Primary Non-small Cell Lung Carcinoma Research Articles

SALTT study: A retrospective analysis of 111 SAlivary gland tumors of Lung and Tracheobronchial Tree

IntroductionPrimary pulmonary salivary gland-type tumours (PPSGT) are rare lung neoplasms arising from submucosal seromucinous glands in the central airway. Methods and resultsWe retrospectively analysed the clinicopathological features of 111 PPSGTs diagnosed at our institute between 2003 and 2021. The mean age at diagnosis was 43.8 years(range 6–78 years) and a male-to-female ratio of 2:1. On imaging, 92 % of cases had centrally located tumours and 37.3 % were early stage. The histopathological types included 70 cases (63 %) of mucoepidermoid carcinoma (MEC), 31 cases (27.7 %) of adenoid cystic carcinoma (ADCC), two cases of myoepithelial carcinoma, one case each of acinic cell carcinoma (ACC), clear cell carcinoma (CCC), epithelial myoepithelial carcinoma (EMC) and 5 others [including adenocarcinoma of minor salivary gland origin(n = 3), carcinoma with sebaceous differentiation(n = 1) and poorly differentiated carcinoma of salivary gland type(n = 1)]. The size of the tumours found in the resection specimens ranged from 1 cm to 13 cm, with an average size of 4.9 cm. High-risk attributes such as lymphovascular invasion (LVI), perineural invasion (PNI), pleural involvement, positive resection margins, and nodal metastasis were identified in 15.3 %, 15.3 %, 13.6 %,15.2 % and 6.7 % of cases, respectively. These attributes were found to be more frequent in ADCC than in MEC. Surgery was the main treatment modality [68/84 (80 %) cases]. ADCC cases had more recurrence and distant metastasis than MEC cases. The 3- year overall-survival (OS) and recurrence-free survival(RFS) were better in patients with age lesser than 60 years(p-value <0.0001), low pT stage (p-value 0.00038) and lower grade of MEC(p-value-0.0067). ConclusionIt is crucial to have an acquaintance with the morphologic spectrum and immunophenotypic characteristics of PPSGT to recognize them in this unusual location. In tandem, it is crucial to differentiate them from conventional primary non-small cell lung carcinoma, as the management protocols and prognostic implications differ significantly.

Concentration of Secreted Frizzled-Related Proteins (SFRPs) in Non-Small Cell Lung Carcinoma Subtypes-A Preliminary Study.

Non-small cell lung carcinoma (NSCLC) is the most common lung cancer worldwide. Secreted frizzled-related proteins (SFRPs) are important tumour suppressors and antagonists of the Wnt signalling pathway, which is linked with cancer development. The aim of this study was to evaluate the concentrations of SFRP1, SFRP2, and SFRP5 proteins in tumour and non-tumour (NT) samples obtained from 65 patients with primary NSCLC. An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of SFRPs in the tissue homogenates. A significantly lower SFRP2 protein concentration was found in the total NSCLC tumour samples and the following NSCLC subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (AC) (p > 0.05, p = 0.028 and p = 0.001, respectively). AC tumour samples had a higher SFRP1 level than NT samples (p = 0.022), while the highest SFRP1 concentration was found in NSCLC samples from patients with clinical stage T4 cancer. Increased concentrations of SFRP1 and SFRP5 were present in stage III NSCLC samples, while the tumour samples with high pleural invasion (PL2) had an increased level of SFRP2. The results from this study suggest that the tumour suppressor or oncogenic roles of SFRPs could be connected with the NSCLC subtype. The levels of SFRPs varied according to the clinicopathological parameters of NSCLC.

Exosomes secreted by metastatic cancer cells promotes epithelial mesenchymal transition in small cell lung carcinoma: The key role of Src/TGF-β1 axis

Exosome-mediated epithelial mesenchymal transition (EMT) is key to cancer metastasis. c-Src is involved in the secretion of exosomes and initiation of EMT. Effects of exosomes from metastatic non-small cell lung carcinoma (NSCLC) cells on the EMT process in primary NSCLC cells were assessed. Levels of c-Src in NSCLC tissues were detected and the influence of exosomes from metastatic NSCLC cells on the exosome secretion and EMT process in primary NSCLC cells was assessed. The expression of c-Src was modulated, and the influence on the secretion of exosomes and EMT initiation was evaluated. The level of c-Src was higher in NSCLC specimen and NSCLC cells with promoted EMT process. The suppression of c-Src inhibited secretion of exosomes. Exosomes from metastatic NSCLC cells enhanced migration and invasion abilities of primary NSCLC cells, which had identical effects to c-Src overexpression. The suppression of c-Src inhibited growth and metastasis of solid tumors as well as secretion of exosomes, while the injection of exosomes with c-Src overexpression promoted lung metastasis. TGF-β1 restored the invasion and migration abilities even with c-Src knockdown. The exosomes from metastatic NSCLC cells with high c-Src expression of can increase c-Src level in primary NSCLC cells, contributing to the promoted EMT process through TGF-β1 pathway.

