Metastatic Tumor Samples Research Articles

PAX9 Determines Epigenetic State Transition and Cell Fate in Cancer.

Abnormalities in genetic and epigenetic modifications can lead to drastic changes in gene expression profiles that are associated with various cancer types. Small cell lung cancer (SCLC) is an aggressive and deadly form of lung cancer with limited effective therapies currently available. By utilizing a genome-wide CRISPR-Cas9 dropout screen in SCLC cells, we identified paired box protein 9 (PAX9) as an essential factor that is overexpressed in human malignant SCLC tumor samples and is transcriptionally driven by the BAP1/ASXL3/BRD4 epigenetic axis. Genome-wide studies revealed that PAX9 occupies distal enhancer elements and represses gene expression by restricting enhancer activity. In multiple SCLC cell lines, genetic depletion of PAX9 led to significant induction of a primed-active enhancer transition, resulting in increased expression of a large number of neural differentiation and tumor-suppressive genes. Mechanistically, PAX9 interacted and cofunctioned with the nucleosome remodeling and deacetylase (NuRD) complex at enhancers to repress nearby gene expression, which was reversed by pharmacologic HDAC inhibition. Overall, this study provides mechanistic insight into the oncogenic function of the PAX9/NuRD complex epigenetic axis in human SCLC and suggests that reactivation of primed enhancers may have potential therapeutic efficacy in treating SCLC expressing high levels of PAX9. SIGNIFICANCE: A genome-wide screen in small cell lung cancer reveals PAX9/NuRD-mediated epigenetic enhancer silencing and tumor progression, supporting the development of novel personalized therapeutic approaches targeting the PAX9-regulated network.

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 2874: Understanding small cell lung cancer metastasis using circulating tumor cell (CTC)-derived tumor explant (CDX) models

Abstract Small cell lung cancer (SCLC) accounts for about 13% of all malignant lung tumors and is characterized by an extremely low survival rate and widespread early dissemination. Over two-thirds of SCLC patients are diagnosed with metastases in multiple organs; typically liver, lymph nodes and brain. The number and site of secondary lesions at diagnosis correlates with unfavorable disease outcome. Studying metastasis in SCLC is extremely challenging, as metastatic tumor samples are rarely obtained from these patients. Our laboratory has developed a patient-faithful biobank of &amp;gt;45 SCLC circulating tumor cell (CTC)-derived explants (CDX) models with which to study SCLC1. We implemented a resection protocol using these models to study the metastatic process in SCLC by interrogating the genetic and phenotypic components of metastatic cells using combinations of tissue pathology and next generation sequencing methods. Upon resection of the subcutaneous tumor, we detect the presence of overt macrometastatic lesions in multiple models, which in most cases retain matched organ tropism to that observed in the patient. Whereas all models so far tested disseminated to the lungs, the ATOH1 subtye2 models CDX17, CDX17P, CDX25 and CDX30P predominantly grew in the liver, and CDX3 and CDX3P (ASCL1 subtype3) preferentially colonized the brain despite having a much slower growth. CDX SCLC cells can be isolated from subcutaneous tumors and orthotopically implanted, recapitulating the same tropism. Finally, CDX cells labelled ex vivo can be re-implanted directly into the bloodstream or into the brain parenchyma giving rise to SCLC secondary tumors, providing us with an excellent tool to monitor and study several steps of the metastatic cascade, including extravasation and brain colonization. We report for the first time, brain tropic mouse models of SCLC. Molecular studies are underway to investigate the underlying genetic determinants and mechanisms underpinning metastatic spread of SCLC via transcriptomic studies of matched subcutaneous and metastatic tissues and CTCs in CDX. In parallel, CDX3P is being used to identify markers of pre-metastatic disease and brain colonization at the single cell level to identify novel therapeutic strategies and biomarkers that may benefit the significant number of SCLC patients who present with brain lesions. 1Hodgkinson et al., Nature Medicine 20, 897-903 (2014) 2Simpson et al., Nature Cancer 1, pages437-451(2020) Citation Format: Maria Peiris-Pagès, Mitchell Revill, Derrick Morgan, Stewart Brown, Melanie Galvin, Lynsey Priest, Mathew Carter, Sheila K. Singh, Kristopher Frese, Fiona Blackhall, Kathryn Simpson, Caroline Dive. Understanding small cell lung cancer metastasis using circulating tumor cell (CTC)-derived tumor explant (CDX) models [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 2874.

The human melanoma proteome atlas-Defining the molecular pathology.

The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients.

