RESUMEN
Group 2 innate lymphoid cells (ILC2s) regulate inflammation and immunity in mammalian tissues1,2. Although ILC2s are found in cancers of these tissues3, their roles in cancer immunity and immunotherapy are unclear. Here we show that ILC2s infiltrate pancreatic ductal adenocarcinomas (PDACs) to activate tissue-specific tumour immunity. Interleukin-33 (IL33) activates tumour ILC2s (TILC2s) and CD8+ T cells in orthotopic pancreatic tumours but not heterotopic skin tumours in mice to restrict pancreas-specific tumour growth. Resting and activated TILC2s express the inhibitory checkpoint receptor PD-1. Antibody-mediated PD-1 blockade relieves ILC2 cell-intrinsic PD-1 inhibition to expand TILC2s, augment anti-tumour immunity, and enhance tumour control, identifying activated TILC2s as targets of anti-PD-1 immunotherapy. Finally, both PD-1+ TILC2s and PD-1+ T cells are present in most human PDACs. Our results identify ILC2s as anti-cancer immune cells for PDAC immunotherapy. More broadly, ILC2s emerge as tissue-specific enhancers of cancer immunity that amplify the efficacy of anti-PD-1 immunotherapy. As ILC2s and T cells co-exist in human cancers and share stimulatory and inhibitory pathways, immunotherapeutic strategies to collectively target anti-cancer ILC2s and T cells may be broadly applicable.
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Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/inmunología , Linfocitos/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Células Dendríticas/inmunología , Femenino , Humanos , Inmunidad Innata/inmunología , Inmunoterapia , Interleucina-33/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunologíaRESUMEN
Background The impact of transarterial radioembolization (TARE) of breast cancer liver metastasis (BCLM) on antitumor immunity is unknown, which hinders the optimal selection of candidates for TARE. Purpose To determine whether response to TARE at PET/CT in participants with BCLM is associated with specific immune markers (cytokines and immune cell populations). Materials and Methods This prospective pilot study enrolled 23 women with BCLM who planned to undergo TARE (June 2018 to February 2020). Peripheral blood and liver tumor biopsies were collected at baseline and 1-2 months after TARE. Monocyte, myeloid-derived suppressor cell (MDSC), interleukin (IL), and tumor-infiltrating lymphocyte (TIL) levels were assessed with use of gene expression studies and flow cytometry, and immune checkpoint and cell surface marker levels with immunohistochemistry. Modified PET Response Criteria in Solid Tumors was used to determine complete response (CR) in treated tissue. After log-transformation, immune marker levels before and after TARE were compared using paired t tests. Association with CR was assessed with Wilcoxon rank-sum or unpaired t tests. Results Twenty women were included. After TARE, peripheral IL-6 (geometric mean, 1.0 vs 1.6 pg/mL; P = .02), IL-10 (0.2 vs 0.4 pg/mL; P = .001), and IL-15 (1.9 vs 2.4 pg/mL; P = .01) increased. In biopsy tissue, lymphocyte activation gene 3-positive CD4+ TILs (15% vs 31%; P < .001) increased. Eight of 20 participants (40% [exact 95% CI: 19, 64]) achieved CR. Participants with CR had lower baseline peripheral monocytes (10% vs 29%; P < .001) and MDSCs (1% vs 5%; P < .001) and higher programmed cell death protein (PD) 1-positive CD4+ TILs (59% vs 26%; P = .006) at flow cytometry and higher PD-1+ staining in tumor (2% vs 1%; P = .046). Conclusion Complete response to transarterial radioembolization was associated with lower baseline cytokine, monocyte, and myeloid-derived suppressor cell levels and higher programmed cell death protein 1-positive tumor-infiltrating lymphocyte levels. © RSNA, 2022 Online supplemental material is available for this article.