Brain-Tumor Interface-Based MRI Radiomics Models to Determine EGFR Mutation, Response to EGFR-TKI and T790M Resistance Mutation in Non-Small Cell Lung Carcinoma Brain Metastasis.

Preoperative assessment of epidermal growth factor receptor (EGFR) status, response to EGFR-tyrosine kinase inhibitors (TKI) and development of T790M mutation in non-small cell lung carcinoma (NSCLC) patients with brain metastases (BM) is important for clinical decision-making, while previous studies were only based on the whole BM. To investigate values of brain-to-tumor interface (BTI) for determining the EGFR mutation, response to EGFR-TKI and T790M mutation. Retrospective. Two hundred thirty patients from Hospital 1 (primary cohort) and 80 patients from Hospital 2 (external validation cohort) with BM and histological diagnosis of primary NSCLC, and with known EGFR status (biopsy) and T790M mutation status (gene sequencing). Contrast-enhanced T1-weighted (T1CE) and T2-weighted (T2W) fast spin echo sequences at 3.0T MRI. Treatment response to EGFR-TKI therapy was determined by the Response Evaluation Criteria in Solid Tumors. Radiomics features were extracted from the 4 mm thickness BTI and selected by least shrinkage and selection operator regression. The selected BTI features and volume of peritumoral edema (VPE) were combined to construct models using logistic regression. The performance of each radiomics model was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC). A total of 7, 3, and 3 features were strongly associated with the EGFR mutation status, response to EGFR-TKI and T790M mutation status, respectively. The developed models combining BTI features and VPE can improve the performance than those based on BTI features alone, generating AUCs of 0.814, 0.730, and 0.774 for determining the EGFR mutation, response to EGFR-TKI and T790M mutation, respectively, in the external validation cohort. BTI features and VPE were associated with the EGFR mutation status, response to EGFR-TKI and T790M mutation status in NSCLC patients with BM. 3 Technical Efficacy: Stage 2.

Abstract 5616: Optical metabolic imaging of treatment-naive human NSCLC to determine long-term outcome

Abstract Lung cancer remains the leading cause of cancer deaths in the US and worldwide.​ The five-year survival rate of patients with non-small cell lung carcinoma (NSCLC) remains significantly low given that over half present with locally advanced or metastatic disease at time of diagnosis, and experience tumor recurrence following therapeutic intervention.​Current evaluation techniques to assess treatment response are lacking, given they are implemented several weeks after treatment completion and are solely based on anatomical changes in tumor size, forgoing other criteria such as functional or metabolic changes.​ There is a critical need to identify markers early on following diagnosis that are indicative of patient long-term outcome. Two photon microscopy (TPM) techniques are well-suited to provide non-invasive high-resolution information on cell metabolism within three-dimensional tissue. Specifically, two-photon excited fluorescence imaging of autofluorescent cofactors, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), can be used to metabolically characterize cancerous tissue. The goal of this study is to utilize the optical redox ratio (ORR) of FAD/[NADH+FAD] autofluorescence and NADH fluorescence lifetime decay to identify measurable differences in optical metabolic endpoints of human NSCLC that are indicative of their long-term outcome. Twenty-nine NSCLC specimens were obtained from the Lung Cancer Biospecimen Resource Network. They were resected from the lung prior to the patient receiving therapy. Clinical detail reports were used to evaluate the follow-up data of each patient and classify them into groups that reflected their eventual outcomes: responder (N=21), non-responder (N=18), non-metastatic (N=25), and metastatic (N=14). TPM was used to determine the ORR for each sample. NADH lifetime images were collected and fit to a biexponential model to separate the short (A1) and long (A2) components of free and bound NADH. We observed a significantly higher optical redox ratio for tumors within the metastatic group compared to non-metastatic. ​This is consistent with previous findings from our lab that revealed increases in the ORR with increasing metastatic potential in cells, as well as other studies reporting a decrease in NADH concentration in metastatic cancers.​ Additionally, a significantly shorter mean NADH lifetime in the metastatic tumor group was revealed when compared to non-metastatic. This stands in agreement with other studies highlighting decreases in the average lifetime of NADH in metastatic cells compared to non-metastatic. These results demonstrate the feasibility of using optical imaging of autofluorescence and lifetimes of metabolic cofactors to characterize treatment-naïve primary NSCLC tumors and determine differences in their optical metabolic endpoints that are indicative of their long-term outcome. Citation Format: Paola Monterroso Diaz. Optical metabolic imaging of treatment-naive human NSCLC to determine long-term outcome. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5616.

