Paired Metastases Research Articles

Integrated analyses of multi-omic data derived from paired primary lung cancer and brain metastasis reveal the metabolic vulnerability as a novel therapeutic target

BackgroundLung cancer brain metastases (LC-BrMs) are frequently associated with dismal mortality rates in patients with lung cancer; however, standard of care therapies for LC-BrMs are still limited in their efficacy. A deep understanding of molecular mechanisms and tumor microenvironment of LC-BrMs will provide us with new insights into developing novel therapeutics for treating patients with LC-BrMs.MethodsHere, we performed integrated analyses of genomic, transcriptomic, proteomic, metabolomic, and single-cell RNA sequencing data which were derived from a total number of 154 patients with paired and unpaired primary lung cancer and LC-BrM, spanning four published and two newly generated patient cohorts on both bulk and single cell levels.ResultsWe uncovered that LC-BrMs exhibited a significantly greater intra-tumor heterogeneity. We also observed that mutations in a subset of genes were almost always shared by both primary lung cancers and LC-BrM lesions, including TTN, TP53, MUC16, LRP1B, RYR2, and EGFR. In addition, the genome-wide landscape of somatic copy number alterations was similar between primary lung cancers and LC-BrM lesions. Nevertheless, several regions of focal amplification were significantly enriched in LC-BrMs, including 5p15.33 and 20q13.33. Intriguingly, integrated analyses of transcriptomic, proteomic, and metabolomic data revealed mitochondrial-specific metabolism was activated but tumor immune microenvironment was suppressed in LC-BrMs. Subsequently, we validated our results by conducting real-time quantitative reverse transcription PCR experiments, immunohistochemistry, and multiplexed immunofluorescence staining of patients’ paired tumor specimens. Therapeutically, targeting oxidative phosphorylation with gamitrinib in patient-derived organoids of LC-BrMs induced apoptosis and inhibited cell proliferation. The combination of gamitrinib plus anti-PD-1 immunotherapy significantly improved survival of mice bearing LC-BrMs. Patients with a higher expression of mitochondrial metabolism genes but a lower expression of immune genes in their LC-BrM lesions tended to have a worse survival outcome.ConclusionsIn conclusion, our findings not only provide comprehensive and integrated perspectives of molecular underpinnings of LC-BrMs but also contribute to the development of a potential, rationale-based combinatorial therapeutic strategy with the goal of translating it into clinical trials for patients with LC-BrMs.

8576 Genomic and Transcriptomic Characterization of Pediatric Thyroid Cancers

Abstract Disclosure: J.C. Ricarte-Filho: None. E.R. Reichenberger: None. K. Hinkle: None. A.R. Isaza: None. A.J. Bauer: None. A.T. Franco: None. Background: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer in both pediatric and adult populations. Recent genomic profiling of papillary thyroid cancers, including that of the Cancer Genome Atlas, has uncovered the genetic alterations and molecular mechanisms driving thyroid cancer. However, these studies were primarily focused on adult patients. Despite their favorable prognosis, pediatric PTCs have higher rates of metastasis and recurrence than adult PTCs, but the molecular characterization of these tumors have been comparatively lacking. Methods: Here we explore the genomic and transcriptomic landscape of pediatric PTCs from 126 patients, including 107 primary tumors and 19 lymph node metastases (non-paired) using whole-exome sequencing and RNA sequencing. Thirteen primary tumors also had a paired lymph node metastasis analyzed. Results: We found driver alterations in 113 of 126 (90%) of the PTCs investigated, including the identification of a novel receptor tyrosine kinase (RTK) fusion not previously reported in thyroid cancer. Genetic drivers were predominantly kinase fusions (48%), most commonly fusions of RET (22%), NTRK3 (10%), ALK (5%) and NTRK1 (5%). The most common point mutations were BRAF p.V600E (21%), NRAS p.Q61R/K (9%) and hotspot mutations in DICER1 (4%). We found genetic alterations in 12 of the 13 paired lymph node metastasis. In all 12 cases the metastatic tissue harbored the same alterations found in the primary tumor. Eight out of 12 cases had fusions (3 RET, 3 NTRK3 and 2 NTRK1), and the additional 4 cases had point mutations (3 BRAFV600E and 1 TSHRM543T). Fourteen patients (11%) had widely invasive disease with lateral lymph node and distant metastases (N1bM1). Most of these N1bM1 tumors (13/14; 93%) were driven by RTK fusions (5 NTRK3, 3 RET, 3 NTRK1 and 2 ALK). The one case lacking a kinase fusion had a CHEK2 mutation. Conclusion: Our genomic and transcriptomic analysis allowed the detection of driver alterations in ∼90% of the cases, reducing the so-called “dark matter” in our cohort. Our data support that kinase fusions are associated with metastatic disease in pediatric PTC. Patients with RET/NTRK/ALK fusions have worse outcomes than those with BRAF p.V600E mutation and RAS-like alterations (NRAS, DICER1, non-V600 BRAF, PTEN). By using the transcriptional profiling generated in this study, we are currently investigating the molecular mechanisms underlying the metastatic behavior of pediatric PTCs harboring oncogenic fusions, including changes in MAPK transcriptional output, differentiation score and recruitment of signaling pathways potentially associated with the metastatic behavior of these tumors. Presentation: 6/3/2024