Abstract 127: Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer

Abstract The adoptive transfer of tumor-infiltrating lymphocytes (TILs) expanded in vitro can mediate regressions of established tumors, yet only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells. This complexity underpins the need to phenotypically characterize T cells infiltrating the tumor microenvironment. In this study, we performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 45,000 T cells isolated from ten archival metastatic tumor samples whose primary histologies included colon, rectal, breast, and melanoma. By transcriptomic clustering of the T cells and mapping TCR identity data onto these blueprints, we observed a diversity of T cell phenotypes spanning from memory-like to dysfunctional effector-like, with clonal expansion occurring more widely in the effector-like cells. Through sequencing of peripheral blood lymphocytes (PBL) obtained at the time of tumor resection, we found T cell clusters that were either tumor enriched or distinctly PBL enriched, potentially representing recent tumor immigrants. Prior knowledge of 30 CD4 and CD8 tumor neoantigen-reactive TCRs (NeoTCRs) from eight patients allowed us to project these NeoTCRs onto the TIL transcriptomic map where we observed 380 total T cells bearing these NeoTCRs. The majority (&amp;gt;85%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, marked by expression of CXCL13, ENTPD1 (CD39), TOX, and PDCD1 (PD-1), indicating a dysfunctional state. We reasoned that other dominant T cell clonotypes within the NeoTCR clusters could contain additional novel NeoTCR clonotypes. We expressed these predicted NeoTCRs in donor PBL and screened them in vitro with antigen presenting cells (APCs) pulsed with peptides or tandem minigenes encoded by patient-specific tumor mutations, yielding 24 new NeoTCR clones (251 cells), which included neoantigen reactivities undetected in the same patients' fragment TIL screens. Using a NeoTCR signature score derived from the archival samples, three prospective patients' TIL were analyzed by scRNA/TCRseq and clusters scored according to their NeoTCR signature. TCRs reconstructed from these clusters and tested against patients' tumor-specific reagents yielded novel CD4 and CD8 NeoTCRs. Altogether, this study enabled successful enrichment and detection of tumor-specific NeoTCRs in the sequenced TIL of 12/12 patients for whom reactivity was either previously assessed or prospectively identified. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapy, as prospective NeoTCR isolation based on transcriptional phenotype of TIL will allow for rapid therapeutic development in the form of T cells expressing these tumor-specific TCRs. Citation Format: Frank J. Lowery, Sri Krishna, Rami Yossef, Praveen D. Chatani, Neilesh B. Parikh, Yong-Chen Lu, Nikolaos Zacharakis, Noam Levin, Ken-ichi Hanada, James C. Yang, Paul F. Robbins, Maria R. Parkhurst, Steven A. Rosenberg. Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human 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 127.

Abstract 2828: The prognostically significant immunological indices for patients who underwent curative resection of pulmonary metastases of colorectal cancer

Abstract Background: In recent years, it has been revealed that the immunological state of the host affects with the effect of cancer treatments. The neutrophil-to-lymphocyte ratio (NLR) as a systemic inflammatory marker and the density of tumor-infiltrating lymphocytes (TILs) at cancer microenvironment have been reported to be useful prognostic indices in various cancers, including colorectal cancer (CRC). The aim of this study is to evaluate the prognostic significance of immunological indices in patients who underwent curative resection of pulmonary metastases of CRC. Patients and Methods: The medical records of 17 patients who underwent potentially curative resection for pulmonary metastases of CRC were retrospectively reviewed. The correlation between the relapse-free survival (RFS) or the overall survival (OS) after curative resection of pulmonary metastases of CRC and the clinicopathological factors, NLR or TILs were evaluated. The NLR was calculated from the blood sample which was obtained within 2 weeks before pulmonary resection by dividing the absolute neutrophil count by the absolute lymphocyte count. According to the receiver operating characteristic (ROC) curve analysis, patients with an NLR of ≥ 2.5 were classified as high-NLR group. The density of TILs was examined by immunohistochemistry of pulmonary metastatic tumor sample, by staining anti-CD3 and anti-CD8 antibody. The number of CD3+TILs and CD8+TILs at the border of tumor was counted in a randomly selected field at a magnification of ×400. The mean of the values obtained in five different areas was used. According to the ROC curve analysis, the patients with the number of CD3+TILs ≥ 31 were classified as high- CD3+TILs group and patients with the number of CD8+TILs ≥ 21 were classified as high- CD8+TILs group. Results: The RFS and OS after resection of pulmonary metastases were significantly worse in the high-NLR group than in the low-NLR group (p=0.001, p=0.003, respectively). The RFS after resection of pulmonary metastases was significantly better in the high-CD3+TILs group than in the low-CD3+TILs group (p=0.041). Multivariate analysis revealed that the number of pulmonary metastases and the NLR before resection of pulmonary metastases were independent prognostic factors of the RFS (p=0.007, p=0.002, respectively) and the NLR before resection of pulmonary metastasis was the independent prognostic factor of the OS (p=0.009). Conclusion: The immunological indices such as the NLR and the density of TILs, may have prognostic significance in patients who underwent curative resection of pulmonary metastases of CRC. Citation Format: Yuki Okazaki, Masatsune Shibutani, En Wang, Hisashi Nagahara, Tatsunari Fukuoka, Yasuhito Iseki, Shinichiro Kashiwagi, Hiroaki Tanaka, Kiyoshi Maeda, Kosei Hirakawa, Masaichi Ohira. The prognostically significant immunological indices for patients who underwent curative resection of pulmonary metastases of 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 2828.