GATA3 expression in primary lung carcinomas: correlation with histopathologic features and TTF-1, napsin A, and p40 expression

This study assessed the expression of GATA3 in primary lung carcinomas and correlated it with tumor histology and immunostains routinely utilized in the work up of primary lung cancers. Tissue microarrays (TMAs) were constructed from a cohort of 184 non-small cell carcinomas, stained with GATA3, p40, TTF-1, and napsin A, and analyzed semi-quantitatively. All TMA cases with GATA3 expression were further analyzed using corresponding whole slide sections. Positive GATA3 staining was present in 16 cases (9%), including 7 squamous cell carcinomas (SqCCs) (4%), 4 adenocarcinomas (AdCs) (2%), 2 adenosquamous carcinomas (AdSqCs) (1%), 2 large cell carcinomas (LCCs) (1%), and 1 sarcomatoid carcinoma (SC) (<1%). Among tumor histotypes, SqCC was more likely to stain with GATA3 (7/49, 14%), while AdC was less likely (4/111, 4%) (p=0.04). In GATA3-positive cases, high-level expression was observed in 9 cases (56%), including 5 p40-positive SqCCs (3 were nonkeratinizing), 1 p40-positive AdSqC (negative for TTF-1 and napsin A), and 1 AdC (solid), 1 LCC, and 1 SC, each negative for p40, TTF-1, and napsin A). Low-level GATA3 expression was found in 3 AdCs (1 was lepidic and 2 were acinar predominant), 2 SqCCs (keratinizing), 1 AdSqC, and 1 LCC. These findings indicate that GATA3 expression occurs in a minor but significant proportion of primary non-small cell lung carcinomas, most often involves SqCC, and tends to show increasing levels of expression in more poorly differentiated subtypes. Caution should be exercised when interpreting GATA3 expression, and a panel of immunostains should be utilized when assigning tumor origin.

Epidermal Growth Factor Receptor Mutation Profiling Among the Primary and Metastatic Non-small Cell Lung Carcinomas: A Prospective Comparative Analysis on the Cytological Samples

Epidermal Growth Factor Receptor Mutation Profiling Among the Primary and Metastatic Non-small Cell Lung Carcinomas: A Prospective Comparative Analysis on the Cytological Samples

Planning Tumor Volume and Local Control Following Stereotactic Body Radiation Therapy of Early-Stage NSCLC

<h3>Purpose/Objective(s)</h3> Stereotactic body radiation therapy (SBRT) is often used to treat primary early-stage non-small cell lung carcinoma (NSCLC) as an alternative to surgical management. The planning methods used for delivering SBRT differs across institutions with various planning target volume (PTV) margins utilized. The purpose of this study is to evaluate the impact of PTV margin and other factors on local control following SBRT. <h3>Materials/Methods</h3> A total of 283 patients with single lesion early-stage NSCLC treated with SBRT at our institution between 2017-2019 were included in this retrospective analysis. The median age was 75 years, with 57% female gender. Of the treated tumors, 68.6% were peripheral while 31.4% were central. Adenocarcinoma was the most common histology at 35.3% followed by 27.2% squamous cell carcinoma with other/unknown histology in the remaining 37.5% of patients. Mean gross tumor volume (GTV) was 9.7 cc (0.3-92.1) and mean PTV was 33.5 cc (5.3-170). The most common fractionation scheme used was 54 Gy in 3 fractions followed by 48 Gy in 4 fractions with mean biologically effective dose at α/β=10 (BED10) of 149.5 Gy. Local recurrence was based on recurrence of the treated lesion on follow-up imaging, defined as either significant enlargement not consistent with post-treatment radiation fibrosis or significant residual FDG-avidity if a PET/CT scan was obtained. Univariate analysis was performed via Kaplan-Meier analysis to analyze the association of local control with GTV volume, ratio of PTV to GTV volume, BED10, histology, and tumor location. <h3>Results</h3> The median follow-up was 1.6 years. Overall, 30 of the 283 patients (10.6%) developed a local recurrence. Kaplan-Meier analysis of local control and patients with a PTV to GTV ratio greater than the median of 4.74 showed statistically significant better local control at 3-years of 90.9% versus 67.7% (p=0.004). Local control was also significantly associated with GTV volume with 3-year local control of 91.9% for tumors <5 cc versus 67.7% for those >5 cc (p=0.007). The BED10 was significantly associated with local control with a 3-year local control of 82.2% for >150 Gy BED10 versus 78.3% for 100-150 Gy BED10 (p=0.016). Local control was not associated with histology (p=0.146) or central versus peripheral location (p=0.25). <h3>Conclusion</h3> Larger PTV to GTV ratio, smaller tumor volume, and a BED10 of greater than 150 Gy were significantly associated with better local control in patients treated with SBRT for early-stage NSCLC. These factors should be considered when treatment planning for SBRT in these patients.

