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Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li–Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies. Significance: New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li–Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are “basal” p53 activities. See related commentary by Stieg et al., p. 1046. See related article by Choe et al., p. 1250. This article is highlighted in the In This Issue feature, p. 1027

Chromosomal microarray (CMA) is a testing modality frequently used in pediatric patients; however, published data on its utilization are limited to the genetic setting. We performed a database search for all CMA testing performed from 2010 to 2020, and delineated the diagnostic yield based on patient characteristics, including sex, age, clinical specialty of providers, indication of testing, and pathogenic finding. The indications for testing were further categorized into Human Phenotype Ontology categories for analysis. This study included a cohort of 14,541 patients from 29 different medical specialties, of whom 30% were from the genetics clinic. The clinical indications for testing suggested that neonatology patients demonstrated the greatest involvement of multiorgan systems, involving the most Human Phenotype Ontology categories, compared with developmental behavioral pediatrics and neurology patients being the least. The top pathogenic findings for each specialty differed, likely due to the varying clinical features and indications for testing. Deletions involving the 22q11.21 locus were the top pathogenic findings for patients presenting to genetics, neonatology, cardiology, and surgery. Our data represent the largest pediatric cohort published to date. This study is the first to demonstrate the diagnostic utility of this assay for patients seen in the setting of different specialties, and it provides normative data of CMA results among a general pediatric population referred for testing because of variable clinical presentations.

Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li–Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.Significance:New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li–Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are “basal” p53 activities.See related commentary by Stieg et al., p. 1046.See related article by Choe et al., p. 1250.This article is highlighted in the In This Issue feature, p. 1027

Table S1 shows predicted off target sites for p53TD mutant mice. Table S2 shows upregulated PPAR target genes in p53AD vs p53-null. Table S3 shows ATAC-seq and RNA-seq congruent genes in p53AD vs p53 null. Table S4 shows p53half site analysis in PPAR target genes Table S5 shows control sequences used for p53 half site analysis Table S6 shows altered genes in p53AD-derived thymic lymphomas Table S7 shows primers used for genotyping of p53 mutant mice Table S8 shows primers used for qRT-PCR Table S9 shows antibodies used for flow cytometry Figure S1 shows extended data for generation of knock-in mouse models and features of p53TD mutant proteins and mice. Figure S2 shows homozygous mutant mice survival curves as well as tumor data. Figure S3 shows exome sequencing results for thymic lymphomas and hemangiosarcomas derived from p53 monomer and dimer mutant mice. Figure S4 shows characterization of stress responses of p53TD mutants. Figure S5 shows RNA-sequencing results for p53AD and p53RP, and Mdm2 genetic deletion rescue experiments. Figure S6 shows RNA-seq results of basal p53AD vs p53-null thymi, upstream regulator analysis, and ATAC-seq results for specific loci. Figure S7 shows extended data for p53 dimer mutant novel activities (co-IP, motif analysis, other dimer mutants activities, and tumor data) Figure S8 shows ferroptosis and scRNA-seq extended data Figure S9 shows diagram summarizing findings within this study.

Pancreatic ductal adenocarcinoma (PDAC) has few effective treatments. Immunotherapy, an attractive alternative strategy, remains challenging with the lack of knowledge on the tumor-infiltrating lymphocyte (TIL) landscape in PDAC. To generate a reference of T-cell subpopulations, we profiled 80,000 T cells from 57 PDAC samples, 22 uninvolved/normal samples, and cultured TIL using single-cell transcriptomic and T-cell receptor analysis. These data revealed 20 cell states and heterogeneous distributions of TIL populations. The CD8+ TIL contained a putative transitional GZMK+ population based on T-cell receptor clonotype sharing, and cell-state trajectory analysis showed similarity to a GZMB+PRF1+ cytotoxic and a CXCL13+ dysfunctional population. Statistical analysis suggested that certain TIL states, such as dysfunctional and inhibitory populations, often occurred together. Finally, analysis of cultured TIL revealed that high-frequency clones from effector populations were preferentially expanded. These data provide a framework for understanding the PDAC TIL landscape for future TIL use in immunotherapy for PDAC.Significance:To improve the efficacy of immunotherapy in PDAC, there is a great need to understand the PDAC TIL landscape. This study represents a reference of PDAC TIL subpopulations and their relationships and provides a foundation upon which to base future immunotherapeutic efforts.This article is highlighted in the In This Issue feature, p. 2221

