Primary Tumor Tissue Samples Research Articles

Activation of the YY1-UGT2B7 Axis Promotes Mammary Estrogen Homeostasis Dysregulation and Exacerbates Breast Tumor Metastasis.

Metastasis is the most common pathway of cancer death. The lack of effective predictors of breast cancer metastasis is a pressing issue in clinical practice. Therefore, exploring the mechanism of breast cancer metastasis to uncover reliable predictors is very important for the clinical treatment of breast cancer patients. In this study, tandem mass tag quantitative proteomics technology was used to detect protein content in primary breast tumor tissue samples from patients with metastatic and nonmetastatic breast cancer at diagnosis. We found that the high expression of yin-yang 1(YY1) is strongly associated with poor prognosis in high-grade breast cancer. YY1 expression was detected in both clinical tumor tissue samples and tumor tissue samples from mammary-specific polyomavirus middle T antigen overexpression mouse model mice. We demonstrated that upregulation of YY1 expression was closely associated with breast cancer metastasis and that high YY1 expression could promote the migratory invasive ability of breast cancer cells. Mechanistically, YY1 directly binds to the UGT2B7 mRNA initiation sequence ATTCAT, thereby transcriptionally regulating the inhibition of UGT2B7 expression. UGT2B7 can regulate the development of breast cancer by regulating estrogen homeostasis in the breast, and the abnormal accumulation of estrogen, especially 4-OHE2, promotes the migration and invasion of breast cancer cells, ultimately causing the development of breast cancer metastasis. In conclusion, YY1 can regulate the UGT2B7-estrogen metabolic axis and induce disturbances in estrogen metabolism in breast tumors, ultimately leading to breast cancer metastasis. Disturbances in estrogen metabolism in the breast tissue may be an important risk factor for breast tumor progression and metastasis SIGNIFICANCE STATEMENT: In this study, we propose for the first time a regulatory relationship between YY1 and the UGT2B7/estrogen metabolism axis and explore the molecular mechanism. Our study shows that the YY1/UGT2B7/estrogen axis plays an important role in the development and metastasis of breast cancer. This study further elucidates the potential mechanisms of YY1-mediated breast cancer metastasis and the possibility and promise of YY1 as a predictor of cancer metastasis.

BTN1A1 is a novel immune checkpoint mutually exclusive to PD-L1

BackgroundWhile Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing...

First-in-human study of SBRT and adenosine pathway blockade to potentiate the benefit of immunochemotherapy in early-stage luminal B breast cancer: results of the safety run-in phase of the Neo-CheckRay trial

BackgroundLuminal B breast cancer (BC) presents a worse prognosis when compared with luminal A BC and exhibits a lower sensitivity to chemotherapy and a lower immunogenicity in contrast to non-luminal...

THU484 Distinct Genetic Characteristics In Follicular Thyroid Cancer With Different Metastatic Potentials

Abstract Disclosure: S. Kwak: None. E. Lee: None. Y. Park: None. Although most follicular thyroid cancers (FTCs) show excellent prognosis, more than 5% accompany distant metastasis, often diagnosed unexpectedly in the advanced stage. Genetic or genomic markers have been shown to be useful as a prognostic tool in many cancers, but it is unclear whether genetic markers predicting the metastatic potential of FTCs exist. To investigate, 11 pairs of matched formalin fixed paraffin embedded tissue samples of primary (PTM1) and metastatic tumors (MT) from FTC patients with distant metastasis were analyzed by whole exome sequencing. For validation, 78 unmatched FTC samples including 49 well-differentiated tumors, 15 poorly-differentiated tumors and 14 anaplastic thyroid cancer (ATC) samples were analyzed by targeted sequencing. Among the 11 paired PTM1 and MT tumors, 10 showed concordant driver mutations (NRAS, HRAS, DICER1, EIF1AX), and the TERT promoter mutation was concordantly found in 6 pairs. However, the concordance rate of other somatic mutations in paired samples were found to be only 16.2%. Other mutational characteristics such as mutational signatures and CNVs also differed, suggesting the independent subclonal evolution of the paired MT from the PTM1 after metastasis. Interestingly, the frequency of mutations including driver, TERT promoter, and other mutations was lower in primary tumors without metastases (PTM0). Mutational characteristics of PTM0 also differed from those of PTM1 and MT, suggesting that the mutational or molecular characteristics of PTs at the time of diagnosis may distinguish metastatic potential. In a pooled analysis including published data of follicular patterned tumors, we found that metastatic or aggressive tumors were more likely to have the RAS mutation, which tended to be coupled with TERT, DICER1, EIF1AX, and TP53, gradually from minimally invasive FTC, widely invasive FTC to poorly differentiated thyroid cancer (PDTC) and ATC. Transcriptomic profiles from The Cancer Genome Atlas (TCGA) and our data (SNUH) showed RAS(+)/TERT(+) mutated tumors and RAS(+)/TERT(-) tumors had differences in several intracellular signal pathways. Therefore, when follicular neoplasm is diagnosed, NRAS and HRAS mutation tests followed by TERT promoter mutation tests may help diagnose FTCs with metastatic potential. Other genetic mutations could be candidates for a diagnostic or prognostic marker, and large-scale validation studies are warranted.This study was funded by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning (NRF-2019R1A2C2084332). Presentation: Thursday, June 15, 2023

