Paired Primary Tumors Research Articles

BIOM-20. TUMOR-INTRINSIC AND PERIPHERAL FEATURES ASSOCIATE WITH SURVIVAL AFTER POLIO VIROTHERAPY IN RECURRENT GBM

Abstract BACKGROUND PVSRIPO is a live-attenuated recombinant rhino:poliovirus that mediates antitumor efficacy by engaging antitumor immunity. A subset (~20%) of patients with recurrent GBM (rGBM) survive >24 months after therapy. We previously reported that low tumor mutation burden (TMB) is associated with longer survival after PVSRIPO and immune checkpoint blockade therapy in rGBM, and that low TMB associates with higher inflammatory gene expression signatures in rGBM tumors. METHODS Clinical features were tested for association with survival after PVSRIPO therapy. Whole exome sequencing and RNA-sequencing of tumors were used to correlate mutational landscape, tumor mutation burden (TMB), and gene expression signatures of patient tumors with survival. An in vitro assay that measures inflammatory responses of patient PBMCs to PVSRIPO was performed. An independent cohort of paired primary and recurrent GBM tumors was used to assess longitudinal changes in TMB and gene expression signatures after standard of care treatment. RESULTS A short time to recurrence and low TMB associated with longer survival after PVSRIPO therapy; these features were not prognostic for longer survival in immunotherapy naïve rGBM cohorts. Unexpectedly, higher pre-treatment polio neutralizing antibody titers were also associated with longer survival after PVSRIPO therapy in two independent clinical cohorts. PBMCs from patients surviving longer after PVSRIPO therapy mounted higher TNF, but lower IFN-a responses after in vitro challenge with PVSRIPO. In analysis of paired primary vs recurrent GBM tumors, we discovered that patients with low TMB upon recurrence were more likely to experience increased tumor inflammation and suppression of overall TMB. Low TMB in rGBM tumors was also associated with neoantigen depletion. Collectively, these observations imply that patients with low TMB and/or shorter duration of standard of care therapy may have intact immune surveillance, and that pre-treatment immunological status may dictate survival response to polio virotherapy.

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Genomic landscape of metastatic lung adenocarcinomas from large-scale clinical sequencing.

Background: Metastases are responsible for over 70% of deaths from lung adenocarcinomas. Previous large-scale studies of LUAD mainly focused on primary diseases. We aimed to comprehensively analyze the genomic landscape of metastatic LUADs and elucidate its clinical implications in the context of precision medicine.Methods: We performed retrospective analyses on targeted sequencing data of 3,743 primary tumors and 934 metastases from 4,480 patients with lung adenocarcinomas, and PD-L1 immunohistochemical data of 1,336 primary tumors and 252 metastases from 1,588 LUAD patients.Results: Metastases generally manifested significantly higher mutational burdens and chromosomal instability than primary lung adenocarcinomas. Clinically actionable alterations, including ALK mutations, ALK and ROS1 fusions, and MET copy number gains, were enriched in metastases, particularly metastases to some specific organs/tissues, such as lymph nodes, liver, and brain. PD-L1 expression decreased as the approximate metastatic distance increased. Additional data of paired primary tumors and metastases to lymph nodes and brain validated patterns of actionable alterations and candidates for metastatic drivers. Two evolutionary modes of metastatic dissemination, common origins and distinct origins, were identified in both types of primary-metastasis pairs.Conclusions: Our study showed heterogenous patterns of clinically actionable alterations, PD-L1 expressions, metastatic driver candidates, and evolutionary patterns among multiple types of metastases of lung adenocarcinomas, which may advise the planning of treatments and the identification of novel therapeutic targets.

MED12 exon 2 and TERT promoter mutations in primary and recurrent breast fibroepithelial lesions.

The genetic alterations in the recurrent breast fibroepithelial tumors are poorly understood. In the present study, we aimed to investigate mediator protein complex subunit 12 (MED12) exon 2 and telomerase reverse transcriptase (TERT) promoter mutations in a series of primary and recurrent fibroepithelial tumors. Sanger sequencing for MED12 exon 2 and TERT promoter was performed in 26 pairs of primary and recurrent fibroepithelial tumors (19 pairs of phyllodes tumors and seven pairs of fibroadenomas). The relationship between the genotypes and clinicopathological variables was also analyzed. MED12 mutation was identified in 19 primary tumors (12 phyllodes tumors and 7 fibroadenomas) and 17 recurrences (14 phyllodes tumors and three fibroadenomas). Most recurrent phyllodes tumors retained the original MED12 variants (17/19). Six recurrent fibroadenomas showed different MED12 variants from their paired primary tumors (6/7). TERT promoter mutation was identified in 13 primary phyllodes tumors (13/19) and 15 recurrent phyllodes tumors (15/19). However, it was only identified in one primary fibroadenoma (1/7). Recurrent phyllodes tumors often retained the original MED12 and TERT promoter mutations, while recurrent fibroadenomas often acquired new MED12 mutations. Our findings suggest that recurrent phyllodes tumors may be "true recurrence," and TERT mutant "benign fibroepithelial tumors" should be treated as phyllodes tumors.