Abstract 196: In vitro modeling of pediatric solid tumor lung metastases

Abstract Metastasis remains the main cause of death in pediatric cancer patients with the lung being one of the most common affected organs. Our inability to cure these patients is largely due to insufficient knowledge about tumor and metastatic niche plasticity, while existing models are not fully effective in mirroring the response seen in clinical setting. Our goal is to overcome these limitations by establishing in vitro lung metastasis models using tumor/lung organoid co-cultures to study lung infiltration of pediatric solid tumors. We successfully established patient-derived lung organoids from pediatric lung tissue and characterized them according to the presence and the localization of the respective lung epithelial cell types. Co-culture conditions of lung organoids with PDX-derived Ewing sarcoma and osteosarcoma cells were optimized and models established, while the establishment of models for other pediatric solid tumor entities is ongoing. We have observed infiltration of the lung organoids by tumor cells, and our preliminary findings suggest distinct patterns of infiltration among different tumor entities. Furthermore, we have performed single-cell RNA sequencing analysis on these co-cultures in comparison to the parental lung organoids and tumor cells, aiming to uncover the specific signaling pathways activated in the lung metastatic niche in response to tumor cell invasion. Additionally, experiments using conditioned medium suggest that the lung organoid secretome is sufficient to induce homing of osteosarcoma and Ewing sarcoma cells to the organoids, with the CXCL12/CXCR4 signaling axis as a prime candidate driver. To ascertain the extent to which our in vitro model recapitulates the in vivo lung metastatic niche, we performed spatial transcriptomics (using VISIUM/10X Genomics) on formalin-fixed paraffin-embedded (FFPE) tissue blocks of paired primary tumors and lung metastasis samples for Ewing sarcoma and osteosarcoma. Comprehensive data analysis is currently ongoing, which will instruct a stepwise incorporation of additional factors into our modular organoid co-culture model, resulting in a better representation of the lung metastatic niche. Further, the analysis of patient-derived material will elucidate the spatial organization of solid tumor lung metastases and contribute to the identification of signaling pathways implicated in lung infiltration. In conclusion, our study not only unravels the complexities of pediatric solid tumor metastasis, but also lays the foundation for exploring the anti-metastatic potential of pathway-specific compounds targeting tumor/lung interactions using the established in vitro lung metastasis models. This study is supported by grant 35353-B of the Austrian Science Fund. Citation Format: Martha Zylka, Christoph Hafemeister, Lukas Watzke, Ulrike Mann, Didier Surdez, E. Alejandro Sweet-Cordero, Bernadette Liegl-Atzwanger, Matthias Dettmer, Matthias Farlik, Florian Halbritter, Martin Metzelder, Heinrich Kovar, Branka Radic-Sarikas. In vitro modeling of pediatric solid tumor lung metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 196.

DNA Methylome and Transcriptome Maps of Primary Colorectal Cancer and Matched Liver Metastasis

Sequencing-based genome-wide DNA methylation, gene expression studies and associated data on paired colorectal cancer (CRC) primary and liver metastasis are very limited. We have profiled the DNA methylome and transcriptome of matched primary CRC and liver metastasis samples from the same patients. Genome-scale methylation and expression levels were examined using Reduced Representation Bisulfite Sequencing (RRBS) and RNA-Seq, respectively. To investigate DNA methylation and expression patterns, we generated a total of 1.01 × 109 RRBS reads and 4.38 × 108 RNA-Seq reads from the matched cancer tissues. Here, we describe in detail the sample features, experimental design, methods and bioinformatic pipeline for these epigenetic data. We demonstrate the quality of both the samples and sequence data obtained from the paired samples. The sequencing data obtained from this study will serve as a valuable resource for studying underlying mechanisms of distant metastasis and the utility of epigenetic profiles in cancer metastasis.