Genomic alterations of cerebrospinal fluid cell-free DNA in leptomeningeal metastases of gastric cancer.

e16022 Background: Leptomeningeal metastases (LM) were rare in gastric cancer (GC), and GC patients with LM (GCLM) generally suffer from poor prognosis. Nevertheless, the clinical utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) was under-investigated in GCLM. Methods: We retrospectively studied 15 GCLM patients, and all patients had paired primary tumor tissue samples and post-LM CSF samples while 5 patients also had post-LM plasma samples. All samples were analyzed using next-generation sequencing (NGS), and the molecular and clinical features were correlated with clinical outcomes. Results: CSF had higher mutation allele frequency ( P=0.015), more somatic mutations ( P=0.032), and more copy-number variations ( P<0.001) than tumor or plasma samples. Multiple genetic alterations and aberrant signal pathways were enriched in post-LM CSF, including CCNE1 amplification and cell cycle-related genes, and CCNE1 amplification was significantly associated with patients’ overall survival ( P=0.0062). More potential LM progression-related markers were detected in CSF samples than in tumor samples, including PREX2 mutation ( P=0.014), IGF1R mutation ( P=0.034), AR mutation ( P=0.038), SMARCB1 deletion ( P<0.001), SMAD4 deletion ( P=0.0034), and TGF-beta pathway aberration ( P=0.0038). Additionally, improvement in intracranial pressure ( P<0.001), improvement in CSF cytology ( P=0.0038), and relatively low levels of CSF ctDNA ( P=0.0098) were significantly associated with better PFS. Lastly, we reported a GCLM case whose CSF ctDNA dynamic changes were well correlated with his clinical assessment. Conclusions: CSF ctDNA could more sensitively detect molecular markers and metastasis-related mechanisms than tumor tissues in GCLM patients, and our study sheds light on utilizing CSF ctDNA in prognostic estimation and clinical assessment in GCLM.

Molecular and phenotypic characteristics influencing the degree of cytoreduction in high-grade serous ovarian carcinomas.

High-grade serous ovarian carcinoma (HGSOC) is the deadliest ovarian cancer subtype, and survival relates to initial cytoreductive surgical treatment. The existing tools for surgical outcome prediction remain inadequate for anticipating the outcomes of the complex relationship between tumour biology, clinical phenotypes, co-morbidity and surgical skills. In this genotype-phenotype association study, we combine phenotypic markers with targeted DNA sequencing to discover novel biomarkers to guide the surgical management of primary HGSOC. Primary tumour tissue samples (n = 97) and matched blood from a phenotypically well-characterised treatment-naïve HGSOC patient cohort were analysed by targeted massive parallel DNA sequencing (next generation sequencing [NGS]) of a panel of 360 cancer-related genes. Association analyses were performed on phenotypic traits related to complete cytoreductive surgery, while logistic regression analysis was applied for the predictive model. The positive influence of complete cytoreductive surgery (R0) on overall survival was confirmed (p = 0.003). Before surgery, low volumes of ascitic fluid, lower CA125 levels, higher platelet counts and relatively lower clinical stage at diagnosis were all indicators, alone and combined, for complete cytoreduction (R0). Mutations in either the chromatin remodelling SWI_SNF (p = 0.036) pathway or the histone H3K4 methylation pathway (p = 0.034) correlated with R0. The R0 group also demonstrated higher tumour mutational burden levels (p = 0.028). A predictive model was developed by combining two phenotypes and the mutational status of five genes and one genetic pathway, enabling the prediction of surgical outcomes in 87.6% of the cases in this cohort. Inclusion of molecular biomarkers adds value to the pre-operative stratification of HGSOC patients. A potential preoperative risk stratification model combining phenotypic traits and single-gene mutational status is suggested, but the set-up needs to be validated in larger cohorts.

