An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Pan-cancer T cell atlas links a cellular stress response state to immunotherapy resistance.

Tumor-infiltrating T cells offer a promising avenue for cancer treatment, yet their states remain to be fully characterized. Here we present a single-cell atlas of T cells from 308,048 transcriptomes across 16 cancer types, uncovering previously undescribed T cell states and heterogeneous subpopulations of follicular helper, regulatory and proliferative T cells. We identified a unique stress response state, TSTR, characterized by heat shock gene expression. TSTR cells are detectable in situ in the tumor microenvironment across various cancer types, mostly within lymphocyte aggregates or potential tertiary lymphoid structures in tumor beds or surrounding tumor edges. T cell states/compositions correlated with genomic, pathological and clinical features in 375 patients from 23 cohorts, including 171 patients who received immune checkpoint blockade therapy. We also found significantly upregulated heat shock gene expression in intratumoral CD4/CD8+ cells following immune checkpoint blockade treatment, particularly in nonresponsive tumors, suggesting a potential role of TSTR cells in immunotherapy resistance. Our well-annotated T cell reference maps, web portal and automatic alignment/annotation tool could provide valuable resources for T cell therapy optimization and biomarker discovery.

Neoadjuvant chemotherapy plus nivolumab with or without ipilimumab in operable non-small cell lung cancer: the phase 2 platform NEOSTAR trial.

Neoadjuvant ipilimumab + nivolumab (Ipi+Nivo) and nivolumab + chemotherapy (Nivo+CT) induce greater pathologic response rates than CT alone in patients with operable non-small cell lung cancer (NSCLC). The impact of adding ipilimumab to neoadjuvant Nivo+CT is unknown. Here we report the results and correlates of two arms of the phase 2 platform NEOSTAR trial testing neoadjuvant Nivo+CT and Ipi+Nivo+CT with major pathologic response (MPR) as the primary endpoint. MPR rates were 32.1% (7/22, 80% confidence interval (CI) 18.7-43.1%) in the Nivo+CT arm and 50% (11/22, 80% CI 34.6-61.1%) in the Ipi+Nivo+CT arm; the primary endpoint was met in both arms. In patients without known tumor EGFR/ALK alterations, MPR rates were 41.2% (7/17) and 62.5% (10/16) in the Nivo+CT and Ipi+Nivo+CT groups, respectively. No new safety signals were observed in either arm. Single-cell sequencing and multi-platform immune profiling (exploratory endpoints) underscored immune cell populations and phenotypes, including effector memory CD8+ T, B and myeloid cells and markers of tertiary lymphoid structures, that were preferentially increased in the Ipi+Nivo+CT cohort. Baseline fecal microbiota in patients with MPR were enriched with beneficial taxa, such as Akkermansia, and displayed reduced abundance of pro-inflammatory and pathogenic microbes. Neoadjuvant Ipi+Nivo+CT enhances pathologic responses and warrants further study in operable NSCLC. (ClinicalTrials.gov registration: NCT03158129 .).

Pathogenesis of Tobacco-Associated Lung Adenocarcinoma Is Closely Coupled with Changes in the Gut and Lung Microbiomes.

Microbial dysbiosis has emerged as a modulator of oncogenesis and response to therapy, particularly in lung cancer. Here, we investigate the evolution of the gut and lung microbiomes following exposure to a tobacco carcinogen. We performed 16S rRNA-Seq of fecal and lung samples collected prior to and at several timepoints following (nicotine-specific nitrosamine ketone/NNK) exposure in Gprc5a-/- mice that were previously shown to exhibit accelerated lung adenocarcinoma (LUAD) development following NNK exposure. We found significant progressive changes in human-relevant gut and lung microbiome members (e.g., Odoribacter, Alistipes, Akkermansia, and Ruminococus) that are closely associated with the phenotypic development of LUAD and immunotherapeutic response in human lung cancer patients. These changes were associated with decreased short-chain fatty acids (propionic acid and butyric acid) following exposure to NNK. We next sought to study the impact of Lcn2 expression, a bacterial growth inhibitor, given our previous findings on its protective role in LUAD development. Indeed, we found that the loss of Lcn2 was associated with widespread gut and lung microbiome changes at all timepoints, distinct from those observed in our Gprc5a-/- mouse model, including a decrease in abundance and diversity. Our overall findings apprise novel cues implicating microbial phenotypes in the development of tobacco-associated LUAD.

The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma.

Tumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD. SIGNIFICANCE: While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy. This article is highlighted in the In This Issue feature, p. 2483.

Cell-by-Cell: Unlocking Lung Cancer Pathogenesis.