Clinicopathological features and survival outcomes of luminal-like breast tumors with estrogen receptor loss at metastatic recurrence: A case-control study

IntroductionEstrogen receptor (ER) loss at metastatic relapse occurs in up to 20% of luminal-like primary breast tumors. Data about clinicopathological features associated with ER loss and its prognostic significance are limited. MethodsIn a nested-case-control study, we compared clinicopathological characteristics and clinical outcomes between a cohort of 51 patients with primary ER+ /HER2- and paired triple-negative metastasis (LUM-TN) and two control cohorts of paired early-metastatic ER+ /HER2- (LUM-LUM, n = 50) and triple-negative (TN-TN, n = 49) breast cancers. Stromal tumor-infiltrating lymphocytes (TILs) were assessed according to the TILs Working Group recommendations as continuous and discrete variables with cutoffs (20%, 40%). ResultsLUM-TN tumors had lower ER expression than LUM-LUM tumors, but lower grade and Ki67 than TN-TN cases. Median distant-metastasis free survival was similar for LUM-TN and LUM-LUM cohorts, but significantly longer than in TN-TN cases (log-rank P < 0.001). LUM-TN and TN-TN cohorts had a comparable survival from the time of metastatic recurrence, which was significantly shorter than in patients with LUM-LUM tumors (log-rank P < 0.001). High TILs were associated with worse outcomes in patients with ER loss (P < 0.001). ConclusionsBreast tumors with ER loss at metastatic relapse have intermediate features and outcomes compared with metastatic luminal-like and ab initio triple-negative tumors. Further investigation on the biological mechanisms underpinning the loss of ER expression is ongoing.

Clinical impact of mixed pulmonary carcinoma and carcinoid: the driver from their mono-clonal origin.

The combination of neuroendocrine/non neuroendocrine lung tumors (CNNELT) mentioned in the last edition of the World Health Organization (WHO) of Thoracic Tumors refers to small cell carcinoma (SCLC) or large cell neuroendocrine carcinoma (LCNEC) mixed with any other non-small cell lung carcinoma (NSCLC). Typical Carcinoid (TC)/Atypical Carcinoid (AC) combined with NSCLC is not included among this category. However, case reports of TC/AC combined with NSCLC have been described. We previously reported 2 cases of lung adenocarcinoma (LUA) mixed with carcinoid sharing mutations in both components supporting the hypothesis of a clonal origin. We extended our analysis to other four cases of mixed NSCLC-carcinoid by performing targeted-DNA and RNA-based NGS analysis in both primary and their paired lymph nodes metastasis. In all cases, LUA and AC components shared at least 1 common mutation (KRAS driver mutation p.Gly12Val in cases 1 and 3, AKAP13-RET fusion in case 2, and missense KRAS driver mutation p.Gly12Ala in case 4, reinforcing the hypothesis of a clonal origin. Moreover, the same mutation was detected in the metastasis constituted only by AC (cases 2 and 4). Although it is a rare malignancy in the lung, mixed LUA and TC/AC could be included among the histotypes for which a deep molecular characterization of both components is needed to identify the presence of potential druggable genetic alterations.

Gene expression comparison between primary aggressive luminal breast cancer and paired axillary and sentinel lymph node metastasis.

e13060 Background: More aggressive and therapy-resistant hormone receptor (HR)-positive breast cancer (hereafter luminal breast cancer, LBC) remains a great challenge. Few studies examine the genomics of axillary lymph node (ALN) metastasis in LBC. The aim of this study was to characterize and compare the mRNA expression patterns of primary breast cancers and paired ALN metastases. Methods: Aggressive pT1-2N2-3M0 LBC patients aged over 18 years who were treated at Shandong Provincial Hospital Affiliated to Shandong First Medical University and had available matched primary and metastatic tumor samples for gene expression analyses were retrospectively recruited. Bulk mRNA expression levels were determined by RNA sequencing (Illumina NovaSeq 6000 system) and calculated using featureCounts. Differential gene expression analysis was performed using DESeq2. All analyses were carried out with differentially expressed genes (DEGs) with an FDR &lt;0.05. Changes of intrinsic subtypes from primary to metastatic disease were assessed. Results: Ten LBC patients with paired tumor samples were included. Compared with the primary tumor, ALN metastatic tumor had 1509 statistically significant DEGs, including 743 upregulated genes and 766 downregulated genes. Notably, there was an upregulation of genes related to immune response-activating signal transduction, T cell receptor signaling pathway, B cell receptor signaling pathway, and cytokine-cytokine receptor interaction in the ALN metastasis. The downregulation of genes were mainly enriched in the PI3K-Akt signaling pathway, ECM-receptor interaction, Wnt signaling pathway, and TGF-beta signaling pathway. Intrinsic subtype distribution in primary tumors was different from those in ALN metastatic tumors, such as Luminal A (30% vs. 50%), Luminal B (10% vs. 20%), HER2-enriched (20% vs. 10%), Basal-like (10% vs. 10%), and Normal-like (30% vs. 10%). These results suggest that LBC is a highly heterogeneous disease. Conclusions: In aggressive pT1-2N2-3M0 LBC, ALN metastases have a distinct gene expression profile and show upregulation of immune-related genes ( CR2, BLK, PAX5, CD19, FCRL1, TNFRSF13B). Genes associated with signaling pathways are involved in the development of breast cancer ( FST, TNN, LAMC3, SFRP2, FGF18), and are downregulated when compared to autologous primary cancer. These pathways are potential targeted pathways in the treatment of breast cancer. LBC cells metastatic to lymph nodes undergo a change to metastasize and survive in the new microenvironment, which may provide insights into the metastatic process. Besides, a switch towards more aggressive intrinsic subtype from primary to advanced disease is also observed.