Genomic alterations of cerebrospinal fluid cell-free DNA in leptomeningeal metastases of gastric cancer

BackgroundLeptomeningeal metastases (LM) were rare in gastric cancer (GC), and GC patients with LM (GCLM) generally suffer from poor prognosis. Nevertheless, the clinical utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) was underinvestigated in GCLM.MethodsWe retrospectively studied 15 GCLM patients, and all patients had paired primary tumor tissue samples and post-LM CSF samples while 5 patients also had post-LM plasma samples. All samples were analyzed using next-generation sequencing (NGS), and the molecular and clinical features were correlated with clinical outcomes.ResultsCSF had higher mutation allele frequency (P = 0.015), more somatic mutations (P = 0.032), and more copy-number variations (P < 0.001) than tumor or plasma samples. Multiple genetic alterations and aberrant signal pathways were enriched in post-LM CSF, including CCNE1 amplification and cell cycle-related genes, and CCNE1 amplification was significantly associated with patients’ overall survival (P = 0.0062). More potential LM progression-related markers were detected in CSF samples than in tumor samples, including PREX2 mutation (P = 0.014), IGF1R mutation (P = 0.034), AR mutation (P = 0.038), SMARCB1 deletion (P < 0.001), SMAD4 deletion (P = 0.0034), and TGF-beta pathway aberration (P = 0.0038). Additionally, improvement in intracranial pressure (P < 0.001), improvement in CSF cytology (P = 0.0038), and relatively low levels of CSF ctDNA (P = 0.0098) were significantly associated with better PFS. Lastly, we reported a GCLM case whose CSF ctDNA dynamic changes were well correlated with his clinical assessment.ConclusionsCSF ctDNA could more sensitively detect molecular markers and metastasis-related mechanisms than tumor tissues in GCLM patients, and our study sheds light on utilizing CSF ctDNA in prognostic estimation and clinical assessment in GCLM.

Abstract 6642: Rapid tissue donation identifies LAG3 as a potential therapeutic target for ALK fusion positive lung cancer

Abstract Introduction: The rapid tissue donation (RTD) program provides post-therapy primary and metastatic tumor tissue samples at the time of death for the study of resistance mechanisms and the tumor microenvironment to inform therapeutic opportunities. NCCN guidelines (NCCN v5.2022) recommend against PD-1/PD-L1 inhibitor monotherapy in the second-line setting for ALK fusion+ non-small cell lung cancer (NSCLC) due to lack of efficacy, irrespective of tumoral PD-L1 expression. We observed high LAG3 protein expression by multiplex immunofluorescence (mIF) in postmortem ALK fusion-positive tumor tissue samples from 3 RTD donors who were treated with ALK inhibitors. Further studies are recommended to investigate the therapeutic potential of LAG3 inhibitors in this patient population. Case Series: Of the 38 patients with lung cancer tissue collected by RTD, 3 patients had ALK fusion and were treated with ALK inhibitors. Despite the initial response, all 3 patients succumbed to the disease. Tissue samples were collected from primary and multiple metastatic sites after death. Immune checkpoint markers, including LAG3, were quantitated via multiplex immunofluorescence staining, and genomic analyses were performed. Results: Analysis of two tissue microarrays comprised of 130 tumor cores from multiple tissue sites of the first 10 NSCLC RTD donors revealed that 66% of tumor cores had &amp;gt;1% LAG3 expression in CD3+ T cells. Among the three patients with ALK fusion-positive NSCLC, 83% (5 of 6 cores), 85% (6 of 7 cores), and 100% (18 of 18 cores) of the tissue cores had &amp;gt;1% LAG3 expression in T cells. The median LAG3 expression in T cells measured by mIF was 1.8% in 99 tumor cores from the ALK fusion-negative study cohort versus 7.3% in the 31 cores from the ALK fusion-positive subset (p=0.004). Postmortem genomic analyses revealed an ALK p.L1196M resistance mutation in the first patient and an AGK-BRAF fusion in the 2nd patient. No known ALK-TKI resistance mutations were detected in the 3rd patient. Discussion: High-quality post-therapy tumor samples collected by the RTD program enable the investigation of genomic and potential immune-related resistance mechanisms. Such studies can inform potential targets for drug development and the study of new therapies to overcome treatment resistance. Our observation of high LAG3 expression in 3 RTD donors with ALK fusion-positive NSCLC suggests the need for further studies to explore the potential for LAG3 inhibitors in patients with NSCLC whose tumors develop resistance to ALK inhibitors. Citation Format: Hilal Ozakinci, Gina Nazario, Karina Colossi Furlan, Dung-Tsa Chen, Trevor Rose, Matthew B. Schabath, Eric B. Haura, Bruna Pellini, Amer Beg, Theresa A. Boyle. Rapid tissue donation identifies LAG3 as a potential therapeutic target for ALK fusion positive lung cancer. [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 6642.