For lung cancers, cellular trajectories and fates are strongly pruned by cell intrinsic and extrinsic factors. Over the past couple of decades, the combination of comprehensive molecular and genomic approaches, as well as the use of relevant pre-clinical models, enhanced micro-dissection techniques, profiling of rare preneoplastic lesions and surrounding tissues, as well as multi-region tumor sequencing, have all provided in-depth insights into the early biology and evolution of lung cancers. The advent of single-cell sequencing technologies has revolutionized our ability to interrogate these same models, tissues, and cohorts at an unprecedented resolution. Single-cell tracking of lung cancer pathogenesis is now transforming our understanding of the roles and consequences of epithelial-microenvironmental cues and crosstalk during disease evolution. By focusing on non-small lung cancers, specifically lung adenocarcinoma subtype, this review aims to summarize our knowledge base of tumor cells-of-origin and tumor-immune dynamics that have been primarily fueled by single-cell analysis of lung adenocarcinoma specimens at various stages of disease pathogenesis and of relevant animal models. The review will provide an overview of how recent reports are rewriting the mechanistic details of lineage plasticity and intra-tumor heterogeneity at a magnified scale thanks to single-cell studies of early- to late-stage lung adenocarcinomas. Future advances in single-cell technologies, coupled with analysis of minute amounts of rare clinical tissues and novel animal models, are anticipated to help transform our understanding of how diverse micro-events elicit macro-scale consequences, and thus to significantly advance how basic genomic and molecular knowledge of lung cancer evolution can be translated into successful targets for early detection and prevention of this lethal disease.

Game of clones: Battles in the field of carcinogenesis.

Recent advances in bulk sequencing approaches as well as genomic decoding at the single-cell level have revealed surprisingly high somatic mutational burdens in normal tissues, as well as increased our understanding of the landscape of "field cancerization", that is, molecular and immune alterations in mutagen-exposed normal-appearing tissues that recapitulated those present in tumors. Charting the somatic mutational landscapes in normal tissues can have strong implications on our understanding of how tumors arise from mutagenized epithelium. Making sense of those mutations to understand the progression along the pathologic continuum of normal epithelia, preneoplasias, up to malignant tissues will help pave way for identification of ideal targets that can guide new strategies for preventing or eliminating cancers at their earliest stages of development. In this review, we will provide a brief history of field cancerization and its implications on understanding early stages of cancer pathogenesis and deviation from the pathologically "normal" state. The review will provide an overview of how mutations accumulating in normal tissues can lead to a patchwork of mutated cell clones that compete while maintaining an overall state of functional homeostasis. The review also explores the role of clonal competition in directing the fate of normal tissues and summarizes multiple mechanisms elicited in this phenomenon and which have been linked to cancer development. Finally, we highlight the importance of understanding mutations in normal tissues, as well as clonal competition dynamics (in both the epithelium and the microenvironment) and their significance in exploring new approaches to combatting cancer.

Chronic Exposure to Waterpipe Smoke Elicits Immunomodulatory and Carcinogenic Effects in the Lung.

Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using an experimental animal model that is prone to tobacco carcinogens and an exploratory observational analysis of human waterpipe smokers and nonsmokers. Mice exhibited elevated incidence of lung tumors following heavy WPS exposure (5 days/week for 20 weeks) compared to littermates with light WPS (once/week for 20 weeks) or control air. Lungs of mice exposed to heavy WPS showed augmented CD8+ and CD4+ T cell counts along with elevated protumor immune phenotypes including increased IL17A in T/B cells, PD-L1 on tumor and immune cells, and the proinflammatory cytokine IL1ß in myeloid cells. RNA-sequencing (RNA-seq) analysis showed reduced antitumor immune gene signatures in animals exposed to heavy WPS relative to control air. We also performed RNA-seq analysis of airway epithelia from bronchial brushings of cancer-free waterpipe smokers and nonsmokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe nonsmokers, harbored gene programs that were associated with poor clinical outcomes in patients with lung adenocarcinoma, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Our findings support the notion that WPS exhibits carcinogenic effects and constitutes a possible risk factor for lung cancer as well as warrant future studies that can guide evidence-based policies for mitigating waterpipe smoking. PREVENTION RELEVANCE: Potential carcinogenic effects of waterpipe smoking are very poorly understood despite its emergence as a socially acceptable form of smoking. Our work highlights carcinogenic effects of waterpipe smoking in the lung and, thus, accentuate the need for inclusion of individuals with exclusive waterpipe smoking in prevention and smoking cessation studies.

Resolving the Spatial and Cellular Architecture of Lung Adenocarcinoma by Multiregion Single-Cell Sequencing.

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand-receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. SIGNIFICANCE: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception.This article is highlighted in the In This Issue feature, p. 2355.

Early Diagnosis and Screening for Lung Cancer.