Supplementary Table from Single-Cell Sequencing Reveals Trajectory of Tumor-Infiltrating Lymphocyte States in Pancreatic Cancer

Supplementary Figure from Single-Cell Sequencing Reveals Trajectory of Tumor-Infiltrating Lymphocyte States in Pancreatic Cancer

Figures S1, S2, S3 and S4. APC and KRAS genotyping of crypts and bulk biopsies in adenomas from MAP, UPF, LS and cMMRD cases. Figures S5, S6 and S7. APC and KRAS genotyping of crypts and bulk biopsies in adenomas from SPO cases. Figure S8, S9 and S10. Hyerarchical clustering analysis of crypts based on KRAS genotyping results. Figure S11. Comparative analysis of mutant allele discrimination by (A) Light Cycler 480 (Roche) and (B) nanofluidic PCR using Dynamic array (Fluidigm). Figure S12. A. Comparative analysis of KRAS genotyping using whole genome amplified (WGA) DNA or pre-amplified DNA using 15 cycles of a nested PCR. Figure S13. Genotyping results including water controls of random KRAS hotspot mutation analysis after performing a pre-amplification step using a nested PCR apporach. Figure S14. Independent validation of KRAS hotspot mutation that have been detected by Nanofluidic Dynamic array using the Nanofluidic Digital PCR genotyping as an alternative approach. Figure S15. Independent validation of KRAS hotspot mutation that have been detected by Nanofluidic Dynamic array using the Nanofluidic Digital PCR genotyping as an alternative approach.

Purpose: The majority of genomic alterations causing intratumoral heterogeneity (ITH) in colorectal cancer are thought to arise during early stages of carcinogenesis as a burst but only after truncal mutations in APC have expanded a single founder clone. We have investigated if the initial source of ITH is consequent to multiple independent lineages derived from different crypts harboring distinct truncal APC and driver KRAS mutations, thus challenging the prevailing monoclonal monocryptal model.Experimental Design: High-depth next-generation sequencing and SNP arrays were performed in whole-lesion extracts of 37 familial adenomatous polyposis colorectal adenomas. Also, ultrasensitive genotyping of hotspot mutations of APC and KRAS was performed using nanofluidic PCRs in matched bulk biopsies (n = 59) and crypts (n = 591) from 18 adenomas and seven carcinomas and adjacent normal tissues.Results: Multiple co-occurring truncal APC and driver KRAS alterations were uncovered in whole-lesion extracts from adenomas and subsequently confirmed to belong to multiple clones. Ultrasensitive genotyping of bulk biopsies and crypts revealed novel undetected APC mutations that were prominent among carcinomas, whereas abundant wild-type APC crypts were detected in adenomas. KRAS mutational heterogeneity within crypts was evident in both adenomas and carcinomas with a higher degree of concordance between biopsy and crypt genotyping in carcinomas. Nonrandom heterogeneity among crypts was also observed.Conclusions: The striking degree of nonrandom intercrypt heterogeneity in truncal and driver gene mutations observed in adenomas and carcinomas is consistent with a polycryptal model derived from multiple independent initiation linages as the source of early ITH in colorectal carcinogenesis. Clin Cancer Res; 23(19); 5936–47. ©2017 AACR.

Figures and Tables

Purpose:Aggressive variant prostate cancer (AVPC) represents a clinical subset distinguished by therapy resistance and poor prognosis, linked to combined losses of the tumor suppressor genes (TSG) PTEN, RB1, and TP53. Circulating tumor cells (CTC) provide a minimally invasive opportunity for identification and molecular characterization of AVPC. We aimed to evaluate the incidence and clinical significance of compound (2+)TSG losses and genomic instability in prostate cancer CTC, and to expand the set genomic biomarkers relevant to AVPC.Experimental Design:Genomic analysis of chromosomal copy-number alterations (CNA) at single-cell resolution was performed in CTC from patients with and without AVPC before initiating chemotherapy with cabazitaxel or cabazitaxel and carboplatin. We evaluated associations between single-CTC genomics and clinical features, progression-free survival, and overall survival.Results:A total of 257 individual CTC were sequenced from 47 patients (1–22 CTC/patient). Twenty patients (42.6%) had concurrent 2+TSG losses in at least one CTC in association with poor survival and increased genomic instability, inferred by high large-scale transitions scores. Higher LST in CTC were independent of CTC enumerated, clinically more indicative of aggressive behavior than co-occurring TSG losses, and molecularly associated with gains in chromosomal regions including PTK2, Myc, and NCOA2; increased androgen receptor expression; and BRCA2 loss. In 57 patients with matched cell-free tumor DNA data, CTC were more frequently detectable and evaluable for CNA analysis (in 73.7% vs. 42.1%, respectively).Conclusions:Our findings suggest that genomic instability in CTC is a hallmark of advanced prostate cancer aggressiveness, and support single-CTC sequencing as a compelling tool to noninvasively characterize cancer heterogeneity.

