Intratumoral Genetic Heterogeneity Research Articles

Abstract 1620: Multiregion spatial sequencing of early-stage head and neck squamous cell carcinoma revealed theranostic intratumor heterogeneity

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) is characterized by substantial intratumor heterogeneity (ITH) at the molecular level that may foster tumor evolution, adaptation, and therapeutic failure. Personalized-medicine strategies mostly rely on single tumor-biopsy samples which might not capture the mutational burden of heterogeneous tumors. We investigated genetic ITH portrayed from multiregion spatial sequencing in early-stage HNSCC patients. Patients and Method: Seventy-eight early-stage HNSCC patients treated with upfront curative-intent surgery in the prospective biobanking SCANDARE study (NCT03017573) were included. We developed a pathological measure of ITH, based on the relative contribution of multiple poorly and well-differentiated intratumor regions (n = 199) in resected tumor samples. To characterize genetic ITH, we performed targeted next-generation sequencing on multiple spatially separated intratumor regions by using a custom panel of 571 genes (named DRAGON), in combination with 3’-Tag RNA sequencing and pathway enrichment analysis. Results: Fifty-nine patients displaying a minimum of two sequenced intratumor regions (total, n = 180) were analyzed. We observed genetic ITH in 22/59 patients (37%), of whom 6/22 (27%) based on theranostic molecular alterations. Three of the six patients exhibited molecular alterations in PIK3CA (n = 2) and TSC2 (n = 1) that have supporting clinical evidence of actionability in some cancer types (OncoKB level 1) and three patients exhibited actionable molecular alterations in PTEN (n = 2) and CDKN2A/B (n = 1) supported by substantial research evidence (OncoKB level 4). Two patients exhibited molecular alterations in SMARCA4 that is likely oncogenic, albeit not yet annotated by OncoKB. The pathological measure of ITH accounted for a significant source of variation in gene expression. Notably, we identified 346 differentially expressed genes imposing p-values ≤ 0.05 and |log fold-change| ≥ 1 involved in several Reactome’s signaling pathways, including the cell junction organization (R-HSA-446728) and the tight junction interaction (R-HSA-420029) pathways. Conclusions: We reported clinically relevant genetic ITH in early-stage HNSCC, which can lead to underestimation of the mutational landscape portrayed from single tumor biopsy and may present major challenges to precision medicine. Our findings uncovered the biology behind ITH and may have translational implications for biomarkers discovery in HNSCC. Citation Format: Grégoire Marret, Sophie Vacher, Constance Lamy, Ladidi Ahmanache, Laura Courtois, Choumouss Kamoun, Maral Halladjian, Zakhia El Beaino, Jerzy Klijanienko, Charlotte Martinat, Emmanuelle Jeannot, Joey Martin, Guillaume Rougier, Nathalie Badois, Maria Lesnik, Antoine Dubray-Vautrin, Olivier Choussy, Wahib Ghanem, Rabah Taouachi, Julien Masliah Planchon, Nicolas Servant, Maud Kamal, Ivan Bièche, Christophe Le Tourneau. Multiregion spatial sequencing of early-stage head and neck squamous cell carcinoma revealed theranostic intratumor heterogeneity [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 1620.

Abstract 5665: Native nanopore sequencing of multiple tumor sites reveals genetic and epigenetic intra-tumor heterogeneity in canine osteosarcoma