Population pharmacokinetic study of pemetrexed in chinese primary advanced non-small cell lung carcinoma patients.

The purposes of this study were to identify physiological and genetic factors that contributed to variability of pemetrexed (PEM) exposure and to optimize the dosing regimens for Chinese non-small cell lung carcinoma patients. A prospective population pharmacokinetics (PPK) research was performed in this population. The PEM concentrations of 192 plasma samples from 116 in-hospital patients were detected. All patients were genotyped for polymorphisms. The PPK model of PEM was developed. The pharmacokinetic behavior of PEM was described by a two-compartment model with first-order elimination. The population typical values were as follows: clearance (CL) 8.29 L/h, intercompartmental clearance (Q) 0.10 L/h, central volume of distribution (V1) 18.94 L and peripheral volume of distribution (V2) 5.12 L. Creatinine clearance (CrCl) was identified as a covariate to CL, and ERCC1 (rs3212986) and CYP3A5 (rs776746) gene polymorphisms as covariates to Q. By using empirical body surface area (BSA)-based dosing strategy, PEM exposure decreased with the elevation of CrCl. Contrarily, CrCl-based dosing strategy exhibited a satisfactory efficacy of achieving the target PEM exposure. BSA-based dosing regimen in current clinic practice is not suitable to achieve the target exposure in PEM chemotherapy of Chinese NSCLC patients. Alternatively, renal function-based dosing strategy is suggested.

Tumor cell SYK expression modulates the tumor immune microenvironment composition in human cancer via TNF-α dependent signaling

BackgroundThe expression of SYK in cancer cells has been associated with both tumor promoting and tumor suppressive effects. Despite being proposed as anticancer therapeutic target, the possible role of SYK...

Untargeted Lipidomics of Non-Small Cell Lung Carcinoma Demonstrates Differentially Abundant Lipid Classes in Cancer vs. Non-Cancer Tissue.

Lung cancer remains the leading cause of cancer death worldwide and non-small cell lung carcinoma (NSCLC) represents 85% of newly diagnosed lung cancers. In this study, we utilized our untargeted assignment tool Small Molecule Isotope Resolved Formula Enumerator (SMIRFE) and ultra-high-resolution Fourier transform mass spectrometry to examine lipid profile differences between paired cancerous and non-cancerous lung tissue samples from 86 patients with suspected stage I or IIA primary NSCLC. Correlation and co-occurrence analysis revealed significant lipid profile differences between cancer and non-cancer samples. Further analysis of machine-learned lipid categories for the differentially abundant molecular formulas identified a high abundance sterol, high abundance and high m/z sphingolipid, and low abundance glycerophospholipid metabolic phenotype across the NSCLC samples. At the class level, higher abundances of sterol esters and lower abundances of cardiolipins were observed suggesting altered stearoyl-CoA desaturase 1 (SCD1) or acetyl-CoA acetyltransferase (ACAT1) activity and altered human cardiolipin synthase 1 or lysocardiolipin acyltransferase activity respectively, the latter of which is known to confer apoptotic resistance. The presence of a shared metabolic phenotype across a variety of genetically distinct NSCLC subtypes suggests that this phenotype is necessary for NSCLC development and may result from multiple distinct genetic lesions. Thus, targeting the shared affected pathways may be beneficial for a variety of genetically distinct NSCLC subtypes.

The Anti-Non-Small Cell Lung Cancer Cell Activity by a mTOR Kinase Inhibitor PQR620.