TAGLN mediated stiffness-regulated ovarian cancer progression via RhoA/ROCK pathway

BackgroundOvarian cancer (OC) progression is an unmet medical challenge. Since omental metastases were palpated harder than their primary counterparts during cytoreductive surgery of patients with epithelial ovarian cancer (EOC), we were inspired to investigate OC progression from the perspective of biomechanics.MethodsAtomic Force Microscope (AFM) was used to measure the Young’s modulus of tissues. The collagen-coated polyacrylamide hydrogel (PA gel) system was prepared to mimic the soft and stiff substrates in vitro. The effect of TAGLN was evaluated both in vitro and in vivo using transwell assay, immunofluorescence, western blot analysis and immunohistochemistry.ResultsWe quantitatively confirmed that omental metastases were stiffer and more abundant in desmoplasia compared with paired primary tumors, and further demonstrated that matrix stiffness could notably regulate OC progression. Remarkably, TAGLN, encoding an actin cross-linking/gelling protein, was identified as a potent mechanosensitive gene that could form a regulation loop with Src activation reacting to environmental stiffness, thus mediating stiffness-regulated OC progression through regulating RhoA/ROCK pathway.ConclusionsThese data demonstrate that targeting extra-cellular matrix (ECM) stiffness could probably hamper OC progression, and of note, targeting TAGLN might provide promising clinical therapeutic value for OC therapy.

PD37-02 FOXF1 IS A NOVEL REGULATOR OF NODAL METASTASIS IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-02 FOXF1 IS A NOVEL REGULATOR OF NODAL METASTASIS IN BLADDER CANCER Anirban P. Mitra, Andrea Kokorovic, Tanner S. Miest, Vikram M. Narayan, Debasish Sundi, Amy Lim, Sharada Mokkapati, and Colin P. N. Dinney Anirban P. MitraAnirban P. Mitra More articles by this author , Andrea KokorovicAndrea Kokorovic More articles by this author , Tanner S. MiestTanner S. Miest More articles by this author , Vikram M. NarayanVikram M. Narayan More articles by this author , Debasish SundiDebasish Sundi More articles by this author , Amy LimAmy Lim More articles by this author , Sharada MokkapatiSharada Mokkapati More articles by this author , and Colin P. N. DinneyColin P. N. Dinney More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Members of the forkhead transcription factor (FOX) family mediate embryonic development and are altered in many cancers. The functional role of FOXF1 in bladder cancer progression is unclear thus far. We investigated the clinical implications of differential FOXF1 expression in bladder cancer and potential mechanisms by which its alteration can lead to metastasis. METHODS: Whole genome expression profiling performed on paired primary tumors and nodal metastases from a radical cystectomy discovery cohort using Illumina HT12 v3-4 BeadChip arrays identified FOXF1 as a top differentially expressed gene. Prognostic role of differential FOXF1 expression was validated in two independent cystectomy cohorts. Differential FOXF1 expression was also evaluated in murine orthotopic xenografts. Small interfering RNA was used to knock down FOXF1 in RT112 and UC6 bladder cancer cell lines to develop an in vitro model for assessment of metastatic potential. Next-generation sequencing and hierarchical clustering analysis were used to identify differentially altered genes secondary to FOXF1 knockdown. 186 biologically curated pathways were interrogated with internal validation to elucidate downstream biologic mechanisms of metastasis. RESULTS: FOXF1 was a top differentially expressed gene in the discovery cohort with 3.6-fold lower expression in nodal metastases than paired primary tumors (n=33, p<0.001). Multivariable analyses in two validation cohorts (total n=128) indicated that FOXF1 underexpression was associated with worse cancer-specific (p=0.046) and overall survival (p=0.006). Murine orthotopic xenografts (n=13) established from human bladder cancer cell lines (UC3, UC6, UC14) showed FOXF1 underexpression in metastatic deposits compared with primary tumors (p=0.004). Hierarchical clustering identified 40 differentially expressed genes between FOXF1-knockdown bladder cancer cell lines and their corresponding controls. Biological pathway interrogation showed differential enrichment for genes associated with mitogen-activated protein kinase signaling, focal adhesion and other carcinogenic pathways in FOXF1-knockdown cells compared with controls (normalized enrichment score ≥1.3). CONCLUSIONS: We identify and characterize FOXF1 as a novel regulatory molecule that drives bladder cancer metastasis. This may be modulated through alterations in intracellular signaling and cellular adhesion. FOXF1 may serve as a prognostic biomarker that can identify patients at impending risk for metastasis who may benefit from more aggressive management. Source of Funding: The University of Texas MD Anderson SPORE in Genitourinary Cancer P50CA091846, NIH/NCI under P30CA016672 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e655-e655 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Anirban P. Mitra More articles by this author Andrea Kokorovic More articles by this author Tanner S. Miest More articles by this author Vikram M. Narayan More articles by this author Debasish Sundi More articles by this author Amy Lim More articles by this author Sharada Mokkapati More articles by this author Colin P. N. Dinney More articles by this author Expand All Advertisement Loading ...