Abstract Osteosarcoma (OS) is an aggressive bone malignancy affecting children and young adults. Implementation and optimization of neoadjuvant chemotherapy led to significant improvements in outcome, but patients with recurrence or metastases at diagnosis continue to have poor prognosis. Whole genome sequencing (WGS) revealed that a hallmark of OS is a highly rearranged genome with complex copy number aberrations rather than point mutations. A better understanding of the key genomic and epigenetic factors driving clonal evolution may provide insights into the tumor progression and the therapy response. Canine OS recapitulates the majority of the hallmark genetic and molecular factors of human OS and is recognized as an informative model. A significant difference is that initial surgery in canine OS allows us to explore tumor evolution unperturbed by treatment, providing invaluable resources to study the genetic and molecular landscape of tumor evolution. Here, we present the first study to date in which we performed bulk WGS of multiple tumor sites and distal normal tissue in two dogs using nanopore long-read sequencing to analyze the genetic and epigenetic evolution of OS. In both subjects, somatic structural variation detection using Severus revealed chromothripsis-like rearrangements in chr11, leading to the deletion of Cdkn2a, a known cell cycle regulator and tumor suppressor. Interestingly, in one of the subjects, the majority of the SVs (n = 131) shared across all the samples were localized on chr5, coinciding with perturbed copy number profiles and bi-allelic frequency of the SNVs, with all mutations identified using complementary bioinformatics analysis of the same native nanopore reads. The multi-site sample analysis identified subclonal structural alterations in TP53 and ATRX genes. With phased nanopore reads, we observed subject-specific copy-neutral loss-of-heterozygosity events and long identical-by-descent blocks. Haplotype-specific copy-number analysis of tumors showed different aneuploidy patterns in both subjects, highlighted clonal compositions of samples from distinct sites, and identified regions of copy-number heterogeneity. Furthermore, phased nanopore reads also revealed haplotype-specific 5mC and 5hmC methylation differences across tumor samples, where samples from different sites of the same tumor demonstrate distinct methylation patterns that overlap DLG2, RUNX2, KDR, and other genes known as mutational hotspots in canine/human OS. Overall, we showcase how a single sequencing assay allows for a joint analysis of genetic and epigenetic tumor alterations, thus providing a richer understanding of the drivers of somatic evolution and their association with the molecular landscape in the canine OS. These findings can be translated into human disease and shed light on tumor progression and therapy response. Citation Format: Ayse Keskus, Sergey Aganezov, Tanveer Ahmad, Xiaoguang Dai, Robert Walker, John Beaulaurier, Scott Hickey, Sissel Juul, Amy Kathleen LeBlanc, Paul S. Meltzer, Mikhail Kolmogorov. Native nanopore sequencing of multiple tumor sites reveals genetic and epigenetic intra-tumor heterogeneity in canine osteosarcoma [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 5665.

Abstract 1621: Integrated analysis of genetic, transcriptional and TME evolution of ccRCC: TRACERx Renal

Abstract Background: Genetic evolution of clear cell renal cell carcinoma (ccRCC) follows distinct trajectories, with varying levels of intratumor heterogeneity (ITH) and chromosomal complexity (WGII). While these patterns associate with clinical outcomes, it remains unknown whether they fully reconcile tumor behavior and how genetic and transcriptional features co-evolve in relation to the tumor microenvironment (TME). Methods: To analyze the patterns of transcriptional and TME heterogeneity, we performed bulk whole-transcriptome sequencing on 244 samples, including 22 metastatic and 12 tumor-adjacent normal samples, from 79 ccRCC patients recruited to the TRACERx Renal study. We integrated transcriptional data with previously published genetic, phylogenetic, spatial and clinical information. Results: Transcriptional distances between paired samples from the same primary tumor mirrored but were not fully determined by genetic distance (p-value < 0.001); and increased from primary-primary to primary-metastasis and primary-normal pairs. Within primary-metastasis pairs, metastasis-seeding primary tumor regions were transcriptionally closest to their matched metastasis (p-value < 0.001), suggesting that an important fraction of metastatic transcriptional traits were acquired in the primary tumor. Regarding the tumor clonal structure, transcriptional evolution followed a conserved path through increasing cell proliferation and oxidative phosphorylation and downregulating DNA repair from earlier to later clones. Further, within tumors with increasing WGII we observed upregulation and downregulation of repressors and downstream effectors, respectively, of the canonical cGAS-STING pathway. Combining the presence of this transcriptional pattern with WGII predicted shorter PFS in TRACERx Renal (p-value < 0.001) and in TCGA-KIRC (p-value < 0.001). Clonal evolution was also linked to changes in TME, with each of the previously defined genetic evolutionary trajectories associated to a specific TME (p-value < 0.001). For example, ccRCCs on a PBRM1-SETD2 trajectory demonstrated increased infiltration of cytotoxic immune cells. TME ITH was pervasive and associated with shorter PFS (p-value = 0.03). A recurrent trend from earlier to later clones was progressive T cell depletion (p-value < 0.001). The evolution of the TCR repertoire mirrored the tumor clonal structure (p-value = 0.002), suggesting the thus far elusive antigenic source in ccRCC is heritable. Accordingly, the TCR repertoire in metastasis-seeding primary tumor regions resembled the closest the TCR repertoire of matched metastasis (p-value = 0.06). Conclusion: Integrated analysis of genetic and transcriptional data in TRACERx Renal showed i) transcriptional and TME ITH not fully recapitulated by genetic ITH, ii) conserved paths of transcriptional and TME evolution and iii) a heritable nature of part of the ccRCC antigen source. Citation Format: Ángel Fernández Sanromán, Lewis Au, Benjy Jek Yang Tan, Charlotte Spencer, Anne-Laure Catin, Irene Lobon, Husayn Pallikonda, Kevin Litchfield, Fiona Byrne, James Larkin, Annika Fendler, Samra Turajlic. Integrated analysis of genetic, transcriptional and TME evolution of ccRCC: TRACERx Renal [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 1621.