Genetic and immune divergence in pancreatic cancer lesions at the time of metastatic relapse compared to primary tumor.

4140 Background: Relapse of pancreatic ductal adenocarcinoma (PDA) is common even after complete resection and adjuvant therapy. Compared to the resected tumor, the biological characteristics of metastatic tumors at the time of first relapse are poorly understood. Methods: Whole-exome sequencing (WES) (250x) and bulk RNA sequencing were performed on samples from 30 patients with PDA. Paired primary tumor samples were obtained after R0 or R1 resection, and metastatic tumor samples were obtained by biopsy at the time of first relapse. 74.1% of patients had received adjuvant chemotherapy and radiation therapy, 7.4% had received adjuvant chemotherapy only, and 3.7% had received adjuvant radiation therapy only. Most common metastatic sites were liver and lung. The cohort was 60% male with a median age at diagnosis of 64 years. The vast majority of patients had stage IIA or IIB disease at diagnosis. Median disease-free survival was 481 days. Analysis used Freebayes (somatic variant calling), Kallisto (transcript quantification), Danaher et al. (cell type deconvolution), and antigen.garnish (neoantigen prediction). Results: High-quality WES and/or RNA sequencing were available for 27/30 patients. Among these were 16 pairs of primary and metastatic samples for WES and 15 paired samples for RNA sequencing. Median tumor purity was 32% (primary) and 42% (metastatic). KRAS mutations were present in 43/48 evaluable samples, with conserved KRAS mutations in 14/16 primary-metastatic pairs. Tumors were otherwise highly variable, with 13/16 patients developing oncogenic mutations in metastatic tumors that were undetected in primary tumors (BRCA1 [3/16], AKT3 [3/16], TP53 [2/16], ROS1 [2/16]). Overall, primary and metastatic tumors had similar tumor mutation burden and neoantigen production rate. However, neoantigens were highly variable at the peptide and gene level, with conservation rates of 2.73% and 11.57%, respectively, across primary-metastatic pairs. PDA transcriptomic subtype also differed across primary-metastatic pairs in all cases. Furthermore, metastatic tumors contained lower immune suppressive signal by transcripts and deconvolution (CTLA4: p = 0.0012, FOXP3: p = 0.0026, PDCD11: p = 0.012, regulatory T cells: p = 0.012), while myeloid cells were higher (CD33: p = 0.0067). Conclusions: With the exception of KRAS, metastatic PDA tumors at relapse contain new oncogenes, distinct neoantigens, and lower immune-suppressive signal compared to primary PDA tumors. These data suggest a potential clinical utility for tumor biopsies at the time of first metastatic relapse and caution against clinical decisions for relapsed, metastatic patients based solely on sequencing of the originally resected tumor.

EARN: an ensemble machine learning algorithm to predict driver genes in metastatic breast cancer

BackgroundToday, there are a lot of markers on the prognosis and diagnosis of complex diseases such as primary breast cancer. However, our understanding of the drivers that influence cancer aggression is limited.MethodsIn this work, we study somatic mutation data consists of 450 metastatic breast tumor samples from cBio Cancer Genomics Portal. We use four software tools to extract features from this data. Then, an ensemble classifier (EC) learning algorithm called EARN (Ensemble of Artificial Neural Network, Random Forest, and non-linear Support Vector Machine) is proposed to evaluate plausible driver genes for metastatic breast cancer (MBCA). The decision-making strategy for the proposed ensemble machine is based on the aggregation of the predicted scores obtained from individual learning classifiers to be prioritized homo sapiens genes annotated as protein-coding from NCBI.ResultsThis study is an attempt to focus on the findings in several aspects of MBCA prognosis and diagnosis. First, drivers and passengers predicted by SVM, ANN, RF, and EARN are introduced. Second, biological inferences of predictions are discussed based on gene set enrichment analysis. Third, statistical validation and comparison of all learning methods are performed by some evaluation metrics. Finally, the pathway enrichment analysis (PEA) using ReactomeFIVIz tool (FDR < 0.03) for the top 100 genes predicted by EARN leads us to propose a new gene set panel for MBCA. It includes HDAC3, ABAT, GRIN1, PLCB1, and KPNA2 as well as NCOR1, TBL1XR1, SIRT4, KRAS, CACNA1E, PRKCG, GPS2, SIN3A, ACTB, KDM6B, and PRMT1. Furthermore, we compare results for MBCA to other outputs regarding 983 primary tumor samples of breast invasive carcinoma (BRCA) obtained from the Cancer Genome Atlas (TCGA). The comparison between outputs shows that ROC-AUC reaches 99.24% using EARN for MBCA and 99.79% for BRCA. This statistical result is better than three individual classifiers in each case.ConclusionsThis research using an integrative approach assists precision oncologists to design compact targeted panels that eliminate the need for whole-genome/exome sequencing. The schematic representation of the proposed model is presented as the Graphic abstract.Graphic abstract