RADI-11. Evaluating the Tissue Effects of Dose-escalated Pre-operative Stereotactic Radiotherapy for Resectable Brain Metastasis

BackgroundAlthough the classic radiobiologic principles of radiotherapy are well understood, the unique effects of the large fractional does that characterize stereotactic radiotherapy (SRT), specifically in terms of antitumor immune cellular processes, vascular damage, tumor necrosis, and apoptosis on brain metastasis have yet to be adequately demonstrated. The objective of this study is to provide the first in-human evaluation of the biological effects of SRT in resected brain metastasis.MethodsAll paired primary tumors and metastases for patients who underwent dose-escalated preoperative SRT followed by resection were evaluated for tumor necrosis using hematoxylin-eosin staining. T cells (CD3+, CD4+, CD8+), natural killer cells (CD56+), vessel density (CD31+), and apoptotic factors (caspase-3) were determined by immunohistochemical analysis.ResultsFifteen patients with brain metastases from solid tumors received a median preoperative SRT dose of 18 Gy (range: 15–18 Gy) in 1 fraction, with 2 patients receiving 27–30 Gy in 3–5 fractions, followed by resection within a median interval of 90 hours (Range: 17.1–260 hours). The rate of necrosis was found to be significantly higher in irradiated brain metastases than in non-irradiated primary tumor samples (mean paired difference: 30.47, SD: 29.28, p=0.001). A decrease in all immunomodulatory cell populations was found in irradiated metastasis: CD3 (mean paired difference -19.4, SD: 31.7, p=0.03), CD4 (-10.0, SD: 20, p=0.01), and CD8 (-17.4, SD: 22.1, p=0.008). While irradiated samples had numerically lower CD 31+, CD 56+, and caspase-3 scores, the difference was not statistically significant. Time interval from SRT to surgery had no effect on these parameters.ConclusionsThere is complex interplay between tumor-associated cells and the unique radiobiological effects of SRT on tumor tissue. Although time interval from SRT to surgery was associated with increased tumor necrosis, differences in immunomodulatory factors may be multifactorial, including concurrent corticosteroids or the immunosuppressive effect of SRT.

TMPRSS4 overexpression promotes the metastasis of colorectal cancer and predicts poor prognosis of stage III-IV colorectal cancer.

To study in detail the expression pattern and prognostic significance of TMPRSS4 in colorectal cancer. The expression of TMPRSS4 protein was determined using Western blot in the colorectal cancer tissues and normal tissues. Immunohistochemistry was used to detect the TMPRSS4 expression in colorectal cancer tissues, and the clinicopathologic characteristics and prognostic significance were analyzed. TMPRSS4 overexpression was associated with tumor budding, lymphovascular invasion, perineural invasion, cancerous emboli, infiltration depth, lymph node metastasis, distant metastasis, and tumor node metastasis stage (P < 0.05 for all). Interestingly, TMPRSS4 expression in the tumor budding, tumor emboli, lymph node, and liver metastatic tumor samples was higher than in the paired primary tumors. In contrast, TMPRSS4 overexpression is inversely correlated with both the overall survival and the disease-free survival of the patients with colorectal cancer (P < 0.05 for both). Also, we found that TMPRSS4 is only of significance in predicting the prognosis of stage III and IV colorectal cancer, not stage I and II. TMPRSS4 was shown to be involved in the whole process of metastasis from tumor budding to lymph node and/or distant metastasis in colorectal cancer and predicted the unfavorable prognosis of stage III-IV, indicating that it is a novel target for the precise treatment of colorectal cancer with lymph node or distant organ metastasis.