Abstract 6946: Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids

Abstract Colorectal cancer (CRC) is one of the most diagnosed cancers worldwide. Though several treatment strategies are available today, around a million people die of this disease each year worldwide. Thus, there is still a pivotal need for new and more effective treatments. Genetic and non-genetic intra-tumor heterogeneity has emerged lately as an intrinsic feature of malignancies and as one of the factors responsible for therapy resistance or tolerance. In particular, the nature and role that phenotypically distinct, yet genetically indistinguishable, subpopulations have on tumor biology is far from being understood. Here we investigate phenotypic heterogeneity in CRC by identifying specific transcriptionally well defined cell subpopulations. We aim at deciphering the interactions between populations in terms of differentiation and plasticity, tumor growth and potentially exposed targetable vulnerabilities. To this end, we used patient-derived metastatic CRC (mCRC) organoids, including KRAS wild type (wt) and mutant (kras) models. We performed in total scRNAseq for 7 mCRC samples (4 wt; 3 kras) in different growth media: normal growth medium (supplemented with EGF) (n=1); medium without EGF (noEGF) (n=7); or treated with Cetuximab (CTX, a clinically approved monoclonal antibody against EGFR) (n=7). We found that the wt sample grown in EGF presents 2 distinct subpopulations: one with stemness characteristics (LGR5 high) and the other with a more differentiated phenotype (KRT20 high), in agreement with the current bibliography. Interestingly, in EGF deprived cultures new subpopulations appear. In particular, we were able to identify a subpopulation with distinct expression of WNT-related genes (APCDD1, WNT6, WIF1). This subpopulation is also present, and in cases increasing in fraction, when organoids are treated with CTX (in 3 out of 7 samples). Interestingly, a fraction of the WNT positive subpopulation seems to be able to proliferate under CTX treatment (Ki67 positive). We identified the negative regulator of the WNT pathway, APCDD1, as a potential marker to track this subpopulation. We took one wt and one kras sample as models to validate these findings by flow cytometry. Furthermore, sorting of APCDD1 positive and negative cells, and further RT-qPCR analysis, confirmed good correlation between antibody staining and gene expression. Moreover, we found that both types of cells are able to re-grow as organoids after re-culturing. This suggests that this WNT-associated subpopulation could be contributing, at least in part, to CTX tolerance or resistance. Our results show promising potential to help unravel basic features of intra-tumor heterogeneity both in basal conditions and upon treatment, and possibly to find new therapeutic targets. Citation Format: Sabrina J. Fletcher, Irene Catalano, Elena Grassi, Sofia Borgato, Barbara Peracino, Elena Piretto, Luca Alessandri, Raffaele Calogero, Luca Primo, Livio Trusolino, Andrea Bertotti, Alberto Puliafito. Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids [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 6946.

Extrachromosomal DNA in cancer.

Extrachromosomal DNA (ecDNA) has recently been recognized as a major contributor to cancer pathogenesisthat is identified in most cancer types and isassociated with poor outcomes. When it wasdiscovered over 60 years ago, ecDNA was considered to be rare, and its impact on tumour biology was not well understood. The application of modern imaging and computational techniques has yielded powerful new insights into the importance of ecDNA in cancer. The non-chromosomal inheritance of ecDNA during cell division results in high oncogene copy number, intra-tumoural genetic heterogeneity and rapid tumour evolution thatcontributes to treatment resistance and shorter patient survival. In addition, the circular architecture of ecDNA results in altered patterns of gene regulation that drive elevated oncogene expression, potentially enabling the remodelling of tumour genomes. The generation of clusters of ecDNAs, termed ecDNA hubs, results in interactions between enhancers and promoters in trans, yielding a new paradigm in oncogenic transcription. In this Review, we highlight the rapid advancements in ecDNA research, providing new insights into ecDNA biogenesis, maintenance andtranscription and itsrole in promoting tumour heterogeneity. To conclude, we delve into a set of unanswered questions whose answers will pave the way for the development of ecDNA targeted therapeutic approaches.