Table S1. Summary of adenomas with multiple APC and KRAS somatic hits identified by next-generation sequencing. Table S2. Clinical and pathologic characteristics of patients analyzed in this manuscript. Table S3. Analysis of APC, KRAS and 5q loss using AmpliSeq and SNP array TableS4. Number of normal mucosa, adenomas, tumors and crypts analyzed per case. NM; normal mucosa. Table S5. Analytical sensibility of Light Cycler® 480 (Roche) and dynamic array (Fluidigm) to detect KRAS mutations. Table S6. In silico of the APC somatic hits observed by next-generaiton sequencing that were labelled as VUS. B, benign; N, neutral; D, damaging; P, Probably damaging. Table S7. APC analysis of adenomas and carcinomas in bulk biopsies and isolated crypts. The analysis of adjancent normal mucosa was not performed. Table S8. KRAS genotyping results of adenomas and carcinomas by digital and dynamic array (Fluidigm) in bulk biopsies and isolated crypts Table S9. Primers and probes used in this manuscript.

Supplementary Materials and Methods (Includes reference to Table S9 and Figures S11-15)

Pancreatic ductal adenocarcinoma (PDAC) is considered ‘non-immunogenic’ with trials demonstrating its recalcitrance to PD1 and CTLA4 immune checkpoint therapies (ICTs). Here, we sought to systematically characterize the mechanisms underlying de novo ICT resistance and identify effective therapeutic options for PDAC. We report that agonist 41BB and antagonist LAG3 ICT alone and in combination, increased survival and anti-tumor immunity, characterized by modulating T cell subsets with anti-tumor activity, increased T cell clonality and diversification, decreased immunosuppressive myeloid cells and increased antigen presentation/decreased immunosuppressive capability of myeloid cells. Translational analyses confirmed the presence of 41BB and LAG3 in human PDAC. Since single and dual ICTs were not curative, T cell-activating ICTs were combined with a CXCR1/2 inhibitor, targeting immunosuppressive myeloid cells. Triple therapy resulted in durable complete responses. Given similar profiles in human PDAC and availability of these agents for clinical testing, our findings provide a testable hypothesis for this lethal disease.

The brain tumor immune microenvironment (TIME) continuously evolves during glioma progression, but only a limited view of a highly complex glioma associated immune contexture across isocitrate dehydrogenase mutation (IDH) classified gliomas is known. Herein, we present an unprecedentedly comprehensive view of myeloid and lymphoid cell type diversity based on our single cell RNA sequencing and spectral cytometry-based interrogation of tumor-associated leukocytes from fifty-five IDH stratified primary and recurrent human gliomas and three non-glioma brains. Our analyses revealed twenty-two myeloid and lymphoid cell types within and across glioma subtypes. Glioma severity correlated with microglial attrition concomitant with a continuum of invading monocyte-derived microglia-like and macrophages amongst other infiltrating conventional T and NK lymphocytes and unconventional mucosa associated invariant T (MAIT) cells. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells (DCs). Furthermore, we identified phagocytosis and antigen presentation gene modules enriched in Triggering receptor expressed on myeloid (TREM)-2+cells as a putative anti-glioma axis. Accelerated glioma growth was observed inTrem2deficient mice implanted with CT2A glioma cells affirming the anti-glioma role of TREM2+myeloid cells. In addition to providing a comprehensive landscape of glioma-specific immune contexture, our investigations discover TREM2 as a novel immunotherapy target for brain malignancies.