Abstract 350: Co-expression and predictive value of HER family members for the response to therapy with cetuximab in patients with metastatic colorectal cancer

Abstract Background: Overexpression of the EGFR is common in patients with a wide range of cancers and the EGFR is an important target for therapy with monoclonal antibodies (mAbs) based drugs. Of these, cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer (mCRC). However, therapeutic benefits are seen in a subset of such patients and the duration of response can also be short. Indeed, there is currently no reliable predictive biomarkers for the selection of patients who benefit from therapy with the anti-EGFR antibodies. While KRAS mutation is currently used as an important negative predictive biomarker for the response to therapy with anti-EGFR mAbs, not all patients with wild type KRAS gain benefit from therapy with anti-EGFR mAbs and objective responses have been reported in mCRC patients with KRAS mutations. Aim: The aim of this study was to investigate the predictive value of EGFR and its dimerization partner (i.e. all other members of the HER family) for the response to treatment with cetuximab in patients with wild-type KRAS status mCRC. Methods: Using immunohistochemistry (IHC) we investigated the co-expression and predictive value of all four members of the HER family, for the response to cetuximab in 144 mCRC patients and in 21 paired primary tumours and their metastatic sites. Results: Co-expression of all four members of the HER family were found in 10% of the cases examined. The expression of wtEGFR was an indicator of poorer overall survival and the membranous expression of HER2 and HER3 3+ intensity was associated with a shorter progression free survival (PFS). In contrast, the cytoplasmic expression of HER2 was associated with better PFS. In 48% and 71% of the cases examined, there were discordance in the expression of EGFR or one or more HER family members in paired primary and related metastatic tumours, respectively. Conclusion: Our results implicate the importance of large prospective investigation of the expression level and predictive value of not only the therapeutic target (i.e. EGFR protein) but also its dimerization partners for the response to therapy with anti-EGFR mAbs and other forms of HER inhibitors in both the primary tumours and metastatic sites in colorectal cancer. Citation Format: Said A. Khelwatty, Soozana Puvanenthiran, Sharadah Essapen, Izhar Bagwan, Alan M. Seddon, Helmout Modjtahedi. Co-expression and predictive value of HER family members for the response to therapy with cetuximab in patients with metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 350.

Hypermethylation of Genes in New Long Noncoding RNA in Ovarian Tumors and Metastases: A Dual Effect.

The role of methylation in the regulation of genes of long noncoding RNA (lncRNA) is still poorly understood. We revealed new hypermethylated lncRNA genes in ovarian tumors and their effect on metastasis of ovarian cancer. A multiple and significant (p<0.001) increase in methylation of a group of lncRNA genes (MEG3, SEMA3B-AS1, ZNF667-AS1, and TINCR) was shown by quantitative methylation-specific PCR using the non-parametric Mann-Whitney test. Moreover, methylation of SEMA3B-AS1, ZNF667-AS1, and TINCR genes in ovarian cancer tumors was detected for the first time. Comparative analysis of 19 samples of peritoneal metastases and paired primary tumors showed a significant decrease in the methylation level of the same 4 genes: MEG3 (p=0.004), SEMA3B-AS1 (p=0.002), TINCR (p=0.002), and ZNF667-AS1 (p<0.001). Reduced methylation of suppressor lncRNA genes in peritoneal metastases is probably associated with the involvement of these lncRNA in the regulation of plastic reversion of the epithelial-mesenchymal transition to the mesenchymal-epithelial transition. Thus, the effect of lncRNA and their methylation on the development of tumors and metastases of ovarian cancer was demonstrated, which is important for understanding of the pathogenesis and mechanisms of metastasis of ovarian cancer. New properties of lncRNA can find application in the development of new approaches in the therapy of ovarian cancer.

Genomic and Transcriptomic Analyses of Breast Cancer Primaries and Matched Metastases in AURORA, the Breast International Group (BIG) Molecular Screening Initiative.