Abstract OT3-12-01: Immunological predictors of nodal response in breast cancer patients undergoing neoadjuvant therapy

Abstract Immunological predictors of nodal response in breast cancer patients undergoing neoadjuvant therapy Maria Luisa Gasparri1, Ilary Ruscito2, Filippo Bellati2, Fabio Corsi3, Rosa Di Micco4, Oreste D. Gentilini4, Thorsten Kuehn5, Andrea Papadia1, Donatella Caserta2, Lorenzo Rossi6, Arianna Calcinotto7 1 Department of Gynecology and Obstetrics, Ente Ospedaliero Cantonale, Ospedale Regionale di Lugano, Lugano, Switzerland 2 Department of Medical and Surgical Science and Translational Medicine, Sapienza University of Rome, Azienda Ospedaliera Sant’Andrea, Rome, Italy 3 Breast Unit, Department of Surgery, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy; Department of Biomedical and Clinical Sciences "Luigi Sacco", Università di Milano, Milan, Italy 4 Breast Surgery Unit, San Raffaele University Hospital, Milan, Italy 5 Interdisciplinary Breast Center, Department of Gynecology and Obstetrics, Klinikum Esslingen, Esslingen, Germany 6 Oncology Institute of Southern Switzerland, Bellinzona, Switzerland 7 Cancer Immunotherapy lab, IOR Institute of Oncology Research, Bellinzona, Switzerland Background: Almost 20% of breast cancer patients present at diagnosis with clinically positive nodes. Most of these patients undergo neoadjuvant therapy in order to de-escalate the axillary surgery in case of response (sentinel lymph node biopsy, targeted axillary dissection or targeted axillary dissection, instead of an axillary lymphadenectomy). The conversion from positive to negative nodes after neoadjuvant therpy is expected in approximately the 60% of the cases, depending by tumor subtypes. Several models have been proposed with the goal of identifying predictors of nodal response prior to neoadjuvant treatment. The immune system plays a pivotal role in cancer invasion and progression. Its role in treatment response is currently under investigation in several settings. Primary endpoint: to identify a preoperative immune profiling of breast cancer patients with nodal involvement at diagnosis and to correlate the immune changes after neoadjuvant therapy with the nodal response (macrometastases, micrometastasis, isolated tumor cells, complete response). Trial design: It is an international prospective cohort study including breast cancer patients undergoing standard neoadjuvant therapy, who present initially with biopsy-proven axillary lymph node metastasis. Ten immune markers will be analyzed using immunohistochemistry and tissue microarray in primary tumor and nodal tissue samples (tumor associated neutrophils, CD4 lymphocytes, CD8 lymphocytes, T regulatory cells, Macrophages, Follicular dendritic cells(DC), plasmocytoid DC, interdigitant DC, mature DC, Lysosomal associated membrane protein 3). The tissue analysis will be performed on the biopsy collected at diagnosis (prior to neoadjuvant therapy) and during the axillary surgery (after neoadjuvant therapy). Target accrual/sample size: 210 patients Statistical analysis: To compare the distribution of immune cells according to the state of lymph node metastasis, Student’s t test will be performed. Pearson’s chi-square test will be used to evaluate the correlation between immune profile and nodal response, based on clinic-pathological features. Odds ratios (ORs) and 95% confidence intervals (CIs) will be calculated using logistic regression analysis. Multivariable analysis will be performed using the multivariable logistic regression model. Logistic regression models will be used to identify the clinical, pathologic and immunological variables associated with the nodal response. P-values less than 0.05 will be considered significant. Analyses will be performed using Microsoft IBM SPSS® version 20.0 for Mac. Current status: Recruitment has not started yet. Contact information: marialuisa.gasparri@eoc.ch Citation Format: Maria Luisa Gasparri, Ilary Ruscito, Filippo Bellati, Fabio Corsi, Rosa Di Micco, Oreste Davide Gentilini, Thorsten Kuehn, Andrea Papadia, Donatella Caserta, Lorenzo Rossi, Arianna Calcinotto. Immunological predictors of nodal response in breast cancer patients undergoing neoadjuvant therapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-12-01.

Transcriptomic Analysis Reveals Dysregulation of the Mycobiome and Archaeome and Distinct Oncogenic Characteristics according to Subtype and Gender in Papillary Thyroid Carcinoma.