In non-small-cell lung carcinoma (NSCLC), aberrant activation of mammalian target of rapamycin (mTOR) contributes to tumorigenesis and cancer progression. PQR620 is a novel and highly-potent mTOR kinase inhibitor. We here tested its potential activity in NSCLC cells. In primary human NSCLC cells and established cell lines (A549 and NCI-H1944), PQR620 inhibited cell growth, proliferation, and cell cycle progression, as well as cell migration and invasion, while inducing significant apoptosis activation. PQR620 disrupted assembles of mTOR complex 1 (mTOR-Raptor) and mTOR complex 2 (mTOR-Rictor-Sin1), and blocked Akt, S6K1, and S6 phosphorylations in NSCLC cells. Restoring Akt-mTOR activation by a constitutively-active Akt1 (S473D) only partially inhibited PQR620-induced cytotoxicity in NSCLC cells. PQR620 was yet cytotoxic in Akt1/2-silenced NSCLC cells, supporting the existence of Akt-mTOR-independent mechanisms. Indeed, PQR620 induced sphingosine kinase 1 (SphK1) inhibition, ceramide production and oxidative stress in primary NSCLC cells. In vivo studies demonstrated that daily oral administration of a single dose of PQR620 potently inhibited primary NSCLC xenograft growth in severe combined immune deficient mice. In PQR620-treated xenograft tissues, Akt-mTOR inactivation, apoptosis induction, SphK1 inhibition and oxidative stress were detected. In conclusion, PQR620 exerted potent anti-NSCLC cell activity via mTOR-dependent and -independent mechanisms.

Diagnostic Value of Napsin A in Small Biopsy of Non-Small Cell Lung Carcinoma

Background: Accurate sub-classification of non-small cell lung carcinoma (NSCLC) is crucial for the targeted therapy. In lung cancer most of tumors are un-respectable at diagnosis and small biopsy or cytological specimen is commonly the only available material for histopathology diagnosis. Although most of NSCLC can be diagnosed by routine stains, however diagnosis of poorly differentiated or undifferentiated tumors in small biopsy is challenging. Objective: To evaluate the diagnostic value of Napsin A immunohistochemical (IHC) marker in NSCLC small biopsy. Materials and Methods: Forty-six cases of primary NSCLC diagnosed by biopsy with subsequent resection were enrolled in this study. Subtyping of tumors was done according to 2015 World Health Organization criteria. IHC expression of Napsin A was assessed for all the cases. For large cell carcinoma (LCC) cases, Thyroid Transcription Factor 1 (TTF-1) and P63 expression were also tested. Markers expression was correlated with the histological diagnosis of the tumor. Sensitivity, specificity, positive, negative predictive values, and total accuracy of the markers were calculated. Result: In NSCLC cases, the diagnostic sensitivity and specificity of Napsin A were 85.2% and 75% (respectively) with a PPV of 85.2%, NPV of 75.0% and 81.4% accuracy. In LCC cases TTF1 sensitivity was 57.1% and specificity 100%, PPV 100%, NPV 14.3%, accuracy 60.0%. In LCC P63 marker had a diagnostic sensitivity, specificity, PPV, NPV and accuracy of 100%, 85.7%, and 33.3%, 100.0% and 86.7% respectively. Expression of both Napsin A and TTF 1 in LCC cases raised the accuracy to 86.7%. Conclusions: In small biopsy specimens Napsin A can subtype NSCLC with high sensitivity and moderate specificity. Additions of TTF-1/p63 IHC markers can further enhance the accuracy.

Iterative surgical resections in non-small cell lung cancer.

IntroductionWe reviewed our surgical preferences and the prognosis for recurrent and second primary tumors in patients who underwent surgical treatment for non-small cell lung carcinoma (NSCLC).AimWe report our experience with patients undergoing iterative pulmonary resection for lung cancer.Material and methodsAmong patients who underwent anatomical resection for primary NSCLC, those who underwent a second surgical resection between 2010 and 2020 due to recurrent or second primary tumor were included in the study. Operative mortality, survival, and prognostic factors were investigated.ResultsIn total, 77 cases were included: 31 (40.3%) underwent the second resection for the recurrent disease and 46 (59.7%) underwent the second resection for the second primary tumor. Postoperative mortality occurred in 8 (10.4%) patients. All patients with postoperative mortality were in the group that underwent thoracotomy in both surgical procedures. The 5-year survival rate was 46.5%. The 5-year survival of those operated on for recurrent or second primary tumor was 32.8% and 51.1%, respectively (p = 0.81). The 5-year survival rate was 68.8% in patients under the age of 60 years, while it was 27.5% in patients aged 60 years and above (p = 0.004). The 5-year survival was 21.8% in patients with an interval of 36 months or less between two operations and 72.2% in those with a longer interval (p = 0.028).ConclusionsOur study shows that survival results similar to or better than primary NSCLC surgery can be obtained with lower mortality if more limited resections are performed via video-assisted thoracic surgery, especially in young patients. In addition, the prognosis is better in patients with an interval of more than 36 months between two operations.