AURORA aims to study the processes of relapse in metastatic breast cancer (MBC) by performing multi-omics profiling on paired primary tumors and early-course metastases. Among 381 patients (primary tumor and metastasis pairs: 252 targeted gene sequencing, 152 RNA sequencing, 67 single nucleotide polymorphism arrays), we found a driver role for GATA1 and MEN1 somatic mutations. Metastases were enriched in ESR1, PTEN, CDH1, PIK3CA, and RB1 mutations; MDM4 and MYC amplifications; and ARID1A deletions. An increase in clonality was observed in driver genes such as ERBB2 and RB1. Intrinsic subtype switching occurred in 36% of cases. Luminal A/B to HER2-enriched switching was associated with TP53 and/or PIK3CA mutations. Metastases had lower immune score and increased immune-permissive cells. High tumor mutational burden correlated to shorter time to relapse in HR+/HER2- cancers. ESCAT tier I/II alterations were detected in 51% of patients and matched therapy was used in 7%. Integration of multi-omics analyses in clinical practice could affect treatment strategies in MBC. SIGNIFICANCE: The AURORA program, through the genomic and transcriptomic analyses of matched primary and metastatic samples from 381 patients with breast cancer, coupled with prospectively collected clinical data, identified genomic alterations enriched in metastases and prognostic biomarkers. ESCAT tier I/II alterations were detected in more than half of the patients.This article is highlighted in the In This Issue feature, p. 2659.

Exosome-derived microRNAs in oral squamous cell carcinomas impact disease prognosis

ObjectivesmicroRNA (miRNA) expression patterns have provided new insight as biomarkers of prognosis as well as novel therapeutic targets for several neoplasms. However, the role of exosomal miRNA in the prognosis of oral squamous cell carcinoma (OSCC) has not yet been completely clarified. Paired primary tumor and normal oral epithelial cells from OSCC patients were obtained, and the exosomal miRNA profiles between them were compared by miRNA microarray analysis. The miRNA levels in the serum exosomes of OSCC patients were verified by real-time quantitative reverse transcription PCR (qRT-PCR) analysis. Finally, the biological functions and the potential as a prognostic marker of the selected miRNA candidates were analyzed in the OSCC cells and patients, respectively. ResultsExosomal miR-155 and miR-21 were significantly upregulated, and exosomal miR-126 was dramatically downregulated in the primary OSCC cells and the serum of OSCC patients. In the analysis of oncogenic behaviors, coculture with either miR-155-rich or miR-21-rich exosomes could promote cell proliferation and invasion accompanied with downregulation of PTEN and Bcl-6 tumor suppressors. Moreover, treatment with miR-126-rich exosomes inhibited oncogenic behaviors and oncogene EGFL7 expression in OSCC cells. Finally, exosomal miR-126 was reduced in the serum of the late-staged OSCC patients, and downregulation of blood exosomal miR-126 was associated with poor survival in OSCC patients. ConclusionExosomal miR-155 and miR-21 are oncogenic miRNAs which suppress PTEN and Bcl-6 expression, and exosomal miR-126 acts as a tumor suppressor which downregulates EGFL7 in OSCC. Furthermore, blood exosomal miRNAs may serve as biomarkers for the diagnosis and prognosis of OSCC.

Dissecting Early Relapse in Liver Cancer, One Cell at a Time.

In a recent issue of Cell, Sun Y, Wu L, Zhong Y, et al. Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma. Cell 2021;184:404-421 e416, performed single-cell RNA sequencing (scRNAseq) to study the underlying mechanisms associated with the recurrence of hepatocellular carcinoma (HCC) and address this unmet clinical need (1). The analysis included tumors from 12 treatment-naïve primary liver cancer patients, 6 early-relapsed patients, and 4 patients with paired primary and early-relapsed tumors.

Involvement of Neutrophils in Metastatic Evolution of Pancreatic Neuroendocrine Tumors.

Simple SummaryThe neutrophil-to-lymphocytes (NLR) reflects the systemic inflammation. Based on a cohort of 144 patients treated for localized or metastatic well-differentiated pancreatic neuroendocrine tumors (pNET), we identified the NLR ≥ 4 to be associated with worse overall survival. Using MCP-Counter on a publicly available pNET RNA-sequencing dataset, we inferred the tumor microenvironment composition of 83 primary pNET and 30 liver metastasis. The neutrophils scores were statistically higher in liver metastasis relative to primary pancreatic tumors (p = 0.005). Gene set enrichment analysis further revealed activation of complement pathway signature in liver metastasis. Through combination of the neutrophil and complement pathway genes, we found that pNET can be classified into two clusters: Neu-Comp1 and Neu-Comp2. Notably, the Neu-Compt1 cluster was enriched in neutrophils and complement pathway signatures and was associated with liver metastasis. These data offer new insights into the role of inflammatory factors in the metastatic progression of the pNET.Well-differentiated pancreatic neuroendocrine tumors (pNET) have an unpredictable natural history. The identification of both blood and tumor immune features associated with patients’ outcomes remains limited. Herein, we evaluated the best prognostic value of the neutrophils-to-lymphocyte ratio (NLR) in a cohort of 144 pNETs. The NLR ≥ 4 was associated with worse overall survival in both univariate analysis (HR = 3.53, CI95% = 1.50–8.31, p = 0.004) and multivariate analysis (HR = 2.57, CI95% = 1.061–6.216, p = 0.036). The presence of synchronous liver metastasis was identified as a prognostic factor in multivariate analysis (HR = 3.35, CI95% = 1.411–7.973, p = 0.006). Interestingly, the absolute tumor-associated neutrophils count was higher in liver metastasis as compared to their paired primary tumor (p = 0.048). Deconvolution of immune cells from the transcriptome of 83 primary tumors and 30 liver metastases reveals enrichment for neutrophils in metastasis relative to primary tumors (p = 0.005), and this was associated with upregulation of the complement pathway (NES = 1.84, p < 0.0001). Combining neutrophils signature and complement pathway genes, unsupervised clustering identified two pNETs subgroups, namely Neu-Comp1 and Neu-Comp2. Characterized by neutrophils infiltration and activation of the complement pathway, Neu-Comp1 was highly enriched for metastatic liver samples as compared to Neu-Comp2 (p < 0.0001). These data suggest the possible link between liver metastasis, complement pathway activation, and neutrophils infiltration in well-differentiated pNET and open avenues for targeting complement pathways in these tumors.