Cancer interception refers to actively blocking the cancer development process by preventing progression of premalignancy to invasive disease. The rate-limiting steps for effective lung cancer interception are the incomplete understanding of the earliest molecular events associated with lung carcinogenesis, the lack of preclinical models of pulmonary premalignancy, and the challenge of developing highly sensitive and specific methods for early detection. Recent advances in cancer interception are facilitated by developments in next-generation sequencing, computational methodologies, as well as the renewed emphasis in precision medicine and immuno-oncology. This review summarizes the current state of knowledge in the areas of molecular abnormalities in lung cancer continuum, preclinical human models of lung cancer pathogenesis, and the advances in early lung cancer diagnostics.

Single-Cell Expression Landscape of SARS-CoV-2 Receptor ACE2 and Host Proteases in Normal and Malignant Lung Tissues from Pulmonary Adenocarcinoma Patients.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

Whole Transcriptome Sequencing Analysis of Cancer Stem/Progenitor Cells Obtained from Mouse Lung Adenocarcinomas.

Interrogation and characterization of lung cancer stem cells (CSCs) that are implicated in lung oncogenesis is crucial for our understanding of inter- and intra-tumor heterogeneity and the aggressive nature of the disease, including tumor resistance and relapse in response to conventional therapy. Here, we describe an in vitro surrogate model, namely the "sphere-forming assay," for the derivation, enrichment, and propagation of lung stem/progenitor cells with CSC properties, including self-renewal, tumor initiation capacity and propagation, and differentiation into cells of the tumor bulk, from a murine Kras-mutant lung adenocarcinoma cell line. Self-renewing cancer stem/progenitor cells, in the form of 3D in vitro spheres, can be phenotypically interrogated using downstream techniques such as gene expression analysis (e.g., whole transcriptome sequencing and quantitative real time PCR), which is the focus of this chapter.

Augmented Lipocalin-2 Is Associated with Chronic Obstructive Pulmonary Disease and Counteracts Lung Adenocarcinoma Development.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator Lcn2 (lipocalin-2) was increased in normal airways from mice with knockout of the airway lineage gene Gprc5a (Gprc5a-/-) and that are prone to developing inflammation and LUAD. Yet, the role of LCN2 in lung inflammation and LUAD is poorly understood.Objectives: Delineate the role of Lcn2 induction in LUAD pathogenesis.Methods: Normal airway brushings, uninvolved lung tissues, and tumors from Gprc5a-/- mice before and after tobacco carcinogen exposure were analyzed by RNA sequencing. LCN2 mRNA was analyzed in public and in-house data sets of LUAD, lung squamous cancer (LUSC), chronic obstructive pulmonary disease (COPD), and LUAD/LUSC with COPD. LCN2 protein was immunohistochemically analyzed in a tissue microarray of 510 tumors. Temporal lung tumor development, gene expression programs, and host immune responses were compared between Gprc5a-/- and Gprc5a-/-/Lcn2-/- littermates.Measurements and Main Results:Lcn2 was progressively elevated during LUAD development and positively correlated with proinflammatory cytokines and inflammation gene sets. LCN2 was distinctively elevated in human LUADs, but not in LUSCs, relative to normal lungs and was associated with COPD among smokers and patients with LUAD. Relative to Gprc5a-/- mice, Gprc5a-/-/Lcn2-/- littermates exhibited significantly increased lung tumor development concomitant with reduced T-cell abundance (CD4+) and richness, attenuated antitumor immune gene programs, and increased immune cell expression of protumor inflammatory cytokines.Conclusions: Augmented LCN2 expression is a molecular feature of COPD-associated LUAD and counteracts LUAD development in vivo by maintaining antitumor immunity.

Field Carcinogenesis in Cancer Evolution: What the Cell Is Going On?

Field carcinogenesis describes the prevalence of tumor-related alterations in normal appearing tissues. Here, we summarize recent efforts in profiling molecular field dynamics for resolving early events in cancer evolution. We also highlight gaps in our knowledge of the molecular and cellular heterogeneity of field carcinogenesis and propose directions to tackle these voids using single-cell-based approaches and unique tissue sampling models. By interrogating both the mutagenized epithelium and its microenvironment, we surmise that single-cell-guided studies will help chart the spatiotemporal molecular and cellular "atlas" of field carcinogenesis, will further delineate preneoplastic initiation and progression, and will help identify cancer prevention and early intervention targets.

Anti-Tumor Effects of Biomimetic Sulfated Glycosaminoglycans on Lung Adenocarcinoma Cells in 2D and 3D In Vitro Models.