Abstract B05: mEAK-7 supports self-renewal and radioresistance in metastatic cancer

Abstract Mammalian EAK-7, or MTOR associated protein, eak-7 homolog, (mEAK-7) functions as a crucial regulator of mTOR signaling. Dysregulation of mTOR signaling contributes to the pathogenicity of human carcinomas, so we sought to clarify the role of mEAK-7 in tumorigenesis and metastasis. Bioinformatics analysis revealed that MEAK7 mRNA or gene copy number amplification was observed in a multitude of human carcinomas, including non-small cell lung carcinoma (NSCLC). Tissue microarrays containing sections of primary NSCLC tumors, noncancerous adjacent tissue, and metastatic lymph tissue were used to quantify mEAK-7 and mTOR signaling. Our analyses revealed high mEAK-7 expression and mTOR signaling in metastasized lymph nodes. NSCLC cell lines H1299 and H1975 were used for all in vitro experiments. To examine the role mEAK-7 may play in cancer stem cell (CSC) self-renewal, NSCLC cells were FACS sorted into two groups: CD44+/CD90+ CSCs and non-CSCs. CSCs expressing CD44+/CD90+ demonstrated higher mEAK-7 expression and mTOR activation than non-CSCs, suggesting a role for mEAK-7 in cancer self-renewal and radiation resistance. Following X irradiation-induced DNA damage, 2D clonogenicity and 3D spheroid assays were used to further examine mEAK-7's role in clonogenic potential and self-renewal, respectively. These assays revealed that mEAK-7 is essential for the clonogenic potential and self-renewal of cancer cells in response to DNA damage. Comet assays were used to quantify the DNA damage response, and immunoblot and immunoprecipitation analysis confirmed that mEAK-7 is required for promoting radiation stress. Similarly, siRNA-mediated knockdown of mEAK-7 resulted in reduced DNA damage repair and enhanced cellular apoptosis following genotoxic stress. To do so, mEAK-7 may be interacting with components of the DNA damage response, such as DNA-PK. Immunoprecipitation and mass spectrometry analyses identified that mEAK-7 interacts with DNA-PK, and this interaction is strengthened following treatment with genotoxic stress. Further, DNA-PK interacts with mEAK-7 to regulate S6K2, a crucial target of the mTOR. This suggests crosstalk between the DNA-PK and PI3K pathways, since mEAK-7 interacts with DNA-PK to impact the DNA damage response in human cancer cells. Therefore, DNA-PK inhibitors may be utilized to regulate mEAK-7 and mTOR signaling in specific human carcinomas. Thus, mEAK-7 is a novel marker for NSCLC metastasis; overexpressed in both NSCLC and CSCs; and required for clonogenic potential, spheroid formation, and radiation resistance. Citation Format: Joe T. Nguyen, Fatima S. Haidar, Alexandra L. Fox, Connor Ray, Daniela B. Mendonça, Jin K. Kim, Paul H. Krebsbach. mEAK-7 supports self-renewal and radioresistance in metastatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B05.

Abstract 6060: The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer

Abstract Lung cancer is the most common cause of mortality by cancer. Non-small cell lung carcinoma (NSCLC) accounts for 80% of lung cancers. Although progresses has been realized, resistance to current treatments still remain high and the overall survival is a dismal ~16%. The high expression of TWIST1 in cancers strongly correlates with invasive, resistant, and metastatic cancers, and is attributed to the induction of the epithelial-mesenchymal transition phenotype. Additionally, TWIST1 antagonizes the induction of the oncogene (KrasG12D)-induced senescence in primary NSCLC tumor. As TWIST1 is a pleiotropic transcription factor essential for development, the targeting of the entire TWIST1 protein may cause unintended side effects. Dissecting what are the critical domains of TWIST1 and its crucial downstream transcriptional targets required for TWIST1-dependent radioresistance and pro-tumorigenicity would allow to decipher new therapeutic strategies. In this goal, we generated a transactivation-null TWIST1 mutant by mutation of phenylalanine at position 191 to glycine and aimed to characterize its functions. We generated mouse models utilizing a tetracycline-inducible gene expression system: in this model, treatment with doxycycline allows to control concomitant induction of KrasG12D oncogene expression (R) with TWIST1 (T)/Luciferase expression or with TWIST1F191G mutant (T-F191G)/Luciferase expression, specifically in the lung epithelium using the CCSP promoter-rtTA construct (C). TWIST1 expression in CRT model induced a more aggressive tumor progression and a shorter survival (median= 15,6 weeks) compared to CR model (median= 31 weeks). The expression of TWIST1F191G mutant in CRT-F191G model abrogates these effects (median= 26.7 weeks) showing the importance of the transactivation domain of TWIST1 to fulfill its pro-tumor progression functions. Next, the histological analysis of the lung tumor sections of these models shown that the expression of TWIST1 in CRT mice induced a higher positivity for Ki67 and a reduced positivity for Caspase3. CRT mice also present a decrease for p16 expression compared to CR mice reflecting TWIST1-induced OIS suppression. Additionally, the expression of TWIST1F191G mutant shown a similar profile to CR model potentially indicating a role of TWIST1 transactivation domain in the suppression of OIS. On the other hand, the expression of TWIST1 in NSCLC lines conferred a partial radiation resistance. We also observed that the expression of TWIST1 in AEF line conferred the same resistant profile while the expression of TWIST1F191G increased the sensitivity to radiation. Moreover, the CRT model also presented more resistance to 1 × 15Gy or 10 × 3Gy thoracic irradiation suggesting again that targeting TWIST1 transactivation function would confer a potential new important therapeutic option. Citation Format: Audrey Lafargue, Hailun Wang, Sivarajan T. Chettiar, Rajendra P. Gajula, Kekoa Taparra, Katriana Nugent, Phuoc T. Tran. The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6060.

