | Author(s): Edwin Parra
- Abstract
- Presentation
Background
Small cell lung cancer (SCLC) is an aggressive cancer. Although sensitive to initial therapy, recurrence is almost inevitable. The molecular mechanisms underlying recurrence are unknown. We have previously demonstrated that complex genomic and T cell receptor (TCR) intratumor heterogeneity (ITH) was associated with increased risks of relapse in non-small cell lung cancers (NSCLC). Genomic ITH and TCR architecture of SCLC and its clinical impact have not been well studied, largely due to lack of tumor specimens as surgery is rarely used to treat SCLC.
Method
We performed multiregion whole-exome sequencing and TCR sequencing of 49 tumor samples from 18 resected limited-stage SCLCs to delineate the immunogenomic ITH of SCLC. We compared the results to those in NSCLC and assessed the association of genomic and TCR attributes with patient’s survival.
Result
On average, 544 mutations/sample were detected. The median proportion of trunk mutations (mutations identified in all regions within the same tumors) was 80.4% versus 70% in NSCLC (TRACERx, Jamal-Hanjani, NEJM, 2017, p=0.08) and all TP53 and RB1 mutations were trunk mutations, suggesting these mutations were early events during carcinogenesis of this cohort of SCLCs. A higher non-synonymous tumor mutational burden (TMB) was associated with a higher T cell density (infiltration) in the tumor (r=0.46, p=0.005). Compared to the TCR repertoire of NSCLC (Reuben, WCLC, 2017), these SCLC tumors demonstrated significantly lower T-cell density (0.05 versus 0.24, p<0.0001), richness (diversity, 1,043 versus 3,666, p<0.0001) and clonality (reactivity, average 0.02 versus 0.15, p<0.0001) despite similar non-synonymous TMB (average 187 in SCLC versus 176 mutations/sample in NSCLC). Only 0.2% to 14.6% of T cells were detectable across all regions from the same tumors, suggesting substantial TCR ITH. Jaccard index (JI), a parameter quantifying TCR ITH was significantly lower in SCLC than in NSCLC (0.06 versus 0.1, p<0.0001) implying higher level of TCR ITH in SCLC than NSCLC. Interestingly, higher T-cell density, richness or clonality appeared to be associated with lower risk of recurrence numerically. Furthermore, higher TCR JI (less degree of ITH) was associated with significantly longer overall survival (HR=0.15, p=0.04).
Conclusion
Limited-stage SCLC tumors have distinct TCR repertoire and genomic ITH architecture. Overall, SCLC may have a more pronounced immunosuppressive microenvironment and higher level of TCR repertoire ITH than NSCLC. Nevertheless, higher degree of T cell infiltration and clonal expansion as well as more homogeneous T cell response may be associated with more favorable clinical outcome in patients with limited-stage SCLC.
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P1.04 - Immuno-oncology (ID 164)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Immuno-oncology
- Presentations: 3
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.04-11 - Depicting the Intra-Tumoral Viral and Microbial Landscape of Localized NSCLC Using Standard Next Generation Sequencing Data (ID 1126)
09:45 - 18:00 | Author(s): Edwin Parra
- Abstract
Background
Studies from our group and others have shown that bacteria and viruses present in the tumor may impact therapeutic responses. In the specific context of non-small cell lung cancer (NSCLC), intra-tumoral viral DNA and bacteria have been reported previously to be linked to therapeutic outcomes. However, the interplay between intra-tumoral microorganisms and the host immune response in NSCLC remains unknown. Moreover, the prognostic and predictive therapeutic value of localized NSCLC-specific microbial composition has yet to be defined.
Method
RNA-sequencing (RNA-seq) (n=82) and whole exome sequencing (WES) (n=80) was performed on surgically resected (pTNM I-III) tumors from lung cancer patients enrolled in the ImmunogenomiC prOfiling of NSCLC (ICON) project. Intra-tumoral bacteria, viruses and fungi were queried with MetaPhlAn2, a bioinformatical analysis pipeline which employs unique clade-specific marker genes, using reads from RNA-seq and WES that did not map to the human genome/transcriptome. Generated data were correlated to patients’ clinicopathologic parameters as well as immune profiling using previously validated multiplex IHC panels based on Vectra 3.0™ multispectral microscopy IHC panels and image analysis (InForm™ 2.2.1 software).