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Neoplasias de la Mama , Carcinoma Hepatocelular , Embolización Terapéutica , Neoplasias Hepáticas , Humanos , Femenino , Tomografía Computarizada por Tomografía de Emisión de Positrones , Neoplasias de la Mama/terapia , Proyectos Piloto , Neoplasias Hepáticas/patología , Embolización Terapéutica/métodos , Biomarcadores , Carcinoma Hepatocelular/patología , Estudios Retrospectivos , Melanoma Cutáneo MalignoRESUMEN
Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.
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Antígenos de Neoplasias/inmunología , Proteínas Bacterianas/inmunología , Supervivientes de Cáncer , Reacciones Cruzadas/inmunología , Neoplasias Pancreáticas/inmunología , Linfocitos T Citotóxicos/inmunología , Adenocarcinoma/sangre , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Antígenos de Neoplasias/genética , Proteínas Bacterianas/sangre , Proteínas Bacterianas/genética , Antígeno Ca-125/genética , Antígeno Ca-125/inmunología , Simulación por Computador , Reacciones Cruzadas/genética , Humanos , Inmunoterapia , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/genética , Pronóstico , Análisis de Supervivencia , Linfocitos T Citotóxicos/citología , Secuenciación del ExomaRESUMEN
Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of â¼ 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log2 fold change ≥ 1, false discovery rate ≤ 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.
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Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Transcriptoma , Regulación Neoplásica de la Expresión Génica , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Intraductal tubulopapillary neoplasm is a relatively recently described member of the pancreatic intraductal neoplasm family. The more common member of this family, intraductal papillary mucinous neoplasm, often carries genetic alterations typical of pancreatic infiltrating ductal adenocarcinoma (KRAS, TP53, and CDKN2A) but additionally has mutations in GNAS and RNF43 genes. However, the genetic characteristics of intraductal tubulopapillary neoplasm have not been well characterized. Twenty-two intraductal tubulopapillary neoplasms were analyzed by either targeted next-generation sequencing, which enabled the identification of sequence mutations, copy number alterations, and selected structural rearrangements involving all targeted (≥300) genes, or whole-exome sequencing. Three of these intraductal tubulopapillary neoplasms were also subjected to whole-genome sequencing. All intraductal tubulopapillary neoplasms revealed the characteristic histologic (cellular intraductal nodules of back-to-back tubular glands lined by predominantly cuboidal cells with atypical nuclei and no obvious intracellular mucin) and immunohistochemical (immunolabeled with MUC1 and MUC6 but were negative for MUC2 and MUC5AC) features. By genomic analyses, there was loss of CDKN2A in 5/20 (25%) of these cases. However, the majority of the previously reported intraductal papillary mucinous neoplasm-related alterations were absent. Moreover, in contrast to most ductal neoplasms of the pancreas, MAP-kinase pathway was not involved. In fact, 2/22 (9%) of intraductal tubulopapillary neoplasms did not reveal any mutations in the tested genes. However, certain chromatin remodeling genes (MLL1, MLL2, MLL3, BAP1, PBRM1, EED, and ATRX) were found to be mutated in 7/22 (32%) of intraductal tubulopapillary neoplasms and 27% harbored phosphatidylinositol 3-kinase (PI3K) pathway (PIK3CA, PIK3CB, INPP4A, and PTEN) mutations. In addition, 4/18 (18%) of intraductal tubulopapillary neoplasms had FGFR2 fusions (FGFR2-CEP55, FGFR2-SASS6, DISP1-FGFR2, FGFR2-TXLNA, and FGFR2-VCL) and 1/18 (5.5%) had STRN-ALK fusion. Intraductal tubulopapillary neoplasm is a distinct clinicopathologic entity in the pancreas. Although its intraductal nature and some clinicopathologic features resemble those of intraductal papillary mucinous neoplasm, our results suggest that intraductal tubulopapillary neoplasm has distinguishing genetic characteristics. Some of these mutated genes are potentially targetable. Future functional studies will be needed to determine the consequences of these gene alterations.