Pancreatic ductal adenocarcinoma (PDAC) has few effective treatments. Immunotherapy, an attractive alternative strategy, remains challenging with the lack of knowledge on the tumor-infiltrating lymphocyte (TIL) landscape in PDAC. To generate a reference of T-cell subpopulations, we profiled 80,000 T cells from 57 PDAC samples, 22 uninvolved/normal samples, and cultured TIL using single-cell transcriptomic and T-cell receptor analysis. These data revealed 20 cell states and heterogeneous distributions of TIL populations. The CD8+ TIL contained a putative transitional GZMK+ population based on T-cell receptor clonotype sharing, and cell-state trajectory analysis showed similarity to a GZMB+PRF1+ cytotoxic and a CXCL13+ dysfunctional population. Statistical analysis suggested that certain TIL states, such as dysfunctional and inhibitory populations, often occurred together. Finally, analysis of cultured TIL revealed that high-frequency clones from effector populations were preferentially expanded. These data provide a framework for understanding the PDAC TIL landscape for future TIL use in immunotherapy for PDAC. Significance: To improve the efficacy of immunotherapy in PDAC, there is a great need to understand the PDAC TIL landscape. This study represents a reference of PDAC TIL subpopulations and their relationships and provides a foundation upon which to base future immunotherapeutic efforts. This article is highlighted in the In This Issue feature, p. 2221

Ductal carcinoma in situ (DCIS) is a non-invasive breast neoplasia that accounts for 25% of all screen-detected breast cancers diagnosed annually. Neoplastic cells in DCIS are confined to the ductal system of the breast, although they can escape and progress to invasive breast cancer in a subset of patients. A key concern of DCIS is overtreatment, as most patients screened for DCIS and in whom DCIS is diagnosed will not go on to exhibit symptoms or die of breast cancer, even if left untreated. However, differentiating low-risk, indolent DCIS from potentially progressive DCIS remains challenging. In this Review, we summarize our current knowledge of DCIS and explore open questions about the basic biology of DCIS, including those regarding how genomic events in neoplastic cells and the surrounding microenvironment contribute to the progression of DCIS to invasive breast cancer. Further, we discuss what information will be needed to prevent overtreatment of indolent DCIS lesions without compromising adequate treatment for high-risk patients.

Gliomas are recalcitrant brain tumors. Differential tumor immune reactivity contributes to survival advantage of isocitrate dehydrogenase-mutant (IDHmut) over wild-type (IDHwt) gliomas. Despite this correlative pattern of immunity and survival, only a limited view of a highly complex immune contexture across IDH mutation classified gliomas is known. Herein, we present an unprecedented view of myeloid and lymphoid cell type diversity by single cell RNA sequencing and spectral cytometry-based interrogation of tumor-associated leukocytes from fifty-five IDH stratified primary and recurrent human gliomas and six non-glioma brains. Our analyses revealed twenty-two myeloid and lymphoid cell types within and across glioma subtypes. Glioma severity in relapsed IDHwt correlated with microglial attrition concomitant with a continuum of invading monocyte-derived microglia-like and macrophages amongst other infiltrating conventional T and NK lymphocytes and unconventional mucosa associated invariant T (MAIT) cells. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells (DCs). As tissue macrophages exhibit multifaceted polarization in response to microenvironmental cues, we clarify the existence of microglia/macrophage functional states beyond M1/M2 paradigms exemplified by the presence of palmitic-, oleic- acid, and glucocorticoid responsive polarized states. Immune related gene ontology analysis identified enriched antigen presentation and phagocytosis gene modules in distinct microglia-like clusters. Importantly, the phagocytic immunomodulator; Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) was upregulated in these microglia-like cells. Contrary to tumor promoting role of TREM2 myeloid cells in non-brain cancers, we identify TREM2 mediated anti-glioma axis as a regulator of antigen presentation Accelerated glioma growth was observed in Trem2 deficient mice implanted with CT2A glioma cells affirming the anti-glioma role of TREM2+ myeloid cells. In addition to providing an advanced landscape of glioma-specific immune contexture for immunotherapy applications, our reverse translational investigations discover TREM2 as a novel immunotherapy target for brain malignancies. Citation Format: Pravesh Gupta, Minghao Dang, Shivangi Oberai, Mekenzie Peshoff, Nancy Milam, Aml Ahmed, Krishna Bojja, Tuan M. Tran, Kathryn Cox, Huma Shehwana, Carlos Kamiya Matsuoka, Jianzhuo Li, Joy Gumin, Alicia Goldman, Sameer A. Seth, Atul Maheshwari, Frederick F. Lang, Nicholas E. Navin, Amy B. Heimberger, Karen Clise Dwyer, Linghua Wang, Krishna P. Bhat. Immunophenotyping of human brain tumors reveals myeloid cell mediated anti-glioma axis [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 669.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

Single-cell RNA sequencing methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics assays can profile spatial regions in tissue sections, but do not have single-cell resolution. Here, we developed a computational method called CellTrek that combines these two datasets to achieve single-cell spatial mapping through coembedding and metric learning approaches. We benchmarked CellTrek using simulation and in situ hybridization datasets, which demonstrated its accuracy and robustness. We then applied CellTrek to existing mouse brain and kidney datasets and showed that CellTrek can detect topological patterns of different cell types and cell states. We performed single-cell RNA sequencing and spatial transcriptomics experiments on two ductal carcinoma in situ tissues and applied CellTrek to identify tumor subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas. Our data show that CellTrek can accurately map single cells in diverse tissue types to resolve their spatial organization.