Lung cancer development relies on cell proliferation and migration, which in turn requires interaction with extracellular matrix (ECM) components such as glycosaminoglycans (GAGs). The mechanisms through which GAGs regulate cancer cell functions are not fully understood but they are, in part, mediated by controlled interactions with cytokines and growth factors (GFs). In order to mechanistically understand the effect of the degree of sulfation (DS) of GAGs on lung adenocarcinoma (LUAD) cells, we synthesized sulfated alginate (AlgSulf) as sulfated GAG mimics with DS = 0.0, 0.8, 2.0, and 2.7. Human (H1792) and mouse (MDA-F471) LUAD cell lines were treated with AlgSulf of various DSs at two concentrations 10 and 100 µg/mL and their anti-tumor properties were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue exclusion, and wound healing assays for 2D models and sphere formation assay for the 3D model. The proliferation and number of live MDA-F471 cells at the concentration of 100 µg/mL decreased significantly with the increase in the DS of biomimetic GAGs. In addition, the increase in the DS of biomimetic GAGs decreased cell migration (p < 0.001 for DS = 2.0 and 2.7 compared to control) and decreased the diameter and number of spheres formed (p < 0.001). The increased DS of biomimetic GAGs attenuated the expression of cancer stem cell (CSC)/progenitor markers in the 3D cultures. In conclusion, GAG-mimetic AlgSulf with increased DS exhibit enhanced anti-proliferative and migratory properties while also reducing growth of KRAS-mutant LUAD spheres in vitro. We suggest that these anti-tumor effects by GAG-mimetic AlgSulf are possibly due to differential binding to GFs and consequential decreased cell stemness. AlgSulf may be suitable for applications in cancer therapy after further in vivo validation.

Insights Into Lung Cancer Immune-Based Biology, Prevention, and Treatment.

Lung cancer is the number one cause of cancer-related deaths. The malignancy is characterized by dismal prognosis and poor clinical outcome mostly due to advanced-stage at diagnosis, thereby inflicting a heavy burden on public health worldwide. Recent breakthroughs in immunotherapy have greatly benefited a subset of lung cancer patients, and more importantly, they are undauntedly bringing forth a paradigm shift in the drugs approved for cancer treatment, by introducing "tumor-type agnostic therapies". Yet, and to fulfill immunotherapy's potential of personalized cancer treatment, demarcating the immune and genomic landscape of cancers at their earliest possible stages will be crucial to identify ideal targets for early treatment and to predict how a particular patient will fare with immunotherapy. Recent genomic surveys of premalignant lung cancer have shed light on early alterations in the evolution of lung cancer. More recently, the advent of immunogenomic technologies has provided prodigious opportunities to study the multidimensional landscape of lung tumors as well as their microenvironment at the molecular, genomic, and cellular resolution. In this review, we will summarize the current state of immune-based therapies for cancer, with a focus on lung malignancy, and highlight learning outcomes from clinical and preclinical studies investigating the naïve immune biology of lung cancer. The review also collates immunogenomic-based evidence from seminal reports which collectively warrant future investigations of premalignancy, the tumor-adjacent normal-appearing lung tissue, pulmonary inflammatory conditions such as chronic obstructive pulmonary disease, as well as systemic microbiome imbalance. Such future directions enable novel insights into the evolution of lung cancers and, thus, can provide a low-hanging fruit of targets for early immune-based treatment of this fatal malignancy.

Genome-Wide and Phenotypic Evaluation of Stem Cell Progenitors Derived From Gprc5a-Deficient Murine Lung Adenocarcinoma With Somatic Kras Mutations.

Lung adenocarcinomas (LUADs) with somatic mutations in the KRAS oncogene comprise the most common molecular subtype of lung cancer in smokers and present with overall dismal prognosis and resistance to most therapies. Our group recently demonstrated that tobacco carcinogen-exposed mice with knockout of the airway lineage G-protein coupled receptor, Gprc5a, develop LUADs with somatic mutations in Kras. Earlier work has suggested that cancer stem cells (CSCs) play crucial roles in clonal evolution of tumors and in therapy resistance. To date, our understanding of CSCs in LUADs with somatic Kras mutations remains lagging. Here we derived CSCs (as spheres in 3D cultures) with self-renewal properties from a murine Kras-mutant LUAD cell line we previously established from a tobacco carcinogen-exposed Gprc5a -/- mouse. Using syngeneic Gprc5a -/- models, we found that these CSCs, compared to their parental isoforms, exhibited increased tumorigenic potential in vivo, particularly in female animals. Using whole-transcriptome sequencing coupled with pathways analysis and confirmatory PCR, we identified gene features (n = 2,600) differentially expressed in the CSCs compared to parental cells and that were enriched with functional modules associated with an augmented malignant phenotype including stemness, tumor-promoting inflammation and anti-oxidant responses. Further, based on in silico predicted activation of GSK3ß in CSCs, we found that tideglusib, an irreversible inhibitor of the kinase, exhibited marked anti-growth effects in the cultured CSCs. Our study underscores molecular cues in the pathogenesis of Kras-mutant LUAD and presents new models to study the evolution, and thus high-potential targets, of this aggressive malignancy.