Intratumor genetic heterogeneity and head and neck cancer relapse

BackgroundHead and neck squamous cell carcinomas are treated by surgery, radiotherapy (RT), chemoradiotherapy (CRT) or combinations thereof, but locoregional recurrences (LRs) occur in 30–40% of treated patients. We have previously shown that in approximately half of the LRs after CRT, cancer driver mutations are not shared with the index tumor. AimTo investigate two possible explanations for these genetically unrelated relapses, treatment-induced genetic changes and intratumor genetic heterogeneity. MethodsTo investigate treatment-induced clonal DNA changes, we compared copy number alterations (CNAs) and mutations between primary and recurrent xenografted tumors after treatment with (C)RT. Intratumor genetic heterogeneity was studied by multi-region sequencing on DNA from 31 biopsies of 11 surgically treated tumors. ResultsInduction of clonal DNA changes by (C)RT was not observed in the xenograft models. Multi-region sequencing demonstrated variations in CNA profiles between paired biopsies of individual tumors, with copy number heterogeneity scores varying from 0.027 to 0.333. In total, 32 cancer driver mutations could be identified and were shared in all biopsies of each tumor. Remarkably, multi-clonal mutations in these same cancer driver genes were observed in 6 of 11 tumors. Genetically distinct heterogeneous cell cultures could also be established from single tumors, with different biomarker profiles and drug sensitivities. ConclusionIntratumor genetic heterogeneity at the level of the cancer driver mutations might explain the discordant mutational profiles in LRs after CRT, while there are no indications in xenograft models that these changes are induced by CRT.

Extrachromosomal DNA: Biogenesis and Functions in Cancer

In cancer, oncogenes can untether themselves from chromosomes onto circular, extrachromosomal DNA (ecDNA) particles. ecDNA are common in many of the most aggressive forms of cancer of women and men and of adults and children, and they contribute to treatment resistance and shorter survival for patients. Hiding in plain sight and missing from cancer genome maps, ecDNA was not, until recently, widely recognized to be an important feature of cancer pathogenesis. However, extensive new data demonstrate that ecDNA is a frequent and potent driver of aggressive cancer growth and treatment failure that can arise early or late in the course of the disease. The non-Mendelian genetics of ecDNA lies at the heart of the problem. By untethering themselves from chromosomes, ecDNA are randomly distributed to daughter cells during cell division, promoting high oncogene copy number, intratumoral genetic heterogeneity, accelerated tumor evolution, and treatment resistance due to rapid genome change. Further, the circular shape of ecDNA, and its high level of chromatin accessibility, promotes oncogene transcription and generates unique enhancer–promoter interactions in cis, as well as cooperative regulatory interactions between ecDNA particles in trans. In this review, we discuss the state of the field and its implications for patients with oncogene-amplified cancers. Expected final online publication date for the Annual Review of Cancer Biology, Volume 8 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Integrating scRNA-seq and bulk RNA-seq to characterize infiltrating cells in the colorectal cancer tumor microenvironment and construct molecular risk models.

Colorectal cancer (CRC) is a malignancy that is both highly lethal and heterogeneous. Although the correlation between intra-tumoral genetic and functional heterogeneity and cancer clinical prognosis is well-established, the underlying mechanism in CRC remains inadequately understood. Utilizing scRNA-seq data from GEO database, we re-isolated distinct subsets of cells, constructed a CRC tumor-related cell differentiation trajectory, and conducted cell-cell communication analysis to investigate potential interactions across cell clusters. A prognostic model was built by integrating scRNA-seq results with TCGA bulk RNA-seq data through univariate, LASSO, and multivariate Cox regression analyses. Eleven distinct cell types were identified, with Epithelial cells, Fibroblasts, and Mast cells exhibiting significant differences between CRC and healthy controls. T cells were observed to engage in extensive interactions with other cell types. Utilizing the 741 signature genes, prognostic risk score model was constructed. Patients with high-risk scores exhibited a significant correlation with unfavorable survival outcomes, high-stage tumors, metastasis, and low responsiveness to chemotherapy. The model demonstrated a strong predictive performance across five validation cohorts. Our investigation involved an analysis of the cellular composition and interactions of infiltrates within the microenvironment, and we developed a prognostic model. This model provides valuable insights into the prognosis and therapeutic evaluation of CRC.

EPCO-32. DISSECTING THE INTRA- AND INTER-TUMORAL HETEROGENEITY UNDERLYING GLIOBLASTOMA PATHOGENESIS UTILIZING MULTI-OMICS ANALYSIS

Abstract Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis. Considering its extensive molecular diversity underlies therapeutic resistance to multimodal treatment, it is imperative to reveal intra- and inter-tumoral heterogeneity, leading to further understanding of GBM pathogenesis. We analyzed 289 whole-genome sequencing data (WGS) including 159 unpublished deep WGS (≥ ×120 coverage) along with RNA-seq, DNA methylation array, whole-genome bisulfite sequencing, and assay for transposase-accessible chromatin with sequencing (ATAC-seq) to uncover the multi-omics molecular heterogeneity of GBM. Along with inter-tumoral heterogeneity of genetic driver alterations, deep WGS enables us to delineate a fine view of clonal architecture where mutational signatures differ across clonal and subclonal mutations, supporting that different mutational processes contribute to GBM pathogenesis depending on the developmental stage. As well, tumor cell differentiation status detected by transcriptional deconvolution analysis is associated with multi-layer profiles including genome, transcriptome, epigenome, and chromatin status. ATAC-seq demonstrated distinct features of genome-wide chromatin accessibility associated with gene expression subtypes of GBM. Motif enrichment analysis detects the differentially accessible regions where the proneural subtypes are enriched with the SOX10 motif, known as a transcription factor for mesenchymal transition whereas the mesenchymal and classical subtypes are enriched with the CREB/ATF motif, a regulator of TGF beta associated with a poor prognosis. Our findings support a model in which GBM evolves through generating genetic and epigenetic intra-tumoral heterogeneity suggesting that specific cell states may have distinct vulnerability to mutational processes and epigenetic modifications. Our analysis reveals the molecular mechanisms underlying the progression of GBM enhancing our understanding of the pathogenesis.