Characterizing the tumor microenvironment of metastatic ovarian cancer by single-cell transcriptomics.

Understanding the cellular composition of the tumor microenvironment and the interactions of the cells is essential to the development of successful immunotherapies in cancer. We perform single-cell RNA sequencing (scRNA-seq) of 9,885 cells isolated from the omentum in 6 patients with ovarian cancer and identify 9 major cell types, including cancer, stromal, and immune cells. Transcriptional analysis of immune cells stratifies our patient samples into 2 groups: (1) high Tcell infiltration (high Tinf) and (2) low Tcell infiltration (low Tinf). TOX-expressing resident memory CD8+ T (CD8+ Trm) and granulysin-expressing CD4+ Tcell clusters are enriched in the high Tinf group. Concurrently, we find unique plasmablast and plasma B cell clusters, and finally, NR1H2+IRF8+ and CD274+ macrophage clusters, suggesting an anti-tumor response in the high Tinf group. Our scRNA-seq study of metastatic tumor samples provides important insights in elucidating the immune response within ovarian tumors.

Collagen molecular phenotypic switch between non-neoplastic and neoplastic canine mammary tissues

In spite of major advances over the past several decades in diagnosis and treatment, breast cancer remains a global cause of morbidity and premature death for both human and veterinary patients. Due to multiple shared clinicopathological features, dogs provide an excellent model of human breast cancer, thus, a comparative oncology approach may advance our understanding of breast cancer biology and improve patient outcomes. Despite an increasing awareness of the critical role of fibrillar collagens in breast cancer biology, tumor-permissive collagen features are still ill-defined. Here, we characterize the molecular and morphological phenotypes of type I collagen in canine mammary gland tumors. Canine mammary carcinoma samples contained longer collagen fibers as well as a greater population of wider fibers compared to non-neoplastic and adenoma samples. Furthermore, the total number of collagen cross-links enriched in the stable hydroxylysine-aldehyde derived cross-links was significantly increased in neoplastic mammary gland samples compared to non-neoplastic mammary gland tissue. The mass spectrometric analyses of type I collagen revealed that in malignant mammary tumor samples, lysine residues, in particular those in the telopeptides, were markedly over-hydroxylated in comparison to non-neoplastic mammary tissue. The extent of glycosylation of hydroxylysine residues was comparable among the groups. Consistent with these data, expression levels of genes encoding lysyl hydroxylase 2 (LH2) and its molecular chaperone FK506-binding protein 65 were both significantly increased in neoplastic samples. These alterations likely lead to an increase in the LH2-mediated stable collagen cross-links in mammary carcinoma that may promote tumor cell metastasis in these patients.

Preoperative immune landscape predisposes adverse outcomes in hepatocellular carcinoma patients with liver transplantation

Immune class in hepatocellular carcinoma (HCC) has been shown to possess immunogenic power; however, how preestablished immune landscapes in premalignant and early HCC stages impact the clinical outcomes of HCC patients remains unexplored. We sequenced bulk transcriptomes for 62 malignant tumor samples from a Korean HCC cohort in which 38 patients underwent total hepatectomy, as well as for 15 normal and 47 adjacent nontumor samples. Using in silico deconvolution of expression mixtures, 22 immune cell fractions for each sample were inferred, and validated with immune cell counting by immunohistochemistry. Cell type-specific immune signatures dynamically shifted from premalignant stages to the late HCC stage. Total hepatectomy patients displayed elevated immune infiltration and prolonged disease-free survival compared to the partial hepatectomy patients. However, patients who exhibited an infiltration of regulatory T cells (Tregs) during the pretransplantation period displayed a high risk of tumor relapse with suppressed immune responses, and pretreatment was a potential driver of Treg infiltration in the total hepatectomy group. Treg infiltration appeared to be independent of molecular classifications based on transcriptomic data. Our study provides not only comprehensive immune signatures in adjacent nontumor lesions and early malignant HCC stages but also clinical guidance for HCC patients who will undergo liver transplantation.