Papillary Thyroid Carcinoma (PTC) is characterized by unique tumor morphology, treatment response, and patient outcomes according to subtype and gender. While previous studies have implicated the intratumor bacterial microbiome in the incidence and progression of PTC, few studies have investigated the potential role of fungal and archaeal species in oncogenesis. In this study, we aimed to characterize the intratumor mycobiome and archaeometry in PTC with respect to its three primary subtypes: Classical (CPTC), Follicular Variant (FVPTC), and Tall Cell (TCPTC), and also with respect to gender. RNA-sequencing data were downloaded from The Cancer Genome Atlas (TCGA), including 453 primary tumor tissue samples and 54 adjacent solid tissue normal samples. The PathoScope 2.0 framework was used to extract fungal and archaeal microbial read counts from raw RNA-sequencing data. Overall, we found that the intratumor mycobiome and archaeometry share significant similarities in CPTC, FVPTC, and TCPTC, although most dysregulated species in CPTC are underabundant compared to normal. Furthermore, differences between the mycobiome and archaeometry were more significant between males and females, with a disproportionate number of fungal species overabundant in female tumor samples. Additionally, the expression of oncogenic PTC pathways was distinct across CPTC, FVPTC, and TCPTC, indicating that these microbes may uniquely contribute to PTC pathogenesis in each subtype. Furthermore, differences in the expression of these pathways were observed between males and females. Finally, we found a specific panel of fungi to be dysregulated in BRAF V600E-positive tumors. This study demonstrates the potential importance of microbial species to PTC incidence and oncogenesis.

Poređenje PD-L1 ekspresije u citološkom i histološkom materijalu seroznog high-grade karcinoma jajnika

Introduction: Neoplastic cells in peritoneal lavage express various proteins with significant prognostic and therapeutic potential. Such expression could differ from the expression in a primary tumor or in metastases. In this research, we compared PD-L1 (programmed cell death ligand-1) expression on ovarian cancer cells in cytological material with its expression on peritoneal metastases and a primary tumor. Materials and methods: The study included 30 patients who had been operated on for high-grade serous ovarian cancer (HGSC) in FIGO IIIC, over the period of one year. Cytoblocks, cytological and tissue microarrays were assembled and immunostained with PD-L1 antibody. For each tumor compartment we determined four PD-L1 expression categories: negative, low, moderate, and strong expression, according to the percentage of membrane positive tumor cells. Moderate and strong positivity together were considered as high PD-L1 expression. Results: Moderate PD-L1 expression was the most frequent pattern in primary HGSC (50%) and in peritoneal metastases (omentum) (60%). Cytological samples mostly showed low PD-L1 expression (57%). Statistical analysis did not show a significant difference in PD-L1 expression between the study groups. We found a positive correlation of PD-L1 expression between different, matched tumor samples in every patient, with statistical significance (p &lt; 0.05) between all the analyzed samples. Conclusion: PD-L1 expression was similar in all three tumor compartments. This could point to similar peritumor regulatory mechanisms of HGSC in primary tumor tissue and cytology tumor samples. Immunohistochemical analysis of the assembled cytoblocks is sufficiently reliable in the assessment of PD-L1 expression on cancer ovarian cells from cytological material.

Association between Intratumoral CD8+ T Cells with FoxP3+ and CD163+ Cells: A Potential Immune Intrinsic Negative Feedback Mechanism for Acquired Immune Resistance.

Acquired immune resistance (AIR) describes a situation in which cancer patients who initially responded clinically to immunotherapies, after a certain period of time, progress with their disease. Considering that AIR represents a feedback response of the tumor against the immune attack generated during the course of immunotherapies, it is conceivable that AIR may also occur before treatment initiation as a mechanism to escape endogenous adaptive antitumor immunity (EAAI). In the present study, we assessed the EAAI in paraffin-embedded breast primary tumor tissue samples and drew correlations with the clinical outcomes. In particular, we analyzed densities of CD8+ cells as elements mediating antitumor cytotoxicity, and of CD163+ and FoxP3+ cells as suppressor elements. We found a direct correlation between the densities of CD8+ cells and of CD163+ and/or FoxP3+ cells in the vast majority of patients' tumors. Importantly, the vast majority of patients whose tumors were overpopulated by CD8+ cells developed AIR, which was characterized by high intratumoral CD163+ and/or FoxP3+ cell densities and reduced overall survival (OS). We also showed that AIR depends on the levels of CD8+ cell-ratios in the tumor center to the invasive margin. Our data suggest that tumors develop AIR only when under a robust endogenous immune pressure.

Ectopic expression of lncRNA MVIH as a potential diagnostic biomarker in cervical cancer.