ScDPN for High-throughput Single-cell CNV Detection to Uncover Clonal Evolution During HCC Recurrence

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution. Unfortunately, classical DNA amplification-based methods have low throughput and introduce coverage bias during sample preamplification. We developed a single-cell DNA library preparation method without preamplification in nanolitre scale (scDPN) to address these issues. The method achieved a throughput of up to 1800 cells per run for copy number variation (CNV) detection. Also, our approach demonstrated a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy for cell line and tumor tissue evaluation. We used this approach to profile the tumor clones in paired primary and relapsed tumor samples of hepatocellular carcinoma (HCC). We identified three clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumor containing additional alterations in chromosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumor, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome heterogeneity and evolution

Tagln Mediated Stiffness-Regulated Ovarian Cancer Progression Via RhoA/ROCK Pathway

Background: Ovarian cancer (OC) progression is an unmet medical challenge. Since omental metastases were palpated harder than their primary counterparts during cytoreductive surgery of patients with epithelial ovarian cancer, we were inspired to investigate OC progression from the perspective of biomechanics. Methods: Atomic Force Microscope (AFM) was used to measure the young’s moduli of tissues. The collagen-coated polyacrylamide hydrogel (PA gel) system was prepared to mimic the soft and stiff substrates in vitro. The effect of TAGLN were evaluated both in vitro and in vivo using transwell, immunofluorescence, western blot analysis and immunohistochemistry. Findings: We quantitatively confirmed that omental metastases were stiffer and more abundant in desmoplasia compared with paired primary tumors, and further demonstrated that matrix stiffness could notably regulate OC progression. Remarkably, TAGLN, encoding an actin cross-linking/gelling protein, was identified as a potent mechanosensitive gene that could form a regulation loop with Src activation reacting to environmental stiffness, thus mediating stiffness-regulated OC progression through regulating RhoA/ROCK pathway. Interpretation: These data demonstrate that targeting extra-cellular matrix stiffness could probably hamper OC progression, and of note, targeting TAGLN might provide promising clinical therapeutic value for OC therapy. Funding: This work was supported by the “973” Program of China (No. 2015CB553903 to Ding Ma and Junbo Hu), Technical Innovation Special Project of Hubei Province (2018ACA138), the Fundamental Research Funds for the Central Universities (2019kfyXMBZ024), National Science and Technology Major Sub- Project (2018ZX10301402-002), and the National Science Foundation of China (81902661, 81772787, 82072889). Declaration of Interest: None to declare. Ethical Approval: Human ovarian cancer tissues were all obtained from patients undergoing surgery at the Department of Obstetrics and Gynecology, Tongji Hospital, Huazhong University of Science and Technology after obtaining written informed consent of the patients and the authorization of the Ethics Committee of Tongji Hospital (TJ-iRB20181103). Fresh tissues were prepared for the Atomic Force Microscope (AFM) measurement, frozen or formalin fixed.

Phase II trial of toripalimab plus chemotherapy as neoadjuvant treatment in resectable stage III non-small cell lung cancer (NeoTPD01 Study).