Clinical implication of genetic intratumor heterogeneity for targeted therapy in head and neck cancer

Background Genomic profiling is increasingly used both in therapeutic decision-making and as inclusion criteria for trials testing targeted therapies. However, the mutational landscape may vary across different areas of a tumor and intratumor heterogeneity will challenge treatments or clinical decisions based on single tumor biopsies. The purpose of this study was to assess the clinical relevance of genetic intratumor heterogeneity in head and neck squamous cell carcinomas (HNSCC) using the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Materials and methods This prospective study included 33 whole tumor specimens from 28 patients with primary or recurrent HNSCC referred for surgery. Three tumor blocks were selected from central, semi-peripheral, and peripheral positions, mimicking biopsies in three different locations. Genetic analysis of somatic copy number alterations (SCNAs) was performed on the three biopsies using Oncoscan, focusing on 45 preselected HNSCC genes of interest. Clinical relevance was assessed using the ESCAT score to investigate whether and how treatment decisions would change based on the three biopsies from the same tumor. Results The SCNAs identified among 45 preselected genes within the three tumor biopsies derived from the same tumor revealed distinct variations. The detected discrepancies could potentially influence treatment approaches or clinical decisions in 36% of the patients if only one tumor biopsy was used. Recurrent tumors exhibited significantly higher variation in SCNAs than primary tumors (p = .024). No significant correlation between tumor size and heterogeneity (p = .7) was observed. Conclusion In 36% of patients diagnosed with HNSCC, clinically significant intratumor heterogeneity was observed which may have implications for patient management. This finding substantiates the need for future studies that specifically investigate the clinical implications associated with intratumor heterogeneity.

Small extrachromosomal circular DNA harboring targeted tumor suppressor gene mutations supports intratumor heterogeneity in mouse liver cancer induced by multiplexed CRISPR/Cas9

BackgroundPrimary liver cancer has significant intratumor genetic heterogeneity (IGH), which drives cancer evolution and prevents effective cancer treatment. CRISPR/Cas9-induced mouse liver cancer models can be used to elucidate how IGH is developed. However, as CRISPR/Cas9 could induce chromothripsis and extrachromosomal DNA in cells in addition to targeted mutations, we wondered whether this effect contributes to the development of IGH in CRISPR/Cas9-induced mouse liver cancer.MethodsCRISPR/Cas9-based targeted somatic multiplex-mutagenesis was used to target 34 tumor suppressor genes (TSGs) for induction of primary liver tumors in mice. Target site mutations in tumor cells were analyzed and compared between single-cell clones and their subclones, between different time points of cell proliferation, and between parental clones and single-cell clones derived from mouse subcutaneous allografts. Genomic instability and generation of extrachromosomal circular DNA (eccDNA) was explored as a potential mechanism underlying the oscillation of target site mutations in these liver tumor cells.ResultsAfter efficiently inducing autochthonous liver tumors in mice within 30–60 days, analyses of CRISPR/Cas9-induced tumors and single-cell clones derived from tumor nodules revealed multiplexed and heterogeneous mutations at target sites. Many target sites frequently displayed more than two types of allelic variations with varying frequencies in single-cell clones, indicating increased copy number of these target sites. The types and frequencies of targeted TSG mutations continued to change at some target sites between single-cell clones and their subclones. Even the proliferation of a subclone in cell culture and in mouse subcutaneous graft altered the types and frequencies of targeted TSG mutations in the absence of continuing CRISPR/Cas9 genome editing, indicating a new source outside primary chromosomes for the development of IGH in these liver tumors. Karyotyping of tumor cells revealed genomic instability in these cells manifested by high levels of micronuclei and chromosomal aberrations including chromosomal fragments and chromosomal breaks. Sequencing analysis further demonstrated the generation of eccDNA harboring targeted TSG mutations in these tumor cells.ConclusionsSmall eccDNAs carrying TSG mutations may serve as an important source supporting intratumor heterogeneity and tumor evolution in mouse liver cancer induced by multiplexed CRISPR/Cas9.