Cervical cancer (CC) is one of the most common cancers in women. Recent advances in screening and vaccination against the papilloma virus (HPV) have increased protection against CC. However, there is no effective diagnostic biomarker and treatment approach during the course of the disease. The current study is thus aimed to evaluate the changes in the expression of lncRNA associated with microvascular invasion in hepatocellular carcinoma (lncRNA MVIH) and its diagnostic value as a biomarker in CC patients. One-hundred and fifteen (n = 115) pairs of CC primary tumor and marginal non-tumor tissue samples were obtained from Tabriz Valiasr International Hospital (Tabriz, Iran). RNA extraction and cDNA synthesis followed by quantitative reverse transcriptase PCR (qRT-PCR) were considered to investigate alterations in the expression levels of MVIH in patients with CC. The associations between MVIH expression changes and clinicopathological features as well as its potential as a diagnostic biomarker were assessed using SPSS and GraphPad prism software and the receiver operating characteristic (ROC). The expression levels of MVIH were significantly higher in CC tumors as compared to marginal non-tumor samples (p < 0.0001). Overexpression of MVIH was significantly associated with younger age (p = 0.033), lymph node metastasis (p = 0.031), tumor invasion depth (p = 0.035), and squamous cell type of CC (p = 0.019). The ROC analysis for MVIH as a diagnostic biomarker revealed the respective sensitivity and specificity of 67.83 and 80. Overexpression of MVIH in CC tumors suggests its oncogenic role during tumorigenesis. Thus, it may serve as a potential diagnostic biomarker.

Molecular Features in Lymphatic Metastases Reflect the Metastasis Mechanism of Lymph Nodes With Non-Small-Cell Lung Cancers.

Lymphatic metastasis influences clinical treatment and prognosis of patients with non-small-cell lung cancer (NSCLC). There is an urgency to understand the molecular features and mechanisms of lymph node metastasis. We analyzed the molecular features on pairs of the primary tumor and lymphatic metastasis tissue samples from 15 NSCLC patients using targeted next-generation sequencing. The potential metastasis-related genes were screened from our cohort based on cancer cell fraction. After filtering with gene functions, candidate metastasis-related events were validated in the MSK cohort with Fisher’s exact test. The molecular signature and tumor mutational burden were similar in paired samples, and the average mutational concordance was 42.0% ± 28.9%. Its metastatic mechanism is potentially a linear progression based on the metastatic seeding theory. Furthermore, mutated ataxia telangiectasia mutated and Rad3-related (ATR) and tet methylcytosine dioxygenase 2 (TET2) genes were significantly enriched in lymphatic metastases (p ≤ 0.05). Alterations in these two genes could be considered metastasis-related driving events. Mutated ATR and TET2 might play an active role in the metastasis of lymph nodes with NSCLC. More case enrollment and long-term follow-up will further verify the clinical significance of these two genes.

Targeted amplicon sequencing for primary tumors and matched lymph node metastases in patients with extrahepatic cholangiocarcinoma.

Lymph node metastasis (LNM) is one of the most adverse prognostic factors in extrahepatic cholangiocarcinoma (EHCC) cases. As next-generation sequencing technology has become more widely available, the genomic profile of biliary tract carcinoma has been clarified. However, whether LNMs have additional genomic alterations in patients with EHCC has not been investigated. Here, we aimed to compare the genomic alterations between primary tumors and matched LNMs in patients with EHCC. Sixteen patients with node-positive EHCCs were included. Genomic DNA was extracted from tissue samples of primary tumors and matched LNMs. Targeted amplicon sequencing of 160 cancer-related genes was performed. Among the 32 tumor samples from 16 patients, 91 genomic mutations were identified. Genomic mutations were noted in 31 genes, including TP53, MAP3K1, SMAD4, APC, and ARID1A. TP53 mutations were most frequently observed (12/32; 37.5%). Genomic mutation profiles were highly concordant between primary tumors and matched LNMs (13/16; 81.3%), and an additional genomic mutation of CDK12 was observed in only one patient. Genomic mutations were highly concordant between primary tumors and matched LNMs, suggesting that genotyping of archived primary tumor samples may help predict genomic mutations of metastatic tumors in patients with EHCC.