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Adenocarcinoma Mucinoso/genética , Adenocarcinoma Papilar/genética , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma Mucinoso/patología , Adenocarcinoma Papilar/patología , Adulto , Anciano , Carcinoma Ductal Pancreático/patología , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/patología , Adulto JovenRESUMEN
In 2010, the World Health Organization reclassified the entity originally described as intraductal oncocytic papillary neoplasm as the 'oncocytic subtype' of intraductal papillary mucinous neoplasm. Although several key molecular alterations of other intraductal papillary mucinous neoplasm subtypes have been discovered, including common mutations in KRAS, GNAS, and RNF3, those of oncocytic subtype have not been well characterized. We analyzed 11 pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms. Nine pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms uniformly exhibited typical entity-defining morphology of arborizing papillae lined by layers of cells with oncocytic cytoplasm, prominent, nucleoli, and intraepithelial lumina. The remaining two were atypical. One lacked the arborizing papilla and had flat oncocytic epithelium only; the other one had focal oncocytic epithelium in a background of predominantly intestinal subtype intraductal papillary mucinous neoplasm. Different components of this case were analyzed separately. Formalin-fixed, paraffin-embedded specimens of all cases were microdissected and subjected to high-depth-targeted next-generation sequencing for a panel of 300 key cancer-associated genes in a platform that enabled the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Fresh frozen specimens of two cases were also subjected to whole-genome sequencing. For the nine typical pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms, the number of mutations per case, identified by next-generation sequencing, ranged from 1 to 10 (median=4). None of these cases had KRAS or GNAS mutations and only one had both RNF43 and PIK3R1 mutations. ARHGAP26, ASXL1, EPHA8, and ERBB4 genes were somatically altered in more than one of these typical 'oncocytic subtype' of intraductal papillary mucinous neoplasms but not in the other two atypical ones. In the neoplasm with flat oncocytic epithelium, the only mutated gene was KRAS. All components of the intestinal subtype intraductal papillary mucinous neoplasms with focal oncocytic epithelium manifested TP53, GNAS, and RNF43 mutations. In conclusion, this study elucidates that 'oncocytic subtype' of intraductal papillary mucinous neoplasm is not only morphologically distinct but also genetically distinct from other intraductal papillary mucinous neoplasm subtypes. Considering that now its biologic behavior is also being found to be different than other intraductal papillary mucinous neoplasm subtypes, 'oncocytic subtype' of intraductal papillary mucinous neoplasm warrants being recognized separately.
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Biomarcadores de Tumor/genética , Mutación , Neoplasias Quísticas, Mucinosas y Serosas/genética , Células Oxífilas , Neoplasias Pancreáticas/genética , Cromograninas/genética , Análisis Mutacional de ADN/métodos , Proteínas de Unión al ADN/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Perfilación de la Expresión Génica/métodos , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Quísticas, Mucinosas y Serosas/clasificación , Neoplasias Quísticas, Mucinosas y Serosas/patología , Proteínas Oncogénicas/genética , Células Oxífilas/clasificación , Células Oxífilas/patología , Neoplasias Pancreáticas/clasificación , Neoplasias Pancreáticas/patología , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas , Secuenciación Completa del GenomaRESUMEN
Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683-691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671-5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12, and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies.
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Apoptosis/genética , Regulación de la Expresión Génica/genética , Genoma Humano/genética , Melanoma/genética , Proteínas Musculares/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Secuencia de Bases , Western Blotting , Cartilla de ADN/genética , Exoma/genética , Vectores Genéticos/genética , Células HEK293 , Humanos , Inmunoprecipitación , Lentivirus , MicroARNs/genética , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular evolution of cancer.