Result
Our analyses revealed that 18.29% (n=15/82) of tumors contained bacterial signatures. The most frequent bacterial signature was related to Escherichia (n=9/15). Moreover, 6.49% (n= 5/77) of tumors had evidence of human viral signatures, including the Epstein-Barr virus (n=1/5). No tumors contained fungal signatures. Preliminary clinicopathologic analyses suggested that patients whose tumors harbor bacterial signatures had a trend towards decreased overall survival (p=0.12). Tumors from former smokers were also more likely to contain bacterial signatures (p=0.11). Preliminary multiplex immune cell IHC analyses did not highlight statistically significant associations with the presence of intra-tumoral bacteria.
Conclusion
Our results suggest that a significant proportion of localized NSCLC tumors may harbor components of the human microbiome. Further studies using larger cohorts and dedicated intra-tumoral microbiome and virome methodologies will be needed to better define these findings and to delineate associations with the local immune infiltrate.
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P1.04-79 - CD73 Expression in Lung Adenocarcinomas and Immunological and Molecular Associations (ID 2412)
09:45 - 18:00 | Author(s): Edwin Parra
- Abstract
Background
Immune checkpoints inhibitors (ICI), in monotherapy or combination with chemotherapy, are the standard of care for lung adenocarcinoma (ADC) patients. Unfortunately, only a restricted number of patients will respond to ICI. Combination therapies such as CD73 inhibitors, are being studied with the goal to achieve synergic effects. CD73 is a membrane-bound protein with immunosuppressive functions. We previously reported that higher immune cell infiltration was associated mainly to CD73 basolateral (BL) expression, in this abstract, we show the correlation of CD73 expression at luminal (L) and BL membrane of ADC malignant cells (MCs), with annotated clinicopathological characteristics, immune and molecular biomarkers.
Method
CD73 IHC expression (clone D7F9A) was evaluated in 106 archived ADCs from patients that underwent surgical treatment without neoadjuvant therapy between February 1999 and February 2012 at MD Anderson Cancer Center (Houston, Texas, USA). We scored % and H-score of CD73 expression at the luminal (L) and basolateral (BL) membrane, we calculated the Total (T) CD73 as the average of L and BL, and classified ADCs in three groups: ‘T High’ (TH) (upper quartile for all tumors); ‘T Low’ (TL); ‘T Neg’ (TN) (<1%). We correlated T, L and BL expression and the three groups with clinicopathological characteristics, mutational status of KRAS and EGFR, TP53, STK11 and Tumor mutation burden (TMB), and cell densities of CD3, CD8, CD68, CD45RO, FOXP3, and Granzyme B, and PD-L1 expression (clone E1L3N) in MCs.
Result
T CD73 expression was found in 76%; BL in 60% and L in 57%; among ADCs with luminal membrane present (n=72), L CD73 was present in 83%. T+ and L+ expression was more frequent in never smokers (p=0.02 and p=0.003). Also higher frequency of L+ was found in older patients (>65) (p=0.01), tumors with non-solid histology patterns (p<0.001), EGFR mutation (p=0.048), non-mutated p53 (p=0.002), negative PD-L1 (p=0.03), and low TMB (<10 mut/MB) (p=0.001). Higher levels of L expression were found in KRAS mutated tumors (p=0.049). Higher BL expression positively correlated with p53 mutated tumors (p=0.038), PD-L1+ in MCs (p=<0.0001), and higher TMB (p=0.040).
Our group analyses revealed that TH and TN were associated with ADCs from patients with >30 pack-year of smoking history (p=0.04), presence of any-solid histology pattern (p=0.03), p53 mutation (p= 0.005) and higher TMB (p=0.003) compared with TL. TH also had higher frequency of PD-L1+ tumors, and a higher cell density of CD3 (p=0.0001), CD8 (p=0.001), CD68 (p=0.048), CD45RO (p=0.036), FOXP3 (p=0.053), and Granzyme B (p=0.024) compared to TL and TN. TN showed higher frequency of STK11 mutation (p=0.034).
Conclusion
Based on the CD73 expression we defined subsets of lung adenocarcinomas that have distinct histological, molecular and immunological characteristics that may play a role in the response to ICI.
Our characterization could help us to understand patient’s response to ICI, and identify patients that could potentially benefit from combination therapies.
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P1.04-83 - Combining Immune Gene Polymorphism and Immune Profile Predicts Brain Metastases and Death in a Brazilian Cohort of Non-Small Cell Lung Carcinoma (ID 2026)
09:45 - 18:00 | Author(s): Edwin Parra
- Abstract
Background
Combining immunotherapies such as anti–PD-1 and anti–CTLA-4 agents, which target complementary pathways in the cancer-immunity cycle, might result in additive or synergistic antitumor activity, and has been suggested to improve survival of patients with advanced non-small cell lung carcinomas (NSCLC). This study was conducted to investigate whether polymorphisms of immune checkpoints genes is associated with the tumor immune cell profile and with the risk of brain metastases and death of patients with advanced stage NSCLC.