P-175 Local inflammatory biomarkers and their association with systemic inflammation as well as overall survival in primary metastatic gastroesophageal cancer patients

Immunotherapy has recently led to a major breakthrough in the treatment of metastatic gastroesophageal cancer patients. Yet, further analyses of large trials investigating checkpoint inhibitor monotherapy or combination with standard chemotherapy suggest, that only a subgroup of patients might benefit from addition of immunotherapy to chemotherapy. Despite these advances, prognosis remains poor, especially in patients with Caucasian ethnicity. Thus, novel prognostic as well as predictive biomarkers are desperately needed to improve patient management. Further knowledge on the complex local inflammatory mechanisms might help to improve our understanding on the efficacy of immunotherapy. Thus, the aim of this study was to further characterize local inflammatory processes in patients with advanced gastroesophageal cancer and investigate their association with systemic inflammation as well as the overall survival (OS). We analyzed local inflammatory biomarkers for T-cells (CD3, CD8), macrophages (CD68) and immune checkpoint inhibition (Lymphocyte-activation gene 3 (LAG3)) in previously untreated, primary tumor tissue samples of advanced gastroesophageal cancer patients treated at the Medical University of Vienna between 2003 and 2016. FFPE tissue was stained using an automated immunohistochemistry slide staining system (Roche Ventana Medical Systems Inc., Tucson, AZ, USA). Analyses of the immunohistochemical staining was performed by Definiens Tissue Studio 4.0 software. Systemic serum inflammatory parameters including leucocyte levels (WBC), C-reactive protein levels (CRP) and albumin (Alb) as well as the OS was evaluated retrospectively by hospital chart review. Local inflammatory parameters were then associated with systemic parameters and the OS using log-rank test and Kruskal-Wallis-test. Primary tumor tissue samples (localization: 29% esopheageal, 25% gastroesophageal junction, 46% stomach; histological subtype: 77% adenocarcinoma, 23% squamous cell carcinoma) of 48 patients (65% male) were analyzed. CD3 was positive in 5.6%, CD8 in 1.8%, CD68 in 2.4% and LAG3 in 0.24% of analyzed cells. Slides were then divided by the median expression into samples with more positive and less positive cells. The median OS of the cohort was 7.6 months (95%CI 5.3-9.9). Local inflammatory biomarker could not be statistically significantly associated with the OS (CD3: p=0.317; CD8: p=0.905, CD68: p=0.369; LAG3: p=0.428). In addition, no clinically significant association with systemic inflammatory parameters could be identified (CD3: CRP p=0.168, WBC p=0.706, Alb p=0.849; CD8: CRP p=0.214, WBC p=0.275, Alb p=0.340; CD68: CRP p=0.499, WBC p=0.992, Alb p=0.181; LAG3: CRP p=0.766, WBC p=0.384), only Alb was associated with LAG3 expression (p=0.048). Local inflammation might play an important role as a prognostic and predictive tool. However, the understanding of local inflammation in gastroesophageal cancer is still scarce and, thus, further research is warranted to identify promising novel biomarkers. Additional staining for further local inflammatory markers as well as expansion of sample size are underway to further characterize the local inflammatory microenvironment in advanced gastroesophageal cancer patients.

Integrated analysis of transcriptome profiles in lung adenocarcinoma with spread through air spaces.

e21103 Background: Spread through air spaces (STAS) is a recently described invasive pattern of lung cancer with a relatively poor prognosis. The current study aims to evaluate and compare the transcriptome patterns and tumor microenvironment (TME) of STAS positive and negative lung adenocarcinoma (LUAD) cases. Methods: Nineteen LUAD patients who underwent radical surgery were enrolled in the current study, of which 11 were STAS-positive and 8 were STAS-negative. We performed RNA sequencing and data analyzes of the patient’s resected primary tumor tissue samples in a College of American Pathologists accredited laboratory (Genecast Biotechnology Co., Ltd, Wuxi). Results: A total of 841 differently expressed genes (DEGs) were identified between STAS positive and negative groups, including 442 up-regulated and 399 down-regulated DEGs in STAS positive tumors. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that the 841 DEGs were significantly enriched in humoral immune response, cell adhesion molecules, and IL-17 signaling pathways, and so on. Moreover, hypoxia VHL targets up was exceedingly up-regulated in STAS positive tumors through gene set enrichment analysis (GSEA). In addition, the abundance of regulatory T cells (Tregs) showed a borderline significant trend to be higher in STAS positive cases. Conclusions: Our study is the first to report the transcriptome profiles and TME of LUAD patients with STAS. These novel findings may add to further understanding underlying biological mechanisms of STAS occurrence and help developing rationale treatments to deliver more survival benefits for STAS positive LUAD patients.