Abstract 1299: Transcriptomic analysis to unveil alveolar rhabdomyosarcoma metastatic process

Abstract Background: Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children and adolescents. It is divided in two major histological subtypes, being the Alveolar (ARMS) the one associated to a poorer prognosis1. The cure rate for these tumors is up to 70% for children with localized disease, but this numbers decrease notably to a 20% 5-year survival rate for patients that show metastatic spreading. However, mechanisms leading to metastatic disease remain poorly understood. Hence, there is an urgent need to better understand this process, which will hopefully lead to novel targeted therapies and improved survival rates for these patients. Methods: ARMS cells (RH4) were injected in the gastrocnemius muscle of Athymic Nude-Foxn1nu mice2. Primary tumors (800mm3) and peritoneal metastases (visible by IVIS®) were collected, and RNA extracted. Xenograft-derived metastatic cell lines were obtained directly as a primary culture from mice metastases. Characterization of paired tumors and metastases was performed using Clariom™ D microarray (Applied Biosystems). After data normalization and comparative analyses (R; RMA and limma), a list of differentially expressed transcripts between sample groups was obtained. These are being validated by qPCR and Western Blot. Results: Transcriptomic profiling of tumors (n=3), paired metastases (n=3), xenograft-derived metastatic cell lines (n=3) and RH4 cell line (n=3) revealed 3420 differentially expressed transcripts (2747 increased in metastases, 673 in tumors). Several transcripts of interest are overexpressed in metastases samples compared to primary tumors as showed by microarray analysis and validated by qPCR. These targets are related in literature to metastasis in other cancers, as well as lymph nodes metastasis, which occurs to happen in our model and in ARMS clinics3. Additionally, some transcripts are overexpressed in tumor samples, which are linked to inhibition of proliferation and motility. Conclusions/Future Work: In vitro phenotypic characterization of the obtained targets will involve proliferation (MTT), migration (Transwell®) and clonogenic assays, including loss-of-function (siRNA and commercial inhibitors). Using stable knock-out and knock-in models of the genes of interest, their role in ARMS metastasis will be further studied in vitro and in vivo. The clinical relevance of these targets will be explored in several cohorts of patients by immunohistochemistry on Tissue Microarrays. Studying the profile of paired tumors and metastases provides a unique tool to decipher the mechanisms of tumor progression. Fully understanding the metastatic process will ultimately lead to the development of better targeted therapies and thus to improved outcomes for ARMS patients. Also, these same steps are being followed in our laboratory for Ewing Sarcoma, another developmental tumor with discouraging survival rates for metastatic patients (&amp;lt;30%)4. Citation Format: Sara Sanchez-Serra, Mariona Chicon-Bosch, Paola Monaco, Laura Santana-Viera, Susana Maqueda-Marcos, Manuel Gris-Lorente, Roser Lopez-Alemany, Oscar M. Tirado. Transcriptomic analysis to unveil alveolar rhabdomyosarcoma metastatic process [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 1299.

Anti-VEGF Therapy Possibly Extends Survival in Patients With Colorectal Brain Metastasis by Protecting Patients From Neurologic Disability

BackgroundColorectal brain metastases (CBMs) are rare with poor prognosis. There is still no standard systemic treatment for multiple or unresectable CBM. our study aimed to explore the impact of anti-VEGF therapy on overall survival, brain-specific disease control, and neurologic symptom burden in patients with CBM. MethodsA total of 65 patients with CBM under treatment were retrospectively enrolled and divided into anti-VEGF based systemic therapy or non–anti-VEGF based therapy. A total of 25 patients who received at least 3 cycles of anti-VEGF agent and 40 patients without anti-VEGF therapy were analyzed by endpoints of overall survival (OS), progression-free survival (PFS), intracranial PFS (iPFS) and neurogenic event-free survival (nEFS). Gene expression in paired primary metastatic colorectal cancer (mCRC), liver, lung and brain metastasis from NCBI data was analyzed using top Gene Ontology (GO) and cBioPortal. ResultsPatients who treated with anti-VEGF therapy had significantly longer OS (19.5 vs. 5.5 months, P = .009), iPFS (14.6 vs. 4.1 months, P < .001) and nEFS (17.6 vs. 4.4 months, P < .001). Patients who received anti-VEGF therapy beyond any disease progression presented with superior OS (19.7 vs. 9.4 months, P = .039). Top GO and cBioPortal analysis revealed a stronger molecular function of angiogenesis in intracranial metastasis. ConclusionsAnti-VEGF based systemic therapy showed favorable efficacy that was reflected in longer overall survival, iPFS and NEFS in patients with CBM.