Method
A total of 75 NSCLC patients were enrolled. The specimens included 37 (50%) adenocarcinomas (ADC), 25 (33%) squamous cell carcinomas (SqCC), and 13 (17%) large cell carcinomas (LCC). Using multiplex immunofluorescence (mIF) and image analysis, we evaluated programmed death ligand 1 (PD-L1) expression in malignant cells (MCs), CD68+ macrophages, and cells expressing the immune markers CD3, CD8, CD57, CD45RO, FOXP3, PD-1, and CD20. We used high-throughput next-generation sequencing (NGS) to evaluate single nucleotide variants (SNVs) in immune checkpoints genes CD274 (PD-L1), CTLA-4, PDCDL1LG2 (PD-L2), LAG3 and B7H4 (VTCN1) in NSCLC by TruSeq Custom Amplicon Panel (Illumina, Inc., San Diego, CA). A total of five single nucleotide polymorphisms (SNPs) were selected: rs2297136G/A (PD-L1), rs231775A/G (CTLA-4), rs7854303C/T (PD-L2), rs870849T/C (LAG3) and rs10754339G/A (B7H4). Cell phenotype data were then integrated with clinicopathologic characteristic and next-generation sequencing gene profiles.
Result
The patients were 47 men (median age, 78yr) and 22 women (median age, 75yr). Tobacco history was present in 22 (30%) patients. Lymph node and brain metastases were observed respectively in 28 (37%) and 8 (10%) patients, and all the patients with brain metastases also presented lymph node metastases. Twenty-three (31%) of patients received adjuvant therapy. The percentage of MCs-PD-L1+ was < 1% (40%), 1-10% (40%) and > 10% (20%). The density of immune cells/mm2 was CD3+ (330), CD3CD8+ (110), CD3-PD1+ (85), CD68+ (58), CD68-PD-L1+ (2.5), CD3CD45RO+ (260), CD3CD45FOXP3+ (260) and CD20+ (46). The frequency of genotypes was: CTLA-4 (AA=40%; AG=25%; GG=10%), CD274 (AA=32%, GA=26%, GG=17%), PDCDL1LG2 (CC=100%), LAG3 (TC=32%, CC=31%, TT=12%) and VTCN1 (AA=57%, GA=13%, GG=5%). A significant association was found between CTLA-4 and CD20+ lymphocytes (R=-0.32; p=0.02), PD-L1and VTCN1 (R=0.26; p=0.02) and PD-L1 polymorphism and MCs-PD-L1 (R=0.26; p=0.04). Different models to predict risk of death were constructed by Cox Regression analysis. Initially, the model was constructed with patient age, histologic type, N stage, brain metastases and adjuvant treatment (p<0.05). Thus CTLA-4, CD274, PDCDL1LG2, LAG3 and VTCN1 genes were included in the model (p<0.05). The second model excluded PDCDL1LG2, VTCN1 genes and introduced MCs-PD-L1 and CD3CD8+, (p<0.05). The final model, controlled for age, histologic types and adjuvant treatment, reliably predicted low risk of brain metastases and death for patients in N1 stage (β coefficient=3.24; p=0.01), CTLA-4 rs231775 AG genotype (β coefficient=-3.71; p=0.04); CD274 rs2297136 GA genotype (β coefficient= -8.55; p=0.03), LAG3 rs870849 TC genotype (β coefficient=2.16; p=0.04), low density of MCs-PD-L1 (β coefficient=6.43, p=0.04) and high density of CD3+CD8+ lymphocytes (B coefficient=-3.06; p=0.04).
Conclusion
Incorporating immune checkpoints genes polymorphisms into immunoprofiling score improves prediction of brain metastases and death in Brazilian NSCLC and may be promise as combining target therapy.
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P1.09 - Pathology (ID 173)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Pathology
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.09-01 - Immunoprofiling Depends on Molecular Determinants to Predict Metastases and Target Therapy in Non-Small Cell Lung Carcinomas (ID 595)
09:45 - 18:00 | Author(s): Edwin Parra
- Abstract
Background
To gain insight into the pathogenesis and progression of non-small cell lung carcinomas (NSCLCs) by 1) characterizing the tumor microenvironment using multiplex immunofluorescence (mIF), image analysis, and genetic mutation analysis and 2) correlating findings with clinicopathologic characteristics and data on tumor progression and prognosis.