Differential Molecular Expression Patterns Associated with Metastasis in Cutaneous Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis

The majority of cutaneous squamous cell carcinomas are treated by surgical removal; however, approximately 4% of tumors will metastasize. Molecular expression testing may improve accuracy in estimating the prognosis and defining the mechanisms important in the disease progression, which may impact response to therapy. Using PubMed (MEDLINE) and EMBASE, a systematic review was performed to evaluate studies published from January 2005 to August 2019 reporting tumor protein or RNA expression along with either outcomes (metastasis or death) or a comparison of primary with metastatic tumor samples. Inclusion criteria were met by 45 studies containing 81 comparisons of 44 distinct proteins and 25 microRNAs. On meta-analysis of studies analyzing primary tumor samples in terms of later outcomes, high primary tumor expression of PD-L1 (OR= 2.34, 95% confidence interval= 1.09-5.02, P= 0.030), EGFR (OR= 2.57, 95% confidence interval= 1.24-5.33, P= 0.011), and podoplanin (OR= 2.33, 95% confidence interval= 1.00-5.41, P= 0.049) conferred increased odds for metastasis. In comparison, metastatic tissue was more likely to have a high expression of PD-L1 than primary tissue (OR= 3.13, 95% confidence interval= 1.00-9.75, P= 0.049). Further studies are needed to confirm whether testing for PD-L1, EGFR, and podoplanin expression aids in cutaneous squamous cell carcinomas prognostic estimation of metastasis or death or predicts response to therapy.

Epigenetic and transcriptional analysis reveals a core transcriptional program conserved in clonal prostate cancer metastases.

The epigenomic regulation of transcriptional programs in metastatic prostate cancer is poorly understood. We studied the epigenomic landscape of prostate cancer drivers using transcriptional profiling and ChIP‐seq in four clonal metastatic tumors derived from a single prostate cancer patient. Our epigenomic analyses focused on androgen receptor (AR), which is a key oncogenic driver in prostate cancer, the AR pioneer factor FOXA1, chromatin insulator CCCTC‐Binding Factor, as well as for modified histones H3K27ac and H3K27me3. The vast majority of AR binding sites were shared among healthy prostate, primary prostate cancer, and metastatic tumor samples, signifying core AR‐driven transcriptional regulation within the prostate cell lineage. Genes associated with core AR‐binding events were significantly enriched for essential genes in prostate cancer cell proliferation. Remarkably, the metastasis‐specific active AR binding sites showed no differential transcriptional output, indicating a robust transcriptional program across metastatic samples. Combined, our data reveal a core transcriptional program in clonal metastatic prostate cancer, despite epigenomic differences in the AR cistrome.

Genomic and Transcriptomic Characterization of Relapsed SCLC Through Rapid Research Autopsy

IntroductionRelapsed SCLC is characterized by therapeutic resistance and high mortality rate. Despite decades of research, mechanisms responsible for therapeutic resistance have remained elusive owing to limited tissues available for molecular studies. Thus, an unmet need remains for molecular characterization of relapsed SCLC to facilitate development of effective therapies. MethodsWe performed whole-exome and transcriptome sequencing of metastatic tumor samples procured from research autopsies of five patients with relapsed SCLC. We implemented bioinformatics tools to infer subclonal phylogeny and identify recurrent genomic alterations. We implemented immune cell signature and single-sample gene set enrichment analyses on tumor and normal transcriptome data from autopsy and additional primary and relapsed SCLC data sets. Furthermore, we evaluated T cell-inflamed gene expression profiles in neuroendocrine (ASCL1, NEUROD1) and non-neuroendocrine (YAP1, POU2F3) SCLC subtypes. ResultsExome sequencing revealed clonal heterogeneity (intertumor and intratumor) arising from branched evolution and identified resistance-associated truncal and subclonal alterations in relapsed SCLC. Transcriptome analyses further revealed a noninflamed phenotype in neuroendocrine SCLC subtypes (ASCL1, NEUROD1) associated with decreased expression of genes involved in adaptive antitumor immunity whereas non-neuroendocrine subtypes (YAP1, POU2F3) revealed a more inflamed phenotype. ConclusionsOur results reveal substantial tumor heterogeneity and complex clonal evolution in relapsed SCLC. Furthermore, we report that neuroendocrine SCLC subtypes are immunologically cold, thus explaining decreased responsiveness to immune checkpoint blockade. These results suggest that the mechanisms of innate and acquired therapeutic resistances are subtype-specific in SCLC and highlight the need for continued investigation to bolster therapy selection and development for this cancer.