Genetic Intratumor Heterogeneity Remodels the Immune Microenvironment and Induces Immune Evasion in Brain Metastasis of Lung Cancer

IntroductionBrain metastasis, with the highest incidence in patients with lung cancer, significantly worsens prognosis and poses challenges to clinical management. To date, how brain metastasis evades immune elimination remains unknown. MethodsWhole-exome sequencing and RNA sequencing were performed on 30 matched brain metastasis, primary lung adenocarcinoma, and normal tissues. Data from The Cancer Genome Atlas primary lung adenocarcinoma cohort, including multiplex immunofluorescence, were used to support the findings of bioinformatics analysis. ResultsOur study highlights the key role of intratumor heterogeneity of genomic alterations in the metastasis process, mainly caused by homologous recombination deficiency or other somatic copy number alteration–associated mutation mechanisms, leading to increased genomic instability and genomic complexity. We further proposed a selection model of brain metastatic evolution in which intratumor heterogeneity drives immune remodeling, leading to immune escape through different mechanisms under local immune pressure. ConclusionsOur findings provide novel insights into the metastatic process and immune escape mechanisms of brain metastasis and pave the way for precise immunotherapeutic strategies for patients with lung cancer with brain metastasis.

Abstract PR012: SETD2 safeguards the genome against isochromosome formation

Abstract Chromosome mis-segregation during cell division occurs in diverse tumor types, promoting aneuploidy and intratumoral genetic heterogeneity. While the mechanisms that govern chromosome segregation are well-established, how changes in the activities of tumor suppressors or oncogenes drive mitotic errors remain poorly understood. Loss of the tumor suppressor SETD2, the methyltransferase responsible for tri-methylation of histone 3 lysine 36 (H3K36me3), correlates with clear cell renal cell carcinoma (ccRCC) tumors exhibiting extensive intratumoral heterogeneity. Yet, how loss of H3K36me3 or SETD2 promotes heterogeneity is unknown. Here, we show that loss of SETD2 promotes chromosome mis-segregation during mitosis and interphase DNA bridges driven by the formation of dicentric chromosomes. Cytogenetic analyses revealed that these dicentrics loss were largely comprised of mirror-imaged isodicentric chromosomes that contain two active centromeres. In addition to isodicentrics, cells lacking SETD2 or H36K36me3 generated iso- and acentric chromosomes, however loss of SETD2 protein additionally caused chromosome instability. These data directly link loss of a tumor suppressor to these mutable chromatin structures that initiate intratumor heterogeneity by promoting gross chromosomal rearrangements. Citation Format: Frank M. Mason, Anteneh T. Tebeje, Emily S. Kounlavong, Rashmi Dahiya, Logan Vlach, Tiffany Guess, Ruhee Dere, Ryoma Ohi, Peter Ly, Cheryl L. Walker, W. Kimryn Rathmell. SETD2 safeguards the genome against isochromosome formation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR012.

HER2 Genetic Intratumor Heterogeneity Is Associated With Resistance to Trastuzumab and Trastuzumab Emtansine Therapy in Recurrent High-Grade Endometrial Cancer

Anti-HER2 targeted therapies have recently demonstrated clinical activity in the treatment of high-grade endometrial carcinomas (ECs), particularly serous carcinomas with HER2 amplification and/or overexpression. Intratumor heterogeneity of HER2 amplification or HER2 genetic intratumor heterogeneity (G-ITH) has been associated with resistance to anti-HER2 therapies in breast and gastroesophageal cancers; however, its clinical relevance in EC is unknown. To characterize HER2 G-ITH in EC, archival specimens from a clinically annotated cohort of 57 ECs treated with trastuzumab or trasutuzmab emtansine in the recurrent (n = 38) or adjuvant (n = 19) setting were subjected to central pathology review, HER2 assessment by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and next-generation sequencing. HER2 G-ITH, defined as HER2 amplification in 5% to 50% of tumor cells examined by FISH, was identified in 36% (19/53) of ECs and was associated with lower HER2 copy number and levels of protein expression. HER2 IHC revealed spatially distinct areas of strong expression juxtaposed with areas of low/absent expression in tumors with the “cluster” pattern of G-ITH, whereas the “mosaic” pattern was typically associated with a diffuse admixture of cells with variable levels of HER2 expression. HER2 G-ITH was frequently observed in cases with IHC/FISH or FISH/next-generation sequencing discrepancies and/or with an equivocal/negative FISH result (9/13, 69%). Although the objective response rate to anti-HER2 therapy in recurrent ECs was 52% (13/25) for tumors lacking HER2 G-ITH, none (0%, 0/10) of the patients with HER2 G-ITH achieved a complete or partial response (P = .005). HER2 G-ITH was significantly associated with worse progression-free survival (hazard ratio, 2.88; 95% CI, 1.33-6.27; P = .005) but not overall survival. HER2 IHC score, HER2/CEP17 ratio, HER2 copy number, histologic subtype, and other genetic alterations, including PIK3CA hotspot mutations, were not significantly associated with therapeutic response or survival outcomes. Treatment responses were not restricted to serous carcinomas, supporting consideration of anti-HER2 therapy in patients with HER2-positive high-grade ECs of non-serous histology. Our results demonstrate that HER2 G-ITH is an important determinant of response to trastuzumab and trastuzumab emtansine in EC, providing a rationale for the development of novel therapeutic strategies to target HER2-nonamplified resistant tumor subpopulations, such as HER2 antibody-drug conjugates with bystander effects.