8541 Background: Multi-modality treatment provides modest survival benefits for locally advanced non-small-cell lung cancer (NSCLC) patients. Preoperative immunotherapy has continuously been shown to be promising in treating resectable NSCLC. The current study aimed to investigate the activity and safety of the PD-1 inhibitor, toripalimab, with chemotherapy given as neoadjuvant treatment for resectable stage III NSCLC in Asian population. Methods: Eligible patients recruited were aged 18 years or older with histologically confirmed AJCC-defined stage IIIA or T3-4N2 IIIB NSCLC who deemed surgically resectable. Patients received 3 cycles of neoadjuvant treatment with intravenous toripalimab (240 mg), carboplatin (area under curve 5), and pemetrexed (500 mg/m2 for adenocarcinoma) or nab-paclitaxel (260 mg/m2 for others) on day 1 of each 21-day cycle. Surgical resection was performed 4-5 weeks following the first day of the last cycle of treatment. The primary endpoint was major pathological response (MPR; ≤10% viable tumor cells). Secondary endpoints included pathological complete response (pCR), R0 resection rate, disease-free survival and safety. Paired primary tumor +/- lymph node and blood samples at baseline and surgery were obtained for exploratory study. This study is registered with ClinicalTrials.gov, NCT04304248. Results: Between August 2019 and July 2020, 33 patients (median age: 61, IQR: 56-66; female: 6, 18.2%) were enrolled and received neoadjuvant treatment. 18 (54.5%) patients had squamous cell lung cancer, and 13 (39.4%) had T3-4N2 stage IIIB disease. Two patients refused surgery and one had progressive disease after treatment. 30 (91.9%) patients underwent resection (median interval between neoadjuvant treatment and surgery: 36.5 days, IQR 30-42.5) and all except one achieved R0 resection (29/30, 96.7%). 20 patients (20/30, 66.7%) had an MPR, including 15 patients (15/30, 50.0%) had a pCR in the per-protocol population. Surgical complications included three arrhythmias, one prolonged air leak, and one chylothorax. 24 patients (80.0%) had pathological downstaging following treatment, and complete lymph node clearances (ypN0) were seen in 70.0% (21/30) of patients. The most common grade 3-4 treatment-related adverse events in the intention-to-treat population were anemia (2, [6.0%]). Severe treatment-related adverse event included one (3.0%) patient with grade 3 peripheral neuropathy (Guillain-Barré syndrome) and resulted in surgery cancellation. At the time of data cutoff (Feb 7, 2021), the median duration of follow-up was 4.13 months, and there were no treatment-related deaths. Conclusions: Toripalimab plus platinum-based doublet yields a high MPR rate, manageable treatment-related toxicity, and feasible surgical resection in resectable stage III NSCLC. Ongoing analysis of biomarker will be available at the meeting. Clinical trial information: NCT04304248.

Whole-exome sequencing on circulating tumor cells explores platinum-based chemotherapy resistance in advanced non-small cell lung cancer.

e21021 Background: Circulating tumour cells (CTCs) have important applications in clinical practice on early tumour diagnosis, prognostic prediction and treatment evaluation. Platinum-based chemotherapy is the first-line treatment for non-small cell lung cancer (NSCLC) patients without targeted drugs. But most patients progressed after a period of treatment. Therefore, to dissect the genetic information contributing to drug resistance and tumour progression in CTCs is valuable for guidance of treatment adjustment. Methods: In this study, we enrolled 9 NSCLC patients treated with platinum-based chemotherapy. For each patient, 10 CTCs were isolated after progression to perform a single cell-level whole-exome sequencing (WES). Meanwhile the patients’ paired primary-diagnosed FFPE sample and progressive biopsy specimens were also performed WES. Results: Comparisons of WES data between primary and progressive specimens, as well as CTCs revealed that more than half patients’ tumour mutation burden (TMB) increased after progression. Dozes to hundreds of single-nucleotide variants (SNVs) and insertions or deletions (Indels) were detected in the CTCs. Slightly more proportion of SNVs/Indels in CTCs shared with paired primary tumours (1.2%-23.1%) than with progressive samples (0.6%-11.7%). Conclusions: Functional annotation on SNVs/Indels showed that CTCs not only harboured cancer driver gene mutations, such as EGFR and TP53, shared with primary and/or progressive tumours, but also harboured chemotherapy-resistance and stem cell-related gene mutations, including SHKBP1, NUMA1, ZNF143, MUC16, ORC1, PON1, PELP1, etc. which have crucial roles in drug resistance and poor prognosis for NSCLCs. Thus, detection of genetic information in CTCs was necessary for guidance of individual therapy and drug resistance study.

Comparison of immune microenvironment between primary lung tumors and paired brain metastatic tumors.