Concordance of Epidermal Growth Factor Receptor Mutation from Tissue Biopsy and Plasma Circulating Tumor DNA in Treatment-Naïve Lung Adenocarcinoma Patients

Background: Prevalence of Epidermal Growth Factor Receptor (EGFR) mutation in ctDNA in treatment-naïve individuals are not well established in Indonesia. In recent years, ctDNA as a specific and sensitive blood-based biomarker had been developed to detect the mutation. The study was done to understand the concordance and acceptance levels of ctDNA in detecting the gene mutation in lung adenocarcinoma patients. &#x0D; Methods: This study used cross-sectional approach with purposive sampling design in 100 treatment-naïve NSCLC, adenocarcinoma patients. Samples were obtained from bronchoscopy, and blood, which were examined to detect the mutation in formalin-fixed, paraffin-embedded (FFPE) specimens or plasma samples using QIAampDNA Micro Kit. Mutation was calculated by droplet digital PCR (ddPCR). &#x0D; Results: A hundred subjects with primary tumor tissue samples were compared with the plasma samples and mutation was detected in 20 patients (20.0%), 12 (12.0%) on exon 19, 7 (7.0%) on exon 21 and 1 (1.0%) on both exon 19 and 21. Within the plasma samples, mutation was found in 15 patients (15%) with mutation on exon 19 and 21 in 12 (12.0%) and 3 (3.0%) patients, respectively. Within the two samples, concordance of EGFR mutation was 83.0% (83/100, P&lt;0.001; correlation index: 0.42). Assuming presence of mutation as the benchmark, the accuracy of mutation presence in plasma DNA was 60.0% (9/15). Kappa test showed a weak agreement between the mutation in tissues and plasma, with a coefficient of 0.414 (95% CI). &#x0D; Conclusion: Tissue biopsy was still considered as the main option to detect EGFR mutation in lung cancer. More research on ctDNA as the standardized tools to detect the mutation are required.

Expression of Prostate-specific Membrane Antigen (PSMA) in Papillary Renal Cell Carcinoma - Overview and Report on a Large Multicenter Cohort.

Prostate specific membrane antigen (PSMA) is an emerging diagnostic and therapeutic target in prostate cancer. 68Ga-PSMA-labeled hybrid imaging is used for the detection of prostate primary tumors and metastases. Therapeutic applications such as Lutetium-177 PSMA radionuclide therapy or bispecific antibodies that target PSMA are currently under investigation within clinical trials. The expression of PSMA, however, is not specific to prostate-tissue. It has been described in the neovascular endothelium of different types of cancer such as breast cancer, and clear cell renal cell carcinoma (ccRCC). The aim of this study was to analyze PSMA expression in papillary RCC (pRCC) type 1 and type 2, the most common non-ccRCC subtypes, and to evaluate the potential of PSMA-targeted imaging and treatment in pRCC. Formalin-fixed, paraffin-embedded tissue samples of primary tumors were analyzed for PSMA expression by immunohistochemistry. Out of n=374 pRCC specimens from the multicenter PANZAR consortium, n=197 pRCC type 1 and n=110 type 2 specimens were eligible for analysis and correlated with clinical data. In pRCC type 1 PSMA staining was positive in 4 of 197 (2.0%) samples whereas none (0/110) of the pRCC type 2 samples were positive for PSMA in this large cohort of pRCC patients. No significant PSMA expression was detected in pRCC. Reflecting current clinical evaluation of PMSA expression in RCC do not encourage further analysis in papillary subtypes.

Extrachromosomal DNA in HPV-Mediated Oropharyngeal Cancer Drives Diverse Oncogene Transcription.

Human papillomavirus (HPV) plays a major role in oncogenesis and circular extrachromosomal DNA (ecDNA) is found in many cancers. However, the relationship between HPV and circular ecDNA in human cancer is not understood. Forty-four primary tumor tissue samples were obtained from a cohort of patients with HPV-positive oropharynx squamous cell carcinoma (OPSCC). Twenty-eight additional HPV oropharyngeal cancer (HPVOPC) tumors from The Cancer Genome Atlas (TCGA) project were analyzed as a separate validation cohort. Genomic, transcriptomic, proteomic, computational, and functional analyses of HPVOPC were applied to these datasets. Our analysis revealed circular, oncogenic DNA in nearly all HPVOPC, with circular human and human-viral hybrid ecDNA present in over a third of HPVOPC and viral circular DNA in remaining tumors. Hybrid ecDNA highly express fusion transcripts from HPV promoters and HPV oncogenes linked to downstream human transcripts that drive oncogenic transformation and immune evasion, and splice multiple, diverse human acceptors to a canonical SA880 viral donor site. HPVOPC have high E6*I expression with specific viral oncogene expression pattern related to viral or hybrid ecDNA composition. Nonchromosomal circular oncogenic DNA is a dominant feature of HPVOPC, revealing an unanticipated link between HPV and ecDNA that leverages the power of extrachromosomal inheritance to drive HPV and somatic oncogene expression.