Abstract PD14-05: Genomic profiling and clinical outcomes with first-line atezolizumab and nab-paclitaxel in triple-negative breast cancer: An exploratory analysis from the phase 3 IMpassion130 trial

Abstract Background: The clinical implications of genomic alterations in metastatic triple-negative breast cancer (mTNBC) have not been clearly addressed. In IMpassion130, atezolizumab (A) + nab-paclitaxel (nP) showed improved progression-free survival (PFS) and clinically meaningful overall survival (OS) benefit vs placebo (P) + nP in PD-L1+ mTNBC (Schmid, NEJM 2018). The goal of this exploratory study was to evaluate the prevalence of genomic alterations and their relationship with PD-L1 status and to determine whether a prognostic or predictive role for these factors exists for A + nP in a randomized dataset. Methods: Primary or metastatic tumor samples from patients in the IMpassion130 biomarker-evaluable population (BEP) were centrally evaluated for short variants (SV), copy number alteration (CNA) and rearrangements by using a targeted next-generation sequencing (NGS) panel (FoundationOne). PD-L1 status was evaluated using the VENTANA SP142 assay, with PD-L1 positivity defined by PD-L1-expressing immune cells on ≥1% of tumor area. Prognostic effects of genomic alterations were evaluated in the pooled population from both treatment arms. PFS and OS (co-primary endpoints) were analyzed by Cox proportional hazards models. ClinicalTrials.gov identifier: NCT02425891. Results: The BEP comprised 614 patients (68% of intent-to-treat population), 605 of whom received treatment. The most common pathogenic alterations were found in the following genes: TP53 (SV, 85%), MYC (CNA, 21%), PIK3CA (SV, 18%), PTEN (SV/CNA, 18%), RB1 (SV, 7%) and BRCA1 (SV, 9%). Primary tumors (n = 419), compared with metastatic tumors (n = 195), were more likely to bear alterations in BRCA1 (11.9% vs 5.6%) and less likely to have PIK3R1 alterations (4.8% vs 9.2%). TP53 loss-of-function mutations were associated with PD-L1+ status, whereas amplifications in VEGFA and CCND3 genes were significantly less associated; none of these alterations were linked with clinical outcomes favoring A + nP. Loss of RB1 (frequency 5%) was linked with reduced PFS and OS prognosis independent of treatment (hazard ratio [HR], 2.09 [95% CI: 1.42, 3.07] and HR, 1.98 [1.26, 3.11]), respectively), and CNA in CDKN2A (12%) and CDKN2B (11%) were associated with improved PFS and OS clinical activity in the A + nP arm vs the P + nP arm (HR PFS, 0.43-0.44 and HR OS, 0.47, respectively). Three of 584 microsatellite-evaluable samples (0.5%) had MSI-H status, which was not associated with PD-L1 status; all 3 MSI-H patients were from the P + nP arm. A total of 220 of 514 evaluable samples (42.8%) had PIK3CA/AKT1/PTEN-altered status, which was not significantly associated with PD-L1 status or A + nP clinical outcome. Conclusions: In this exploratory analysis using a targeted NGS panel, we show that tumors from patients with previously untreated mTNBC had similar genomic profiles at baseline as those published for early TNBC tumors. The few alterations that were linked to PD-L1 status were not linked to clinical outcome. CNA in cell cycle genes (e.g., RB1) had potential prognostic value, whereas CDKN2A and CDKN2B were potentially predictive of A + nP clinical benefit favoring A + nP. These data are hypothesis generating and require validation in an independent data set. Citation Format: Leisha Emens, Luciana Molinero, Sylvia Adams, Hope S. Rugo, Andreas Schneeweiss, Véronique Diéras, Hiroji Iwata, Carlos Barrios, Eric P. Winer, Ching-Wei Chang, Stephen Y. Chui, Peter Schmid, Sherene Loi. Genomic profiling and clinical outcomes with first-line atezolizumab and nab-paclitaxel in triple-negative breast cancer: An exploratory analysis from the phase 3 IMpassion130 trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD14-05.

Abstract OT-17-01: Solti-1903 hope: Real-world clinical practice study to assess the impact of using comprehensive genomic data on the next treatment decision making-choice in patients with locally advanced or metastatic breast cancer in Spain