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Desdiferenciación Celular/genética , ADN Intergénico , Melanoma/genética , Metástasis de la Neoplasia , Polimorfismo de Nucleótido Simple , Adulto , Variaciones en el Número de Copia de ADN , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Melanocitos/metabolismo , Melanocitos/patología , Cultivo Primario de Células , Secuencias Reguladoras de Ácidos Nucleicos , Células Tumorales CultivadasRESUMEN
Phacomatosis pigmentokeratotica (PPK) is characterized by the co-existence of epidermal nevi and large segmental speckled lentiginous nevi of the papulosa type. PPK, previously explained as 'twin spot' mosaicism due to the postzygotic crossing-over of two homozygous recessive mutations, has recently been shown to derive from one postzygotic activating RAS mutation. Epidermal nevi, including those in PPK, are known to give rise to neoplasms such as trichoblastoma and basal cell carcinoma. Within speckled lentiginous nevi, Spitz nevi and melanoma have been well documented. We report a case of PPK with a combined melanocytic and adnexal neoplasm presenting where the nevi conjoined. Using next-generation sequencing techniques, we were able to identify the same HRAS G13R mutation within both components of the tumor, and to show the absence of additional mutated modifier genes in a panel of 300 cancer-related genes. Given the genetic findings in this rare tumor-type, we suggest that this case may be used as a model for understanding the development of biphenotypic neoplasia or intratumoral heterogeneity in some cases.
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Mutación , Neoplasias Complejas y Mixtas/genética , Neoplasias Complejas y Mixtas/patología , Nevo Pigmentado/genética , Nevo Pigmentado/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Adolescente , Análisis Mutacional de ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Captura por Microdisección con Láser , Melanoma/genética , Melanoma/patología , Neoplasias de las Glándulas Sudoríparas/genética , Neoplasias de las Glándulas Sudoríparas/patologíaRESUMEN
Although immunotherapy with PD-(L)1 blockade is routine for lung cancer, little is known about acquired resistance. Among 1,201 patients with non-small cell lung cancer (NSCLC) treated with PD-(L)1 blockade, acquired resistance is common, occurring in >60% of initial responders. Acquired resistance shows differential expression of inflammation and interferon (IFN) signaling. Relapsed tumors can be separated by upregulated or stable expression of IFNγ response genes. Upregulation of IFNγ response genes is associated with putative routes of resistance characterized by signatures of persistent IFN signaling, immune dysfunction, and mutations in antigen presentation genes which can be recapitulated in multiple murine models of acquired resistance to PD-(L)1 blockade after in vitro IFNγ treatment. Acquired resistance to PD-(L)1 blockade in NSCLC is associated with an ongoing, but altered IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance may inform therapeutic strategies to effectively reprogram and reverse acquired resistance.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Transducción de Señal , Inmunoterapia , Presentación de Antígeno , Antígeno B7-H1/metabolismo , Microambiente TumoralRESUMEN
Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Up to date, even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53 (TP53) and Retinoblastoma Transcriptional Corepressor 1 (RB1) mutations in their tumors, no therapeutic strategies are available to prevent or delay histological transformation. Upregulation of the cell cycle kinase Cell Division Cycle 7 (CDC7) occurred in tumors during the initial steps of NE transformation, already after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen (MYC), implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics (cisplatin, irinotecan) in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity, as well as to effectively treat NE tumors de novo or after transformation. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk of transformation.