Heterogeneity of PD-L1 expression and CD8 lymphocyte infiltration in metastatic colorectal cancer and their prognostic significance

PurposeIn recent years, immune checkpoint inhibitors have become a major therapeutic method for the treatment of metastatic colorectal cancer (mCRC). Growing evidence indicates that tumour-infiltrating lymphocytes (TILs) in the tumour microenvironment are a prerequisite for the effectiveness of PD-1/PD-L1 blockade therapy. In this study, we aimed to compare PD-L1 expression and cluster of differentiation 4 (CD4) and CD8 TIL infiltration in primary tumours and paired metastases. Patients and methodsAltogether, 111 patients with mCRC who underwent surgery at our hospital were included. PD-L1, CD4, and CD8 expression were detected by immunohistochemistry in a tissue microarray. PD-L1 expression was assessed using the combined positivity score (CPS), and a score ≥1 was judged as positive. The area proportion of TILs with positive staining ≥10% was classified as “high”, while <10% was classified as “low”. ResultsWe observed the discordance of PD-L1 expression between primary tumours and paired metastases in 35/111 (31.5%) patients (κ = 0.137, P = 0.142). This heterogeneity was significantly correlated with discordance of CD8 TIL infiltration between primary tumours and paired metastases (P = 0.003). Compared with corresponding colorectal cancer tumours, lung metastases showed more CD8 TIL infiltration (P = 0.022, median: 8.5% vs. 5.0%), whereas liver metastases exhibited less CD8 TIL infiltration (P = 0.028, median: 3.0% vs. 5.0%). Area proportion of CD4+ and CD8+ TIL infiltration in lung metastases were all higher than those in liver metastases (P = 0.005, median: 15.0% vs. 9.0%; P = 0.001, median: 8.5% vs. 3.0%). Compared with p MMR (MSI-L/MS-S) subgroup, area proportion of CD8 TIL infiltration in primary tumours and CD4, CD8 TIL infiltration in paired metastases were all higher in d MMR (MSI-H) group (P = 0.026, median: 15.0% vs 5.0%; P = 0.039, median: 15.0% vs 9.0%; P = 0.015, median: 15.0% vs 5.0%). Preoperative chemo/radiotherapy may increase CD8 TIL infiltration in primary tumours (P = 0.045, median: 10.0% vs. 5.0%). CD8 TIL infiltration in primary tumours was an independent predictive factor for overall survival (HR 0.28, 95% CI 0.09–0.93, P = 0.038). ConclusionHeterogeneity in PD-L1 expression and CD8 TIL infiltration was found between primary tumours and paired metastases in mCRC. CD8 TIL infiltration in primary tumours could independently forecast the overall survival of patients with mCRC.

A Transcriptomic-Based Tool to Predict Gemcitabine Sensitivity in Advanced Pancreatic Adenocarcinoma

A Transcriptomic-Based Tool to Predict Gemcitabine Sensitivity in Advanced Pancreatic Adenocarcinoma

Intraindividual Tumor Heterogeneity of Mismatch Repair Status in Metastatic Colorectal Cancer.

Heterogeneous mismatch repair (MMR) status in metastatic colorectal cancer (mCRC) may associate with refractoriness to immunotherapy. We aimed here to study the concordance in MMR status between primary and paired metastasis in mCRC. Our study included 84 patients diagnosed with mCRC with primary and matched metastatic cancers. Immunohistochemistry was used to determine the MMR status of primary lesions and matched metastases. Pooled analysis of 913 cases was used to evaluate the prevalence and organ specificity of MMR status heterogeneity. The correlations between MMR pattern heterogeneity and clinical outcomes were analyzed. MMR status heterogeneity between primary and corresponding metastatic sites was exhibited by 10 (11.9%) patients. The prevalence of the heterogeneous MMR phenotype was significantly higher in primary tumors with deficient MMR (dMMR) than with proficient MMR (pMMR), which was verified in the pooled analysis ( P <0.001). Among patients with a dMMR primary tumor, the discrepancy was detected in liver, lung, ovary, peritoneum, and distant lymph node metastases. However, the discrepancy was confined to liver (26/440) or peritoneum (7/112) ( P =0.02) in patients with a pMMR primary tumor. Patients with or without MMR status heterogeneity experienced comparable overall survival ( P =0.452). Heterogeneous MMR patterns generally existed in a subset of patients with mCRC, particularly those with dMMR primary tumors. Testing the metastatic site may be valuable because the discordance of MMR status may potentially affect immune surveillance and immunotherapy.