Method
Tissue microarrays from 164 primary tumors from patients with stage I–III NSCLC were examined. The specimens included 94 adenocarcinomas, 51 squamous cell carcinomas, and 19 large cell carcinomas. Using mIF and image analysis, we evaluated programmed death ligand 1 (PD-L1) expression in malignant cells (MCs), CD68+ macrophages, and cells expressing the immune markers CD3, CD8, CD57, CD45RO, FOXP3, PD-1, and CD20 (Figure 1). Cell phenotype data were then integrated with clinicopathologic characteristic and next-generation sequencing gene profiles.
Result
PD-L1 expression by MCs and other cells was associated with specific clinicopathologic characteristics and next-generation sequencing profiles (Figure 2). In addition, higher densities of antigen-experienced T-cells were associated with brain metastases. The most frequent microenvironments in the NSCLC tissues were type II (immunologic ignorance) and type IV (tolerance). Multivariate analysis showed that tumors with 1) brain metastasis, 2) lower densities of T-cells, memory T-cells, and natural killer T-cells, and 3) CD276, CTLA4, MMP-2, and TP53 mutations had worse overall survival compared with tumors without 1) brain metastasis, 2) higher densities of T-cells, memory T-cells, and natural killer T-cells, and 3) tumors without CD276, CTLA4, MMP-2, and TP53 mutations.
Conclusion
We detected cell phenotypes and gene mutations associated with tumor metastases in NSCLC.
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P2.04 - Immuno-oncology (ID 167)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Immuno-oncology
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.04-19 - Neoadjuvant Chemotherapy Is Associated with Immunogenic Cell Death and Increased T Cell Infiltration in Early-Stage NSCLC (ID 1122)
10:15 - 18:15 | Author(s): Edwin Parra
- Abstract
Background
Recent success using immune checkpoint blockade (ICB) in the metastatic setting has raised the need to understand the immune microenvironment (IME) in early-stage disease. Moreover, pre-clinical evidence suggests that cytotoxic agents can modulate this IME. A recent study conducted by our group showed that non-small cell lung cancer (NSCLC) patients who received neoadjuvant chemotherapy followed by surgery (NCT), as compared to patients who received upfront surgery (US), had higher densities of CD3+ lymphocytes and CD68+ tumor-associated macrophages (TAMs). CD3+CD4+ lymphocytes and TAMs also correlated with better clinical outcomes. In this study, we explored the relationships between NCT and the IME by harvesting tumor samples of multiple surgical NSCLC cohorts.
Method
The PROSPECT microarray database was queried in NCT (n=45) and US (n=200) patients to investigate differentially expressed genes related to immunogenic cell death (ICD), susceptibility to CD8+ T cell and NK cell cytotoxicity, priming of antigen presenting cells, immunosuppressive enzymes and intra-tumoral cytokines. Available data from the ImmunogenomiC prOfiling of NSCLC (ICON) and other surgical NSCLC cohorts was evaluated to determine: 1) differential immune profiling using FACS (NCT=17; US=39) and multiplex IHC imaging (NCT=10; US=72); 2) plasma circulating cytokines (NCT=18; US=73); 3) tumor mutational burden (TMB) (NCT=40; US=61). Participants who received NCT or US were excluded according to these criteria: 1) concurrent treatment in addition to NCT; 2) sarcomatoid and small cell histologies; 3) clinical or pathological TNM Stage 4 disease; 4) synchronous malignancies other than lung.
Result
PROSPECT NCT patients expressed increased damage-associated molecular pattern (DAMP) genes (HSPA2, HSPA4, HSPE1, and S100A2; p<0.05) and T cell-related chemotaxis and antigen presentation genes (CXCR7, CD1A; p<0.05). Concordantly, the ICON cohort FACS results showed that NCT patients display increases in: 1) infiltration of CD8+ T cells (p=0.004); 2) proliferating Ki67+CD8+ T cells (p=0.02); 3) tissue resident memory CD8+CD103+ (p=0.02) and CD4+CD103+ non-Treg cells (p=0.01). Trends from the ICON multiplex IHC also highlighted increases in CD8+ T cells (p=0.09), CD20+ cells (p=0.08), as well as PD-L1+ malignant cells (p=0.08) and PD-L1+ TAMs (p=0.08) in NCT patients, the latter finding being supported by increased circulating MCP-1 (p=0.03). TMB was similar between NCT and US groups (p=0.912).
Conclusion
Our data provides the first evidence of ICD (i.e., increased DAMP gene expression) following NCT in human early-stage NSCLC. Furthermore, our data highlights the association of NCT with a favorable IME (i.e., increased T cell infiltration), supporting the rationale of NCT and ICB combinations in localized NSCLC.