Prostate cancer is the most commonly diagnosed cancer among men and a major cause of cancer-related deaths in the modern world. Metastatic castration-resistant prostate cancer (mCRPC) is the most lethal form and has complex and heterogeneous genomic patterns. Despite the recent development of life-prolonging therapies, mCRPC remains incurable since most patients develop resistance over time. A better understanding of the evolution of resistance to therapy in mCRPC is urgently needed. A major logistical challenge is that collecting serial tissue biopsies from the prostate and metastatic sites such as bone is not feasible. To overcome this issue, we developed a minimally invasive liquid biopsy approach to study the genomic basis of resistance through circulating tumor DNA (ctDNA) from longitudinally collected plasma samples. To best account for interpatient tumor heterogeneity and treatment history, we selected 60 mCRPC patients who were serially treated with life-prolonging second generation androgen signaling inhibitors (abiraterone, enzalutamide, or the combination) and taxane-based chemotherapy, and prospectively collected longitudinal blood samples (2 to 10 per patient). ctDNA was isolated from each blood plasma sample for sparse whole-genome sequencing and exome sequencing at 200x coverage depth. Somatic copy number alterations (CNA) and exome mutations were profiled and the resulting data was used to reconstruct the clonal substructure and infer clonal evolutionary dynamics across the treatment timepoints. The mutation signatures and the CNA signatures were also characterized. One of the CNA signatures identified the patients who carry CDK12 mutations through genome-wide focal tandem duplications in ctDNA. The subclones resistant to second-generation androgen signaling inhibitors and taxane-based chemotherapy were also identified, along with their associated genomic aberrations affecting AR, RB1, and PTEN. Furthermore, by analyzing the evolutionary dynamics patterns, we identified three distinct genomic responses to therapy: intrinsic resistance, clonal progression, and clonal responders, which correlated with overall survival outcomes. In summary, our data show that clonal evolutionary dynamics in mCRPC can be tracked over time and in response to treatment with minimally invasive liquid biopsy methods. Our results provide insights into the genomic evolution of life-prolonging therapy resistance in mCRPC and identified candidate resistance biomarkers for subsequent validation. Citation Format: Yuehui Zhao, Naveen Ramesh, Emi Sei, Min Hu, Shanshan Bai, Patricia Troncoso, Paul Corn, Ana Aparicio, Christopher J. Logothetis, Nicholas E. Navin, Amado J. Zurita. Investigating longitudinal therapeutic resistance in serially treated metastatic castration resistant prostate cancer by liquid biopsy genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3791.