37. Epigenetic reprogramming of brain development pathways during non-small cell lung cancer metastasis to brain

Non-small cell lung cancer (NSCLC) is one of the most common and deadliest cancers, with roughly half of all patients initially presenting with both primary and metastatic disease. While the major events in the metastatic cascade have been identified, a mechanistic understanding of how NSCLC routinely, successfully colonizes the brain is largely unknown. To better understand this process, we profiled a combination of genomic, transcriptomic, and methylomic landscapes of 45 paired NSCLC primary and brain metastasis samples. 75 genes displayed recurrent metastasis enriched variants, largely implicated in focal adhesion and extracellular matrix receptor interactions. Variant allele frequencies over a wide range of epigenetic regulators displayed an increase in metastases, suggesting that epigenetic misregulation may be selected for and possibly contribute to NSCLC metastasis to brain. Consistent with these observations, we observed widespread changes in DNA methylation throughout disease progression, many found within brain-specific active enhancers and correlated with increased nearby gene expression. The greatest recurrent methylation changes during metastatic progression occurred over a subset of DNA methylation valleys (DMVs) enriched for H3K9me3 and bivalent marks H3K27me3 and H3K4me1 in normal lung. Mapping EZH2, the catalytic subunit of polycomb repressive complex 2 (PCR2), binding locations in H1299, a lymph node-derived lung cancer cell line, revealed a pervasive loss of EZH2 binding within DMVs accompanied by an increase in DNA methylation, exemplifying epigenetic switching. The vast majority of these DMR-associated DMVs harbor developmental genes, suggesting that altered epigenetic regulation of developmentally important genes may confer a selective advantage during metastatic progression.

Abstract IA029: The origins of leiomyosarcoma: implications for prognosis and defining new therapeutic options

Abstract Introduction: Leiomyosarcomas (LMS) are genetically heterogenous tumors differentiating along smooth muscle lines. Currently, LMS treatment is not informed by molecular subtyping and is associated with highly variable survival. While disease site continues to dictate clinical management, the contribution of genetic factors to LMS subtype, origins, and timing are unknown. Here we analyze 70 genomes and 130 transcriptomes of LMS, including multiple tumor regions and paired metastases, with the goal of defining the molecular origins of LMS and using both computational analysis and functional genomics to define new therapeutic arenas for metastatic disease. Results: Molecular profiling highlight the very early origins of LMS. We uncover three specific subtypes of LMS that likely develop from distinct lineages of smooth muscle cells. Of these, dedifferentiated LMS with high immune infiltration and tumors primarily of gynecological origin harbor genomic dystrophin deletions and/or loss of dystrophin expression, acquire the highest burden of genomic mutation, and are associated with worse survival. Homologous recombination defects lead to genome-wide mutational signatures, and a corresponding sensitivity to PARP trappers and other DNA damage response inhibitors, suggesting a promising therapeutic strategy for LMS. Finally, by phylogenetic reconstruction, we present evidence that clones seeding lethal metastases arise decades prior to LMS diagnosis. Conclusions: In summary, LMS, the catch all-label for any primary malignant smooth muscle neoplasm, encompasses at least three distinct entities. We report that the utility of DNA damage response inhibitors is promising and warrants further exploration. Finally, the molecular evolution and timing of LMS provide a broad window for early intervention and risk stratification for these patients. Citation Format: Rebecca A. Gladdy. The origins of leiomyosarcoma: implications for prognosis and defining new therapeutic options [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA029.