Expression of NEAT1 can be used as a predictor for Dex resistance in multiple myeloma patients

ObjectiveMultiple myeloma is a heterogeneous disorder and the intratumor genetic heterogeneity contributes to emergency of drug resistance. Dexamethasone has been used clinically for decades for MM. Nevertheless, their use is severely hampered by the risk of developing side effects and the occurrence of Dex resistance. LncRNA NEAT1 plays a oncogenic role and participates in drug resistance in many solid tumors. Therefore, we investigated a potential usefulness of this molecular as a biomarker for diagnosis of MM and possible correlations of NEAT1 expression with drug resistance and prognosis.MethodsBone marrow and peripheral blood mononuclear cells samples were collected from 60 newly diagnosed MM patients. The expression of NEAT1expression level were detected by quantitative real-time PCR analyses. The relationship about the expression levels of lncRNA with other clinical and cytogenetic features was analyzed. In addition, we measured to analysis the correlation between the expression of NEAT1 and Dex resistance in MM patients.ResultsIt was found that the expression of NEAT1 is significantly higher in multiple myeloma patients compared to controls and does not change with other clinical features and cytogenetic features. We further discovered that overexpression of NEAT1 was associated with Dex resistance and a poor prognosis in MM patients.ConclusionLncRNA NEAT1 has a significant value that might act as a promoting factor in the development of MM and may be severed as a diagnostic factor in MM. NEAT1 invovled in Dex resistance, which provide a new interpretation during the chemotherapy for MM.

Mass Cytometry reveals unique phenotypic patterns associated with subclonal diversity and outcomes in multiple myeloma

Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.

Integrated analysis of single-cell chromatin state and transcriptome identified common vulnerability despite glioblastoma heterogeneity

In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed invitro and invivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.

Histological and Genetic Diversity in Ovarian Mucinous Carcinomas: A Pilot Study.

Tumor heterogeneity remains an ongoing challenge in the field of cancer therapy. Intratumor heterogeneity significantly complicates the diagnosis of cancer and presents challenging clinical problems due to resistance to drug therapy. This study aimed to elucidate the genetic changes histologically (mucinous cystadenoma (MCA), mucinous borderline tumor (MBT), and mucinous ovarian carcinoma (MOC)) in a portion of mucinous ovarian tumors within the same sample. Seven tumor samples obtained from different patients were used to evaluate the genetic mutations in each component. Intratumor genetic heterogeneity was observed in all patients; among them, BRAF (V600E) and p53 (T118I, P142S, T150I, and T170M) point mutations were observed in the MBT component, while KRAS (G12D and G13D) and PIK3CA (E545K) mutations were found in the MOC component. The current findings suggest that diverse genetic alterations occur in mucinous tumors, according to tumor histology. Tumor heterogeneity and genetic diversity in mucinous ovarian tumors might be the cause of treatment failure. Knowledge of intertumor heterogeneity may lead to an increased understanding of the tumor response to treatment.