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Proteínas de Ciclo Celular , Neoplasias Pulmonares , Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-myc , Humanos , Masculino , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Línea Celular Tumoral , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ratones , Animales , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/patología , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/tratamiento farmacológico , Proteolisis/efectos de los fármacos , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
PURPOSE: The impact of the intratumoral microbiome on immune checkpoint inhibitor (ICI) efficacy in patients with non-small-cell lung cancer (NSCLC) is unknown. Preclinically, intratumoral Escherichia is associated with a proinflammatory tumor microenvironment and decreased metastases. We sought to determine whether intratumoral Escherichia is associated with outcome to ICI in patients with NSCLC. PATIENTS AND METHODS: We examined the intratumoral microbiome in 958 patients with advanced NSCLC treated with ICI by querying unmapped next-generation sequencing reads against a bacterial genome database. Putative environmental contaminants were filtered using no-template controls (n = 2,378). The impact of intratumoral Escherichia detection on overall survival (OS) was assessed using univariable and multivariable analyses. The findings were further validated in an external independent cohort of 772 patients. Escherichia fluorescence in situ hybridization (FISH) and transcriptomic profiling were performed. RESULTS: In the discovery cohort, read mapping to intratumoral Escherichia was associated with significantly longer OS (16 v 11 months; hazard ratio, 0.73 [95% CI, 0.59 to 0.92]; P = .0065) in patients treated with single-agent ICI, but not combination chemoimmunotherapy. The association with OS in the single-agent ICI cohort remained statistically significant in multivariable analysis adjusting for prognostic features including PD-L1 expression (P = .023). Analysis of an external validation cohort confirmed the association with improved OS in univariable and multivariable analyses of patients treated with single-agent ICI, and not in patients treated with chemoimmunotherapy. Escherichia localization within tumor cells was supported by coregistration of FISH staining and serial hematoxylin and eosin sections. Transcriptomic analysis correlated Escherichia-positive samples with expression signatures of immune cell infiltration. CONCLUSION: Read mapping to potential intratumoral Escherichia was associated with survival to single-agent ICI in two independent cohorts of patients with NSCLC.
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INTRODUCTION: Although targeted therapies have revolutionized the therapeutic landscape of lung adenocarcinomas (LUADs), disease progression on single-agent targeted therapy against known oncogenic drivers is common, and therapeutic options after disease progression are limited. In patients with MDM2 amplification (MDM2amp) and a concurrent oncogenic driver alteration, we hypothesized that targeting of the tumor-suppressor pathway (by means of restoration of p53 using MDM2 inhibition) and simultaneous targeting of co-occurring MAPK oncogenic pathway might represent a more durably effective therapeutic strategy. METHODS: We evaluated genomic next-generation sequencing data using the Memorial Sloan Kettering Cancer Center-Integrated Mutation Profiling of Actionable Cancer Targets platform to nominate potential targets for combination therapy in LUAD. We investigated the small molecule MDM2 inhibitor milademetan in cell lines and patient-derived xenografts of LUAD with a known driver alteration and MDM2amp. RESULTS: Of 10,587 patient samples from 7121 patients with LUAD profiled by next-generation sequencing, 6% (410 of 7121) harbored MDM2amp. MDM2amp was significantly enriched among tumors with driver alterations in METex14 (36%, p < 0.001), EGFR (8%, p < 0.001), RET (12%, p < 0.01), and ALK (10%, p < 0.01). The combination of milademetan and the MEK inhibitor trametinib was synergistic in growth inhibition of ECLC5-GLx (TRIM33-RET/MDM2amp), LUAD12c (METex14/KRASG12S/MDM2amp), SW1573 (KRASG12C, TP53 wild type), and A549 (KRASG12S) cells and in increasing expression of proapoptotic proteins PUMA and BIM. Treatment of ECLC5-GLx and LUAD12c with single-agent milademetan increased ERK phosphorylation, consistent with previous data on ERK activation with MDM2 inhibition. This ERK activation was effectively suppressed by concomitant administration of trametinib. In contrast, ERK phosphorylation induced by milademetan was not suppressed by concurrent RET inhibition using selpercatinib (in ECLC5-GLx) or MET inhibition using capmatinib (in LUAD12c). In vivo, combination milademetan and trametinib was more effective than either agent alone in ECLC5-GLx, LX-285 (EGFRex19del/MDM2amp), L13BS1 (METex14/MDM2amp), and A549 (KRASG12S, TP53 wild type). CONCLUSIONS: Combined MDM2/MEK inhibition was found to have efficacy across multiple patient-derived LUAD models harboring MDM2amp and concurrent oncogenic drivers. This combination, potentially applicable to LUADs with a wide variety of oncogenic driver mutations and kinase fusions activating the MAPK pathway, has evident clinical implications and will be investigated as part of a planned phase 1/2 clinical trial.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Mutación , Progresión de la Enfermedad , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/uso terapéutico , Proteínas Proto-Oncogénicas c-mdm2/genética , Factores de Transcripción/genéticaRESUMEN