Introduction: Patients with luminal A and B early-stage ER+/HER2- breast cancer (BrCa) are uniformly treated with adjuvant endocrine therapy (ET) (±chemotherapy). A better understanding of drivers of ET resistance is required as subsequent lethal metastatic disease remains a major clinical problem. Therefore, there is an unmet clinical need to identify biomarkers that select low- and high-risk patients who may benefit from de-escalation of current treatments or alternative therapeutic interventions. In the present study, we hypothesized that while luminal A and B tumors both arise from normal hormone-responsive cells, they are transcriptionally distinct, hence allowing the identification of unique gene signatures that can predict outcomes in each subtype. Methods: Tumors from 10 early-stage ER+/HER2- BrCa patients were subjected to single-cell RNA-sequencing (scRNA-seq) analysis (10X Genomics); 6/10 tumors were classified as luminal A and 4/10 as luminal B based on combined PAM50 and immunohistochemical classification (Ki67 cut-off=20%). We performed a direct transcriptional comparison between luminal A and B tumors, using well-established signatures and unbiased differential gene expression analysis. To identify unique luminal A and B tumor-specific genes, we compared the gene expression profile of each luminal subtype with 10 non-neoplastic breast tissues. A predictive model (LASSO) was applied to select genes with the highest frequency using a training dataset. This resulted in 5- and 4-gene signatures for luminal A and B, respectively, which were used to calculate risk scores that divided each subtype into low- and high-risk groups. The prognostic value of the above signatures was validated in an independent dataset. Results: The integrated scRNA-seq analysis of luminal A and B tumors revealed transcriptionally distinct tumor cell clusters while tumor microenvironment (TME) clusters were well intermixed. Luminal B tumors had higher cell cycle and BrCa-specific scores, low ER pathway-gene expression scores, and increased 8q amplifications. IFNγ, OXPHOS, p53, hypoxia and MYC targets were the most upregulated pathways in the luminal B subtype. The TME of luminal B tumors was comprised of lower CD4+ and CD8+ T cell but higher Treg levels. Comparison with normal breast tissues revealed that early-stage ER+ BrCa arises from hormone-responsive epithelial cells and provided a number of tumor-specific genes that were used to generate prognostic signatures. These signatures were capable of differentiating high- from low-risk patients within each subtype and predicting survival outcomes in two large-scale training and validation cohorts. Conclusions: We developed a novel prognostic tool that can be used to determine duration of adjuvant ET and/or new therapeutic strategies for high-risk luminal A and B patients in the early-stage ER+/HER2- setting. Citation Format: Sofia Mastoraki, Jerome Lin, Xiayu Rao, Sophie R. Liu, Harsh Batra, Maria G. Raso, Edwin R. Parra Cuentas, Akshara S. Raghavendra, Komal S. Rasaputra, Min Yi, Jing Wang, Aysegul Sahin, Debasish Tripathy, Kelly K. Hunt, Nicholas E. Navin, Khandan Keyomarsi. Single-cell transcriptomic analysis of HR+/HER2- breast cancer identifies gene signatures that predict outcomes of luminal A and B subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 515.

This protocol was developed for the human breast cell atlas (HBCA) project to obtain high-viability cell suspensions from freshly dissociated breast tissues from human patients. There are two options for performing this protocol: rapid-dissociation (15-30 min) or exhaustive dissociation (overnight). In our experience the rapid dissociation protocol will provide higher cell viability and better representation of breast cell types, however the total number of cells dissociated may be lower after enumeration. In contrast the exhaustive protocol provides higher cell numbers and maintains good cell viability but may result in skewing of cell type representations over the long time period. The rapid-dissociation protocol was used to generate most of the cell suspensions for the MD Anderson human breast cell atlas project and has been optimized and tested more extensively.

Castration-resistant prostate cancer (CRPC) is a lethal disease. A subset of these patients present with atypical clinical characteristics and aggressive disease behavior. These patients, classified as Aggressive Variant Prostate Cancer (AVPC), may derive benefit from platinum-based chemotherapy. However, the identification of AVPC patients is often challenging due to heterogeneity in clinical presentations and potentially in biological drivers, which may not be fully reflected in a biopsy obtained from a single tumor site. To address these challenges, we developed an unbiased genome sequencing method called PEGASUS (Plasma Exome and Genome Analysis by Size-Selection and Unbiased Sequencing) that enables the simultaneous detection of copy number aberrations and exome mutations from circulating-tumor DNA (ctDNA). We applied this method to plasma specimens obtained from 160 CRPC patients with and without AVPC participating in a randomized trial of cabazitaxel alone or in combination with carboplatin. We were able to successfully isolate ctDNA (>2 ng) for genomic profiling using PEGASUS in 91/160 (57%) CRPC patients. Among these 91 CRPC patients, we detected common prostate cancer mutations as well as genomic copy number changes in genes such as RB1, PTEN and TP53 and several other genes associated with DNA repair. One of the most salient features that distinguished the patients’ outcomes was whether they had diploid or aneuploid genomes detected by whole genome profiling of the ctDNA. Patients with aneuploid ctDNA had worse progression-free and overall survival. Overall, our data shows the feasibility of performing whole-genome and exome profiling of ctDNA in CRPC patients to characterize the molecular features associated with distinct clinical presentations. Candidate markers associated to benefit from platinum-based chemotherapy are being evaluated. These results pave the way for future clinical applications in biomarker discovery to assist treatment decisions in prostate cancer patients. Citation Format: Naveen Ramesh, Emi Sei, Pei Ching Tsai, Christopher Logothetis, Paul Corn, Ana Aparicio, Amado J. Zurita, Nicholas E. Navin. Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 446.