Abstract 314: Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer

Abstract The high incidence of colon cancer (CC) worldwide is a major health concern. Although surgery and adjuvant chemotherapy are effective to a high extent in lymph-node negative colon cancers, there are still some 10-15% of patients that show disease relapse within the next 5-year period after intended curative surgery. We hypothesize that genomic instability and intratumor heterogeneity fuel the formation of subclonal populations in the primary tumor that might promote metastasis. In the current study, we assessed the presence of intratumor heterogeneity in CC using multiplex interphase FISH (miFISH) and whole-exome sequencing (WES) to evaluate samples obtained from primary stage II colon tumors and their patient-matched associated liver metastases using multi-region sampling (N=9). In combination with genome-wide copy-number changes obtained from WES analysis, miFISH allowed the simultaneous quantification of copy-numbers for nine CC relevant genes and a centromeric ploidy control probe in intact tumor nuclei derived from archival patient material. Our preliminary data on the four cases analyzed so far, revealed high similarities regarding copy-number changes and ploidy between primary tumor and metastatic samples for three of the cases. While two of these cases showed either triploid or highly aneuploid genomes with different degrees of genomic instability and subclonality among them, the third case displayed a diploid content for both matched lesions. Intriguingly, as seen by miFISH and WES, loss of CDX2 in the primary tumor might have originated the main clonal population giving rise to metastasis in this latter case. Conversely, the fourth case analyzed exhibited a triploid baseline for the primary tumor while the metastasis revealed a diploid population. Interestingly, despite higher absolute copy numbers in the primary tumor, there was an overlap of five of the gains and losses observed for the major clone populations of the tumor and metastasis by miFISH. However, two of the highly clonal gains observed in the tumor, namely the EGFR and MYC gain, were not present in the metastasis. Altogether, in addition to the ploidy change, results indicated major discrete copy-number changes from primary tumor to metastasis in this patient. In summary, we show here that the combined analysis of WES and miFISH is able to describe the subclonal composition of copy-number alterations in primary tumors and their patientmatched liver metastases. Future perspectives also include the assessment of subclonality affecting single-nucleotide variants to complete the genomic landscape of tumor evolution and clonal development in this patient cohort with the goal to better understand the nature of these early metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Sara Lahoz, Veronica Pablo-Fontecha, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 314.

PD46-11 ASSESSING THE ROLE OF NEOADJUVANT CHEMOTHERAPY IN UPPER TRACT UROTHELIAL CARCINOMA EVOLUTION

You have accessJournal of UrologyCME1 Apr 2023PD46-11 ASSESSING THE ROLE OF NEOADJUVANT CHEMOTHERAPY IN UPPER TRACT UROTHELIAL CARCINOMA EVOLUTION Neeta D'Souza, Andrew B. Katims, Ali Mouzannar, Eugene J. Pietzak, David Solit, Kwanghee Kim, and Jonathan A. Coleman Neeta D'SouzaNeeta D'Souza More articles by this author , Andrew B. KatimsAndrew B. Katims More articles by this author , Ali MouzannarAli Mouzannar More articles by this author , Eugene J. PietzakEugene J. Pietzak More articles by this author , David SolitDavid Solit More articles by this author , Kwanghee KimKwanghee Kim More articles by this author , and Jonathan A. ColemanJonathan A. Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003359.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Systemic neoadjuvant chemotherapy (NAC) may apply selective pressure on tumors and shape cancer evolution. In bladder cancer, intratumor genetic heterogeneity can help predict response to NAC, with RB1, ERCC2, ATM, and FANCC mutations significantly associated with a positive response to NAC. Molecular studies have shown upper tract urothelial carcinoma (UTUC) to be genetically distinct from bladder cancer with a significantly worse response to NAC, yet still harboring significant intratumor heterogeneity. This study aims to assess the effect of NAC in selecting chemotherapy-resistant mutations among the intratumor heterogeneity in UTUC. METHODS: We identified patients with UTUC who underwent platinum-based NAC followed by nephroureterectomy treated at Memorial Sloan Kettering Cancer Center. Patients with complete response to NAC were excluded. Pre-NAC biopsy and post-NAC nephroureterectomy samples underwent next generation targeted gene sequencing through MSK-IMPACT. Mutational frequency among the 505 tested genes was compared in the pre-and post- treatment cohorts to assess for significant changes (p<0.05). RESULTS: We identified 15 patients with UTUC that had an incomplete response/progression of disease after NAC with gemcitabine/cisplatin as determined by final pathology at radical nephroureterectomy. The median age at the time of NAC was 60 (IQR 55-70). The most common mutations are seen in Figure 1. The most common mutated genes in pre-NAC UTUC biopsy were TERT (53.3%), KMT2D (40.0%), and ARID1A (33.3%). Post-chemotherapy, the most common mutations were TERT (56.3%), FGFR3 (31.3%), and KMT2D (31.3%). There were no significant differences between the frequency of mutations pre- and post-NAC. There was no decrease in the frequency of RB1, ERCC2, ATM, and FANCC post-NAC. CONCLUSIONS: Administration of NAC did not select for specific resistant mutations in the global tumor landscape. It is possible that changes to the tumor microenvironment, sub-clonal expansion, or differences in expression may be identified through RNA based studies. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1174 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Neeta D'Souza More articles by this author Andrew B. Katims More articles by this author Ali Mouzannar More articles by this author Eugene J. Pietzak More articles by this author David Solit More articles by this author Kwanghee Kim More articles by this author Jonathan A. Coleman More articles by this author Expand All Advertisement PDF downloadLoading ...