Overexpression in metastatic breast cancer supports Syndecan-1 as a marker of invasiveness and poor prognosis

BackgroundMetastasis is the main cause of breast cancer (BC) mortality. Increasing evidence points to a role of syndecan-1 (CD138) expression as a prognostic marker involved in BC tissue and leptomeningeal metastasis. Aim of this study was to investigate and compare syndecan-1 tissue expression and localization in primary and secondary BC, focusing on brain metastases.MethodsSyndecan-1 expression was determined by immunohistochemistry. Focal vs diffuse (< or > 50% of cancer cells, respectively) pattern of expression, cellular localization (cytoplasm vs membrane) and intensity of immunostaining on neoplastic cells were evaluated. Moreover, the extent and pattern of expression of syndecan-1 were compared between primary tumors and paired metastases and correlated with the tumor intrinsic subtype.ResultsA total of 23 cases, 10 with paired primary and metastatic tumor and 13 brain metastases, were evaluated. Syndecan-1 was expressed in both primary and metastatic BC. A diffuse cytoplasmic expression was observed in most primary BCs; by contrast, all metastatic lesions showed a membrane pattern of expression, suggesting a shift in cellular localization of syndecan-1 during the metastatic process. Concerning the extent of expression, we observed in metastatic lesions, a trend of association between intrinsic subtypes and extent of positivity. In particular, both BC characterized by overexpression of HER2 and triple-negative tumors were correlated with a diffuse pattern of expression with a moderate to strong intensity.ConclusionA diffuse cytoplasmic expression was observed in most primary BCs; by contrast, all metastatic lesions showed a membrane pattern of expression, suggesting a shift in cellular localization of syndecan-1 during the metastatic process.

377P Intraindividual tumor heterogeneity of mismatch repair status in metastatic colorectal cancer

Tumor DNA mismatch repair (MMR) deficiency testing has become an indispensable test in metastatic colorectal cancer (mCRC) due to the high efficacy of immunotherapy in patients with deficient MMR (dMMR) status. However, a significant portion of dMMR CRC tumors exhibit primary or acquired resistance. Heterogeneous MMR status may associate with refractoriness to immunotherapy. We aimed to study the concordance in MMR status between primary and paired metastasis in mCRC.

EP-369 Examining EphA2 expression in Colorectal Cancer

Abstract Aims EphA2 is a potential therapeutic target for the treatment of colorectal cancer (CRC), however reports of EphA2 expression in CRC are inconsistent and often lack the context of a normal tissue comparator. The aim of this investigation is to undertake a detailed assessment of EphA2 protein expression across a range of pathological and normal specimens. Methods 177 patient specimens (100 paired primary and normal colon, 42 paired primary and metastasis and 35 unpaired samples as part of a multi-tumour TMA) underwent immunohistochemical analysis (IHC) probing for EphA2 expression alongside a normal tissue array. Results Weak/moderate EphA2 expression was noted in normal tissues of the proximal GI tract (stomach, small intestine) with little expression in the colon/rectum with a significant upregulation of EphA2 noted in primary lesions compared to matched normal tissue controls (p&amp;lt;0.0001). No difference in expression between stage of disease, sex or effect of patient age, location of samples (edge vs centre), stage of disease, T-stage or N-stage. EphA2 expression was significantly higher in primary lesions associated with M1 disease primaries (p=0.03). No difference in EphA2 expression was noted between matched primary and metastatic lesions (p&amp;gt;0.05). Conclusion In this study, EphA2 protein expression was significantly higher in CRC lesions compared to healthy tissue (p&amp;lt;0.0001) with no diminution noted across a wide range of stages or lesion types. On this basis, diagnostic or therapeutic interventions targeting EphA2 are likely to be useful across a wide spectrum of disease.

EGFR, HER2, and HER3 protein expression in paired primary tumor and lymph node metastasis of colorectal cancer

Due to the difficulty in sampling of metastatic tumors, patient selection is commonly based on results of primary tumor samples when metastatic samples are not available. However, due to tumor heterogeneity, metastatic tumors may be different from primary tumors in their phenotypes. The aim of this study was to investigate the expression of EGFR, HER2, and HER3 between primary and lymph node metastatic lesions of colorectal cancer. Paired primary tumors and lymph node metastases from 79 patients with colorectal cancer were retrospectively collected and analyzed for EGFR, HER2, and HER3 expression. High EGFR, HER2, and HER3 expression (2+ and 3+) was found in 64.2%, 66.0%, and 85.0% of primary tumors, and 56.8%, 46.0%, and 76.0% of lymph node metastases, respectively. Correlation rates between primary and metastatic lesions were 67.1%, 63.3%, and 74.7% for EGFR, HER2, and HER3, respectively. Stage IV tumors (with distant metastasis) had higher correlation rates of HER2 expression compared to stage III tumors (without distant metastasis) (P = 0.050). Moderate correlation rates in EGFR, HER2, and HER3 expression were observed between primary and metastatic lesions of colorectal cancer. Tumor stage or existence of distant metastasis could serve as potential predictive markers for the correlation of HER2 expression between primary tumors and lymph node metastases of colorectal cancer.

PD-L1 expression and FGFR-mutations among Danish patients diagnosed with metastatic urothelial carcinoma: A retrospective and descriptive study.