Abstract BACKGROUND: Metastatic breast cancer (mBC) remains an incurable disease and is the cause of nearly all deaths from breast cancer. Targeted molecular therapies and the evolving role of next-generation sequencing (NGS) technologies are increasing and may improve outcomes in breast cancer patients. However, they are not being routinely used in the clinic. One strategy to overcome the barriers of implementing NGS in the clinic is to promote the active participation of mBC patients in the management of their disease. With this in mind, we designed HOPE (SOLTI-1903), a national real-world study where patients lead their inclusion, participation and follow-up in the study through a digital tool that will guide them in every step of the journey. Our objective is empowering mBC patients and gather real-world data about the utilization of molecular information in the management of mBC. TRIAL DESIGN: Patients diagnosed with mBC who are receiving, have just received, or will receive standard treatment or treatment in a clinical trial (CT) can be included. Demographic data, disease characteristics, treatment history and quality of life data will be collected through a digital tool (DT) by the patient. Patients are encouraged to involve their physician`s in the study journey. The study is complemented by a patient empowerment program including informative workshops and precision medicine video-tutorials. A total of 600 patients will be included in Spain. Patient Journey Once patients request to participate in HOPE through the DT, a dedicated team from SOLTI will assist them in the following steps while validating that eligibility criteria are met according to data introduced by themselves. Then, patients will receive instructions via DT to go to the nearest partner local laboratory, where they will sign the study consent form. A metastatic (preferably) or primary archival tumor sample will be requested to the patient’s reference hospital and analyzed by FoundationOne®CDx. Also, a blood sample will be collected and analyzed by Guardant360. For all patients, two NGS tests will be offered (If no tissue available, only blood test will be performed). Both molecular analyses results will be reviewed during regular meetings by a Molecular Advisory Board (MAB). The MAB, based on their joint experience in clinical oncology, genomics, molecular biology, bioethics and pathology, may add some advice to these reports via DT, making comments about detected molecular alterations and adding further recommendations for specific treatment options or available CT with targeted therapies. From that moment, patients will record their disease evolution in the DT each 3 months for 2 years. The primary objective is to assess the real-world clinical practice integrating molecular profiling in the Standard of Care management of mBC patients connected through a DT. Secondary objectives include to describe genetic mutational profile of mBC, to evaluate the enrollment rate in CT of patients engaged in a patient-centered strategy for molecular tumor assessment, to asses Progression Free Survival, Overall Survival and Quality of life status among patients enrolled in CT according to the tumor’s genomic profile and those receiving standard treatment and to describe logistic feasibility of the study. This study is sponsored by SOLTI and financially supported by Novartis and two non-profit organizations: Asociación Cáncer de Mama Metastásico y Fundación Actitud frente al Cáncer. Roche and Guardant Health provide their test for all patients. Citation Format: Ana Casas, Eva Ciruelos, Mafalda Oliveira, Cristina Saura, Meritxell Bellet, Sonia Pernas, Joaquín Gavilá, Montserrat Muñoz, María Vidal, Blanca Gonzalez Farré, Juan M. Cejalvo, Rafael Lopez, Ana Vivancos, Maurizio Scaltriti, Javier S. Bofill, Isabel Blancas, Emilio Alba, Valentina Boni, Susana De la Cruz, Elena Galve, Antonia Perelló, Mireia Margelí, Jordi Canes, Pamela Céliz, Helena Masanas, Rosa Olmos, Marga Fonts, Pilar Fernandez-Pascual, Patricia Villagrasa, Aleix Prat. Solti-1903 hope: Real-world clinical practice study to assess the impact of using comprehensive genomic data on the next treatment decision making-choice in patients with locally advanced or metastatic breast cancer in Spain [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-17-01.

Abstract PS18-11: Molecular characterization of advanced breast cancer according to site of metastasis