In lung and prostate adenocarcinomas, neuroendocrine (NE) transformation to an aggressive derivative resembling small cell lung cancer (SCLC) is associated with poor prognosis. We previously described dependency of SCLC on the nuclear transporter exportin 1. Here, we explored the role of exportin 1 in NE transformation. We observed up-regulated exportin 1 in lung and prostate pretransformation adenocarcinomas. Exportin 1 was up-regulated after genetic inactivation of TP53 and RB1 in lung and prostate adenocarcinoma cell lines, accompanied by increased sensitivity to the exportin 1 inhibitor selinexor in vitro. Exportin 1 inhibition prevented NE transformation in different TP53/RB1-inactivated prostate adenocarcinoma xenograft models that acquire NE features upon treatment with the aromatase inhibitor enzalutamide and extended response to the EGFR inhibitor osimertinib in a lung cancer transformation patient-derived xenograft (PDX) model exhibiting combined adenocarcinoma/SCLC histology. Ectopic SOX2 expression restored the enzalutamide-promoted NE phenotype on adenocarcinoma-to-NE transformation xenograft models despite selinexor treatment. Selinexor sensitized NE-transformed lung and prostate small cell carcinoma PDXs to standard cytotoxics. Together, these data nominate exportin 1 inhibition as a potential therapeutic target to constrain lineage plasticity and prevent or treat NE transformation in lung and prostate adenocarcinoma.
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Adenocarcinoma , Neoplasias Pulmonares , Neoplasias de la Próstata , Factores de Transcripción SOXB1 , Carcinoma Pulmonar de Células Pequeñas , Humanos , Masculino , Adenocarcinoma/patología , Regulación hacia Abajo , Neoplasias Pulmonares/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Carcinoma Pulmonar de Células Pequeñas/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Animales , Proteína Exportina 1RESUMEN
Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however, little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. We performed whole-genome sequencing of 37 tumor samples from eight patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. We identified subclonal copy number alterations in all but one patient. We observed that in five patients, a subclonal copy number clone from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clone in 6 out of 7 patients with more than one clone. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy number clones. Our study sheds light on intratumor heterogeneity and the potential drivers of treatment resistance in osteosarcoma.
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PURPOSE: We report updated clinical outcomes from a phase II study of pembrolizumab, trastuzumab, and chemotherapy (PTC) in metastatic esophagogastric cancer in conjunction with outcomes from an independent Memorial Sloan Kettering (MSK) cohort. PATIENTS AND METHODS: The significance of pretreatment 89Zr-trastuzumab PET, plasma circulating tumor DNA (ctDNA) dynamics, and tumor HER2 expression and whole exome sequencing was evaluated to identify prognostic biomarkers and mechanisms of resistance in patients treated on-protocol with PTC. Additional prognostic features were evaluated using a multivariable Cox regression model of trastuzumab-treated MSK patients (n = 226). Single-cell RNA sequencing (scRNA-seq) data from MSK and Samsung were evaluated for mechanisms of therapy resistance. RESULTS: 89Zr-trastuzumab PET, scRNA-seq, and serial ctDNA with CT imaging identified how pre-treatment intrapatient genomic heterogeneity contributes to inferior progression-free survival (PFS). We demonstrated that the presence of intensely avid lesions by 89Zr-trastuzumab PET declines in tumor-matched ctDNA by 3 weeks, and clearance of tumor-matched ctDNA by 9 weeks were minimally invasive biomarkers of durable PFS. Paired pre- and on-treatment scRNA-seq identified rapid clearance of HER2-expressing tumor clones with expansion of clones expressing a transcriptional resistance program, which was associated with MT1H, MT1E, MT2A, and MSMB expression. Among trastuzumab-treated patients at MSK, ERBB2 amplification was associated with improved PFS, while alterations in MYC and CDKN2A/B were associated with inferior PFS. CONCLUSIONS: These findings highlight the clinical relevance of identifying baseline intrapatient heterogeneity and serial ctDNA monitoring of HER2-positive esophagogastric cancer patients to identify early evidence of treatment resistance, which could guide proactive therapy escalation or deescalation.