Checkpoint inhibitors have changed the treatment landscape of advanced urothelial carcinoma (mUC), and recently, a fibroblast‐growth‐factor‐receptor (FGFR) inhibitor has been introduced. This study aimed at estimating programmed death‐ligand 1 (PD‐L1) expression in primary tumors (PTs) and the PD‐L1 expression concordance between PTs and paired metastases in 100 patients with UC managed in the real‐world setting. Further, the aim was to investigate FGFR1–3 aberrations and the correlation between FGFR1–3 aberrations and PD‐L1 expression. PD‐L1 immunohistochemistry was performed on 100 formalin‐fixed paraffin‐embedded archival primary UC samples and 55 matched metastases using the 22C3 PD‐L1 assay. PD‐L1 expression was determined by the combined positive score, considered positive at ≥10. Targeted next‐generation sequencing on the S5+/Prime System with the Oncomine Comprehensive Assay version 3 was used to detect FGFR1‐3 aberrations in PTs. We found that 29 of 100 PTs had positive PD‐L1 expression. The PD‐L1 concordance rate was 71%. FGFR1‐3 aberrations were observed in 18% of PTs, most frequently FGFR3 amplifications or mutations. We found no association between FGFR1‐3 aberrations and PT PD‐L1 expression (p = 0.379). Our data emphasize the need for further studies in predictive biomarkers.

Abstract 1603: Genomic transcriptomic evolution in TRACERx lung cancer and metastasis

Abstract Background: Genomic intra-tumor heterogeneity (ITH) drives tumor evolution, leading to immune evasion and resistance to therapy. Emerging evidence implicates the transcriptome as a source of important variation that impacts tumor phenotype. Here, we perform a genomic-transcriptomic analysis of intra-tumor transcriptomic diversity upon 941 tumor regions taken from 357 TRACERx non-small cell lung cancers (NSCLC) across primary and metastatic disease subjected to high-depth bulk DNA and RNA sequencing, as well as 91 tumor-adjacent normal tissue samples. Results: Genomic and transcriptomic diversity are linked across primary and metastatic disease, with expression signatures of proliferation being enriched in the metastasis seeding subclone of the primary tumor relative to non-metastasis seeding subclones. Copy-number independent allele-specific expression (CN-independent ASE), a source of transcriptome-specific diversity, affects 1% (± 0.5%) of genes and is underpinned by aberrant allele-specific methylation (OR=7.58, p≤2.2x10-16), thus providing a window to the NSCLC epigenomic landscape. Driver mutations in chromatin remodellers and histone modifiers, in particular SETD2 and KDM5C, are associated with increased global levels of CN-independent ASE (p=0.0001). In genomically stable tumors, high levels of CN-independent ASE are linked to expression signatures consistent with genomic instability and proliferation (R=0.58, p=0.001), highlighting convergence between the genome and transcriptome in tumor evolution. For the first time, we uncover mutational signatures of RNA editing. Analysis of their activity links the expression of ADAR and APOBEC enzymes to editing processes revealing otherwise hidden APOBEC activity within tumors at sampling (RNA APOBEC activity identified in 188 tumor regions (32%) without evidence of DNA APOBEC activity). RNA editing activity is shared between primary tumors and paired metastasis, but not paired tumor-adjacent normal tissue, suggestive of heritability of this somatic transcriptional process. Finally, we combine multiple measures of genomic and transcriptomic variation in a multi-region approach to define important variation within cancer genes. We illustrate examples that would be missed with a purely genomic focus and demonstrate genomic-transcriptomic parallel evolution, converging on disruption to single cancer genes, such as FAT1 and APC, in different regions of a tumor. Conclusions: This work highlights the importance of the transcriptome during tumor evolution, as well as the power of integrative multi-omic assessments of ITH, and provides novel insight into the role of transcriptomic variation in lung cancer. Citation Format: James R. M. Black, Carlos Martinez-Ruiz, Clare Puttick, Jonas Demeulemeester, Elizabeth Larose Cadieux, Kerstin Thol, Thomas P. Jones, Selvaraju Veeriah, Cristina Naceur-Lombardelli, Andrew Rowan, Sophia Ward, Michelle Dietzen, Ariana Huebner, Maise Al Bakir, Miljana Tanic, Thomas B. Watkins, Emilia L. Lim, Ali M. Al Rashed, Daniel E. Cook, Rachel Rosenthal, Gareth Wilson, Alexander M. Frankell, Nnennaya Kanu, Kevin Litchfield, Nicolai J. Birkbak, Allan Hackshaw, Stephan Beck, Peter Van Loo, Mariam Jamal-Hanjani, the lung TRACERx Consortium, Charles Swanton, Nicholas McGranahan. Genomic transcriptomic evolution in TRACERx lung cancer and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1603.