Abstract Background: There is an increasing need in the clinic to biopsy metastatic disease in patients with advanced breast cancer (ABC). However, the microenvironment and tumor cell biology of breast cancer metastasis is largely unknown. Here, we report a molecular characterization of ABC according to the site of metastasis. Methods: RNA from 184 FFPE metastatic samples were evaluated using the nCounter BC 360 Panel, which includes the expression of 689 BC-related genes and 82 immune-related genes. PAM50 subtypes (Basal-like, HER2-enriched [HER2-E], Luminal A, Luminal B and Normal-like) were also determined. HER2 protein expression was assessed by immunohistochemistry (IHC) in 115 tumor samples, and HER2-low tumors (i.e. 1+ or 2+ and ISH-negative) were identified. Descriptive statistics, significance analysis of microarrays (using False Discovery Rate [FDR]) and logistic regressions were used to identify organ-specific gene expression profiles. Finally, we derived an organ-specific predictor of 209-genes from our cohort, and applied it to the RNAseq-based TCGA PanCancer dataset, which includes 174 glioblastomas multiforme, 424 liver hepatocellular carcinomas and 576 lung adenocarcinomas, among other cancer-types. Results: A single metastatic tumor sample from 184 individual patients with ABC was obtained from bone (18%), liver (17%), skin (15%), brain (12%), breast (13%), lymph nodes (9%), lung (7%), pleura (5%), ovary (2%), muscle (1%) and peritoneum (1%). All PAM50 subtypes were identified across the main organ sites; however, significant differences in subtype distribution were observed (p&amp;lt;0.001). Basal-like subtype was more prevalent in brain, lung and skin metastasis (FDR=0.3%). Unsupervised analysis and principal component analysis revealed brain and liver metastasis as the most distinct. Supervised analysis identified organ-specific genes independently of PAM50 subtype, i.e.: bone-specific genes (WIF1, IBSP, MMP9 and ITGB3); brain-specific genes (CRYAB, SOX10, FGF1 and CHI3L1); liver-specific genes (ALDH1A1, CYP4F3, PCK1, and SFRP2); lung-specific genes (CAV1, WNT5A, PTGS2 and IL6); skin-specific genes (KRT14, KRT5, S100A7 and SERPINB5). Among the organ-specific gene list, 15 (30%) of the 50 PAM50 genes were identified, including up-regulation of FGFR4 in liver, ESR1 in bone, ERBB2 in lung and KRT5 and KRT14 in skin. Regarding ERBB2, a high correlation between ERBB2 mRNA and HER2 IHC expression (0, 1+, 2+ and 3+) was observed (p&amp;lt;0.001). Interestingly, HER2-low disease was identified across all PAM50 subtypes and organ sites, including bone metastasis. Regarding immune-genes, all were found differentially expressed across the main organ sites (FDR&amp;lt;5%) with lung metastasis showing the highest expression (i.e. PDCD1, CD8A, GMZA, IL1B) and liver and brain metastasis the lowest. Finally, the 209-gene organ-specific predictor in PanCancer TCGA identified 96% of glioblastomas multiforme as brain, 98.6% of liver hepatocellular carcinomas as liver and 57.1% of lung adenocarcinoma as lung. Conclusions: The main sites of metastasis in ABC have unique biological features independently of tumor molecular subtype. Our results suggest that treatment strategies based on the site(s) of metastasis should be explored. Citation Format: Fara Brasó-Maristany, Laia Paré, Núria Chic, Olga Martínez-Sáez, Tomás Pascual, Meritxell Mallafré, Blanca González-Farré, Esther Sanfeliu, Débora Martínez, Patricia Galván, Belinda Salinas, Barbara Adamo, Reinaldo Moreno, Maria Vidal, Montserrat Muñoz, Aleix Prat. Molecular characterization of advanced breast cancer according to site of metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-11.

Reconnaissance of tumor immune microenvironment spatial heterogeneity in metastatic renal cell carcinoma and correlation with immunotherapy response.

A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor-stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68+ CD163+ CD206+ ) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet+ T helper type 1 (Th1) cell density was highest at the tumor-stromal interface (P=0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P=0·015) and survival after immunotherapy (P=0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)+ T regulatory cell density correlated with improved survival after immunotherapy (P=0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

Distinct T cell receptor repertoire diversity of clinically defined high-grade serous ovarian cancer treatment subgroups

SummaryIn patients with high-grade serous ovarian cancer (HGSC), it is unclear which genomic features are related to complete gross resection (R0), which is typically associated with better clinical outcomes, or response to neoadjuvant chemotherapy (NACT). In this study, we evaluated T cell receptor (TCR) repertoire diversity in primary and metastatic tumor samples (n = 90) from clinically well-annotated patients with HGSC who achieved R0 or received NACT with excellent or poor response based on a laparoscopic triage algorithm. TCR sequencing followed by an integrative analysis with comprehensive multi-omics data identified higher TCR diversity (e.g., higher number of unique productive sequences and less clonal relatedness) in the R0 than NACT groups. We found enrichment of specific TCRβ genes usage, distinct mutual exclusiveness and co-occurrence pattern of TCRβ genes among the groups. We also found significantly positive correlations between clonal relatedness and neoantigens, copy number variations, and mutation load in the groups.

Aggregation-induced emission luminogens reveal cell cycle-dependent telomerase activity in cancer cells.

Telomerase acts as an important biomarker for tumor identification, and synthesizes telomeric repeats at the end of chromosome telomeres during the replicative phase of the cell cycle; thus, the expression level of telomerase changes as the cell cycle progresses. TERT mRNA expression and telomerase activity were significantly increased in over 80% of human cancers from tissue specimens. Although many efforts have been made in detecting the activity of TERT mRNA and active telomerase, the heterogeneous behavior of the cell cycle was overlooked, which might affect the accuracy of the detection results. Herein, the AIEgen-based biosensing systems of PyTPA-DNA and Silole-R were developed to detect the cellular level of TERT mRNA and telomerase in different cell cycles. As a result, the fluorescence signal of cancer cells gradually increased from G0/G1, G1/S to S phase. In contrast, both cancer cells arrested at G2/M phase and normal cells exhibited negligible fluorescence intensities. Compared to normal tissues, malignant tumor samples demonstrated a significant turn-on fluorescence signal. Furthermore, the transcriptomics profiling revealed that tumor biomarkers changed as the cell cycle progressed and biomarkers of CA9, TK1 and EGFR were more abundantly expressed at early S stage. In this vein, our study presented advanced biosensing tools for more accurate analysis of the cell-cycle-dependent activity of TERT mRNA and active telomerase in clinical tissue samples.