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Neoplasias de la Mama , Neoplasias Esofágicas , Neoplasias Gástricas , Humanos , Femenino , Receptor ErbB-2/metabolismo , Receptor de Muerte Celular Programada 1/uso terapéutico , Radioisótopos/uso terapéutico , Circonio , Biomarcadores de Tumor/metabolismo , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/inducido químicamente , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Trastuzumab/efectos adversos , Neoplasias de la Mama/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversosRESUMEN
Multiple large-scale genomic profiling efforts have been undertaken in osteosarcoma to define the genomic drivers of tumorigenesis, therapeutic response, and disease recurrence. The spatial and temporal intratumor heterogeneity could also play a role in promoting tumor growth and treatment resistance. We conducted longitudinal whole-genome sequencing of 37 tumor samples from 8 patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. Subclonal copy-number alterations were identified in all patients except one. In 5 patients, subclones from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clones in 6 of 7 patients with multiple clones. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy-number clones. A chromosomal duplication timing analysis revealed that complex genomic rearrangements typically occurred prior to diagnosis, supporting a macroevolutionary model of evolution, where a large number of genomic aberrations are acquired over a short period of time followed by clonal selection, as opposed to ongoing evolution. A mutational signature analysis of recurrent tumors revealed that homologous repair deficiency (HRD)-related SBS3 increases at each time point in patients with recurrent disease, suggesting that HRD continues to be an active mutagenic process after diagnosis. Overall, by examining the clonal relationships between temporally and spatially separated samples from patients with relapsed/refractory osteosarcoma, this study sheds light on the intratumor heterogeneity and potential drivers of treatment resistance in this disease. SIGNIFICANCE: The chemoresistant population in recurrent osteosarcoma is subclonal at diagnosis, emerges at the time of primary resection due to selective pressure from neoadjuvant chemotherapy, and is characterized by unique oncogenic amplifications.
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Neoplasias Óseas , Osteosarcoma , Humanos , Osteosarcoma/genética , Secuenciación Completa del Genoma , Genómica , Neoplasias Óseas/genética , Recurrencia , Variaciones en el Número de Copia de ADN , MutaciónRESUMEN
Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T , Células Clonales , Microambiente TumoralRESUMEN
Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis and extreme lethality. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy providing modest benefits in a subset of patients. However, nearly all patients treated with systemic therapy quickly develop resistant disease, and there is an absence of effective therapies for recurrent and progressive disease. Here we conducted CRISPR-Cas9 screens using a druggable genome library in multiple SCLC cell lines representing distinct molecular subtypes. This screen nominated exportin-1, encoded by XPO1, as a therapeutic target. XPO1 was highly and ubiquitously expressed in SCLC relative to other lung cancer histologies and other tumor types. XPO1 knockout enhanced chemosensitivity, and exportin-1 inhibition demonstrated synergy with both first- and second-line chemotherapy. The small molecule exportin-1 inhibitor selinexor in combination with cisplatin or irinotecan dramatically inhibited tumor growth in chemonaïve and chemorelapsed SCLC patient-derived xenografts, respectively. Together these data identify exportin-1 as a promising therapeutic target in SCLC, with the potential to markedly augment the efficacy of cytotoxic agents commonly used in treating this disease. SIGNIFICANCE: CRISPR-Cas9 screening nominates exportin-1 as a therapeutic target in SCLC, and exportin-1 inhibition enhances chemotherapy efficacy in patient-derived xenografts, providing a novel therapeutic opportunity in this disease.
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Carioferinas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/metabolismo , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patología , Ratones , Carcinoma Pulmonar de Células Pequeñas/patología , Proteína Exportina 1RESUMEN
BACKGROUND: Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies. METHODS: We performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment. RESULTS: Our data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib. CONCLUSIONS: Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.