Concordance of PD-L1 expression and CD8+ TIL intensity between NSCLC and synchronous brain metastases.

Programmed death-ligand 1 (PD-L1) is suggested to be a predictive biomarker in non-small-cell lung carcinoma (NSCLC). However, the differential expression of PD-L1 in primary lung tumor vs. synchronous metastases, especially brain metastasis (BM), remains unclear. This study assessed the concordance of PD-L1 expression on tumor cells and tumor-infiltrating lymphocytes (TILs) and CD8+ TIL intensity between primary lung tumors and synchronous BMs from 24 NSCLC patients. PD-L1, CD3, and CD8 positivity was determined by immunohistochemistry (IHC). PD-L1 scoring was based on the proportion of tumor cells with membranous expression of PD-L1 and the cutoff values <1%, 1–49%, and ≥50%. CD3 and CD8 positivity in TILs was evaluated semi-quantitatively and the proportion of CD3+/CD8+ TILs was determined. PD-L1 expression on tumor cells and TILs was evaluated in relation to CD3+/CD8+ TIL proportions and the intensity of CD8+ TILs between the paired primary lung and BM tissues. In the primary lung tumors, PD-L1 positivity was observed in 25%, 37.5%, and 37.5% cases for the cutoff values <1%, 1–49%, and ≥50%, respectively. PD-L1 expression on tumor cells was strongly correlated between the paired primary lung and BM tissues, in all cutoff groups. However, PD-L1 expression on TILs and the proportion of CD3+/CD8+ TILs were not strongly correlated in all three groups between the paired primary lung tumors and BMs. The intensity of CD8+ TILs was concordant in only 54.16% of the paired primary lung tumors and BMs. This study showed a high concordance of PD-L1 expression in neoplastic cells between primary NSCLC and synchronous BMs.

Chromogenic immunohistochemical quadruplex provides accurate diagnostic differentiation of non-small cell lung cancer

Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung malignancies, the vast majority of which are non-small cell lung carcinomas (NSCLC). Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or by immunohistochemistry, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung. Each marker is represented by a distinct color that can be read by a pathologist, using standard, bright field microscopy. We evaluated the ability of pathologists to differentiate NSCLCs using the multiplexed assay as compared to standard, single marker per slide diaminobenzidine (DAB)-based IHC. All cases in a cohort of 264 NSCLCs showed concordance of information (including positivity of stain, intensity of stain and coverage) between single IHC stains and the multiplex assay. This new multiplex IHC offers the capability to accurately diagnose and sub-classify primary lung NSCLCs, while conserving precious tissue for additional testing.