2020 Background: Lung cancer is one of the most common causes of brain metastases (BMs) and is always associated with poor prognosis. To evaluate the characteristics of the tumor immune microenvironment in brain metastases of non-small-cell lung cancer (NSCLC), we investigated the immunophenotype of primary NSCLC and paired brain metastases. Methods: Forty-three Chinese patients with NSCLC who had BMs at presentation or during the course of their disease were admitted to the Sun Yat-Sen University Cancer Center (Guangzhou, China) from 2000 to 2019. RNA sequencing (RNA-seq) of eighty-six formalin-fixed, paraffin embedded (FFPE) samples from primary lung tumors and paired brain metastases of 43 patients was conducted to comprehensively analyze the tumor immune microenvironment. Results: Our data revealed that brain metastases compared with primary lung tumors exhibited reduced tumor infiltrating lymphocytes (TILs) (all 28 immune cell subtypes P &lt; 0.05), lower fraction of activated CD8 T cell and effector memory CD8 T cell in total TILs (P = 0.028, P &lt; 0.001, respectively); higher fraction of macrophage and neutrophil in total TILs (P &lt; 0.001, P &lt; 0.01, respectively). Comparing with the primary lung tumors, the scores of some immune related signatures, including MHC non-class signature, IFN gamma signature and T-cell-inflamed gene-expression profile (GEP) signature, were significantly lower in brain metastases (P = 0.004, P = 0.009, P = 0.004, respectively), while the score of MHC class-II signature was higher in brain metastases (P = 0.045). We found the distributions of tumor microenvironment immune types (TMIT) in brain metastases and primary lung tumors were different. Brain metastases contained significantly lower proportion of TMIT I (high PD-L1/ high CD8A) (23%) than primary lung tumors (47%) (P &lt; 0.05). Besides, we found three immune inhibitory checkpoint molecules, namely C10orf54 (VISTA), CTLA4 and CD274 (PD-L1) were downregulated in brain metastases than in primary lung tumors (P &lt; 0.001, P &lt; 0.001, P = 0.034, respectively). Moreover, there was poor correlation of PD-L1 expression between paired brain metastases and primary lung tumors (R = 0.28, P = 0.068). Unsupervised hierarchic cluster analysis revealed the primary lung tumors had two distinct patterns of immune gene signatures, namely Cluster A and Cluster B, and the tumors in Cluster B were immune rich, but associated with poor prognosis (log-rank P = 0.021). Conclusions: Our work illustrates the immune landscape of brain metastases from NSCLC, and suggests that the tumor immune microenvironment in brain metastases compared with primary lung tumors is further immunosuppressed, that may help guide immunotherapeutic strategies for NSCLC brain metastases.

Myeloid derived suppressor cells and the release of micro-metastases from dormancy.

Metastasis is the primary cause of cancer mortality and an improved understanding of its pathology is critical to the development of novel therapeutic approaches. Mechanism-based therapeutic strategies require insight into the timing of tumor cell dissemination, seeding of distant organs, formation of occult lesions and critically, their release from dormancy. Due to imaging limitations, primary tumors can only be detected when they reach a relatively large size (e.g. > 1 cm3), which, based on our understanding of tumor evolution, occurs approximately 10years and about 30 doubling times following tumor initiation. Genomic profiling of paired primary tumors and metastases has suggested that tumor seeding at secondary sites occurs early during tumor progression and frequently, years prior to clinical diagnosis. Following seeding, tumor cells may enter into and remain in a dormant state, and if they survive and are released from dormancy, they can proliferate into an overt lesion. The timeline of tumor initiation and metastatic dormancy is regulated by tumor interactions with its microenvironment, angiogenesis, and tumor-specific cytotoxic T-lymphocyte (CTL) responses. Therefore, a better understanding of the cellular interactions responsible for immune evasion and/or tumor cell release from dormancy would facilitate the development of therapeutics targeted against this critical part of tumor progression. The immunosuppressive mechanisms mediated by myeloid-derived suppressor cells (MDSCs) contribute to tumor progression and, we posit, promote tumor cell escape from CTL-associated dormancy. Thus, while clinical and translational research has demonstrated a role for MDSCs in facilitating tumor progression and metastasis through tumor escape from adoptive and innate immune responses (T-, natural killer and B-cell responses), few studies have considered the role of MDSCs in tumor release from dormancy. In this review, we discuss MDSC expansion, driven by tumor burden associated growth factor secretion and their role in tumor cell escape from dormancy, resulting in manifest metastases. Thus, the therapeutic strategies to inhibit MDSC expansion and function may provide an approach to delay metastatic relapse and prolong the survival of patients with advanced malignancies.