Functional redundancy of HSPA1, HSPA2 and other HSPA proteins in non-small cell lung carcinoma (NSCLC); an implication for NSCLC treatment

Heat shock proteins (HSPs) are a large group of chaperones considered critical for maintaining cellular proteostasis. Their aberrant expression in tumors can modulate the course of processes defined as hallmarks of cancer. Previously, we showed that both stress-inducible HSPA1 and testis-enriched HSPA2, highly homologous members of the HSPA (HSP70) family, are often overexpressed in non-small cell lung carcinoma (NSCLC). HSPA1 is among the best characterized cancer-related chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously we found that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting possible different roles of these proteins in cancer. Therefore, in this work we investigated the impact of HSPA1 and HSPA2 on NSCLC cell phenotype. We found that neither paralog-selective nor simultaneous knockdown of HSPA1 and HSPA2 gene expression reduced growth and chemoresistance of NSCLC cells. Only blocking of HSPA proteins using pan-HSPA inhibitors, VER-155008 or JG-98, exerted potent anticancer effect on NSCLC cells, albeit the final outcome was cell type-dependent. Pan-HSPA inhibition sensitized NSCLC cells to bortezomib, but not to platinum derivates. Our result suggests the inhibitors of proteasome and HSPAs seem an effective drug combination for pre-clinical development in highly aggressive NSCLC.

Abstract 1180: Impact of the spatial analysis of tumor-associated lymphocytes and tumor-associated macrophages on recurrence at early stage of non-small cell lung carcinoma

Abstract Introduction. The interaction between malignant cells (MCs), stromal cells, tumor-associated lymphocytes (TILs), and tumor-associated macrophages (TAMs) is relevant for non-small cell lung carcinoma (NSCLC) progression. The spatial distribution of those cells may affect the prognosis and can be related to genetic intra-tumor heterogeneity (ITH). The aim of this study was to characterize the immunologic ITH and the spatial distribution of immune cells to MCs in primary NSCLC tumors at early stages using multiplex immunofluorescence (mIF) and image analysis approaches. Material and methods. We studied 33 surgically resected NSCLC cases (adenocarcinomas=23; squamous-cell carcinomas=10) with a history of recurrence in a follow-up of at least 60 months (recurrence, N=13; non-recurrence, N=15). Consecutive FFPE tissue sections were stained with two mIF panels (panel 1: cytokeratin (AE1/AE3), PD-L1, PD-1, CD3, CD8, and CD68; panel 2: AE1/AE3, CD3, CD8, granzyme-B, CD45RO, and FOXP3). Three intra-tumor regions (3mm2 each) per case were selected after gridding the whole tumor section. A total of 99 intratumor regions were scanned and analyzed using Vectra Multispectral-Microscope and InForm-software. From each intratumor region, TILs and TAMs densities, as well as the coefficient of variation, were evaluated. The median distance and the G-Cross area under the curve (AUC) for specific radial distances (10µm, 20µm, and 40µm) were obtained between TILs and TAMs phenotypes to MCs. Results. Recurrence was associated with higher MCs density and TAMs/TILs ratio, and lower TIL densities. A high ITH of cytotoxic T-cells (CTLs) PD-L1+ was associated with worse survival. The distance of TAMs PD-L1+ to MCs PD-L1 negative (60µm vs 25µm) or to MCs PD-L1 positive (25µm vs 13µm) was higher in the non-recurrence group than in recurrence group. Close TAMs PD-L1+ to MCs was associated with worst survival. In a radial distance of 10µm, 20µm, and 40µm, a higher infiltration of CTLs PD-1+, was observed in the group of recurrence than non-recurrence group, surrounding MCs PD-L1 negative (AUC 0.49, 3.80, and 20.03; vs AUC 0.01, 0.16, and 1.29, respectively), and MCs PD-L1 positive (AUC 0.60, 4.35, and 19.90; vs AUC 0.01, 0.20, and 2.20, respectively). A high infiltration of CTLs PD-1+ surrounding MCs, with or without expression of PD-L1, was associated with worse survival. All the differences were statistically significant (P&amp;lt;0.05). Conclusion. Close spatial proximity of antigen-experienced CTLs and TAMs PD-L1+ to MCs are associated with recurrence and poor survival in early stages of NSCLC. We determined that ITH of immune cell densities is associated with recurrence of surgically resected NSCLC. Tumor-immune cell spatial modeling offers a deep understanding of tumor microenvironment that impacts on clinical outcomes. Supported by CPRITRP160668 and UT Lung SPORE grants Citation Format: Alejandro Francisco-Cruz, Edwin R. Parra, Santhoshi N. Krishnan, Souptik Barua, Mei Jiang, Junya Fujimoto, Christine B. Peterson, Priyam Das, Chi-Wan Chow, Jaime Rodriguez-Canales, Carmen Behrens, Neda Kalhor, Annikka Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, Arvind Rao, J. Jack Lee, Cesar Moran, Andrew Futreal, Jianjun Zhang, Ignacio I. Wistuba. Impact of the spatial analysis of tumor-associated lymphocytes and tumor-associated macrophages on recurrence at early stage of non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1180.