RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation. SIGNIFICANCE: Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Técnicas de Cocultura , Transição Epitelial-Mesenquimal , Inflamação , Integrina beta1 , Neoplasias Pancreáticas , Análise de Célula Única , Microambiente Tumoral , Humanos , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Inflamação/patologia , Inflamação/metabolismo , Integrina beta1/metabolismo , Integrina beta1/genética , Organoides/patologia , Organoides/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Comunicação CelularRESUMO
CD8+ T cell dysfunction impedes antitumor immunity in solid cancers, but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional coactivator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend on oncogene-mediated ECM composition and remodeling.
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Linfócitos T CD8-Positivos , Matriz Extracelular , Sarcoma , Microambiente Tumoral , Proteínas de Sinalização YAP , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Animais , Microambiente Tumoral/imunologia , Camundongos , Proteínas de Sinalização YAP/imunologia , Proteínas de Sinalização YAP/genética , Humanos , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Sarcoma/imunologia , Sarcoma/patologia , Sarcoma/genética , Sarcoma/metabolismo , Colágeno Tipo VI/genética , Colágeno Tipo VI/imunologia , Colágeno Tipo VI/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/imunologia , Oncogenes , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/imunologiaRESUMO
Pancreatic cancer is more prevalent in older individuals and often carries a poorer prognosis for them. The relationship between the microenvironment and pancreatic cancer is multifactorial, and age-related changes in nonmalignant cells in the tumor microenvironment may play a key role in promoting cancer aggressiveness. Because fibroblasts have profound impacts on pancreatic cancer progression, we investigated whether age-related changes in pancreatic fibroblasts influence cancer growth and metastasis. Proteomics analysis revealed that aged fibroblasts secrete different factors than young fibroblasts, including increased growth/differentiation factor 15 (GDF-15). Treating young mice with GDF-15 enhanced tumor growth, whereas aged GDF-15 knockout mice showed reduced tumor growth. GDF-15 activated AKT, rendering tumors sensitive to AKT inhibition in an aged but not young microenvironment. These data provide evidence for how aging alters pancreatic fibroblasts and promotes tumor progression, providing potential therapeutic targets and avenues for studying pancreatic cancer while accounting for the effects of aging. SIGNIFICANCE: Aged pancreatic fibroblasts secrete GDF-15 and activate AKT signaling to promote pancreatic cancer growth, highlighting the critical role of aging-mediated changes in the pancreatic cancer microenvironment in driving tumor progression. See related commentary by Isaacson et al., p. 1185.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pancreáticas , Animais , Camundongos , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/uso terapêutico , Proteínas Proto-Oncogênicas c-akt , Neoplasias Pancreáticas/patologia , Pâncreas/patologia , Fibroblastos/patologia , Microambiente Tumoral , Linhagem Celular Tumoral , Fibroblastos Associados a Câncer/patologiaRESUMO
Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in >60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression serve as a mechanism of intrinsic early/adaptive CDK4i/6i resistance. ALM cells that have acquired CDK4i/6i resistance following chronic treatment exposure also exhibit hyperactivation of the MAPK pathway. MEK and/or ERK inhibition increases CDK4i/6i efficacy against therapy naïve and CDK4i/6i-resistant AM cells in xenograft and patient-derived xenograft (PDX) models and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach for patients with metastatic ALM to improve outcomes.
Assuntos
Melanoma , Neoplasias Cutâneas , Animais , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Modelos Animais de Doenças , Ciclo Celular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor.
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BACKGROUND AND AIMS: The treatment of hepatocellular carcinoma (HCC) has been transformed by the use of immune checkpoint inhibitors. However, most patients with HCC do not benefit from treatment with immunotherapy. There is an urgent need to understand the mechanisms that underlie response or resistance to immunotherapy for patients with HCC. The use of syngeneic mouse models that closely recapitulate the heterogeneity of human HCC will provide opportunities to examine the complex interactions between cancer cells and nonmalignant cells in the tumor microenvironment. APPROACH AND RESULTS: We leverage a multifaceted approach that includes imaging mass cytometry and suspension cytometry by time of flight to profile the tumor microenvironments of the Hep53.4, Hepa 1-6, RIL-175, and TIBx (derivative of TIB-75) syngeneic mouse HCC models. The immune tumor microenvironments vary across these four models, and various immunosuppressive pathways exist at baseline in orthotopic liver tumors derived from these models. For instance, TIBx, which is resistant to anti-programmed cell death protein 1 therapy, contains a high proportion of "M2-like" tumor-associated macrophages with the potential to diminish antitumor immunity. Investigation of The Cancer Genome Atlas reveals that the baseline immunologic profiles of Hep53.4, RIL-175, and TIBx are broadly representative of human HCCs; however, Hepa 1-6 does not recapitulate the immune tumor microenvironment of the vast majority of human HCCs. CONCLUSIONS: There is a wide diversity in the immune tumor microenvironments in preclinical models and in human HCC, highlighting the need to use multiple syngeneic HCC models to improve the understanding of how to treat HCC through immune modulation.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Imunoterapia/métodos , Neoplasias Hepáticas/patologia , Microambiente Tumoral , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.
Assuntos
Envelhecimento , Pulmão , Melanoma , Metástase Neoplásica , Células Estromais , Microambiente Tumoral , Idoso , Envelhecimento/patologia , Fibroblastos/patologia , Humanos , Pulmão/patologia , Melanoma/patologia , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Neoplasia Residual , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Pele/patologia , Células Estromais/patologia , Proteína Wnt-5a , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
PURPOSE: Biliary tract cancers (BTCs) are aggressive cancers that carry a poor prognosis. An enhanced understanding of the immune landscape of anatomically and molecularly defined subsets of BTC may improve patient selection for immunotherapy and inform immune-based combination treatment strategies. METHODS: We analyzed deidentified clinical, genomic, and transcriptomic data from the Tempus database to determine the mutational frequency and mutational clustering across the three major BTC subtypes (intrahepatic cholangiocarcinoma [IHC], extrahepatic cholangiocarcinoma, and gallbladder cancer). We subsequently determined the relationship between specific molecular alterations and anatomical subsets and features of the BTC immune microenvironment. RESULTS: We analyzed 454 samples of BTC, of which the most commonly detected alterations were TP53 (42.5%), CDKN2A (23.4%), ARID1A (19.6%), BAP1 (15.5%), KRAS (15%), CDKN2B (14.2%), PBRM1 (11.7%), IDH1 (11.7%), TERT (8.4%), KMT2C (10.4%) and LRP1B (8.4%), and FGFR2 fusions (8.7%). Potentially actionable molecular alterations were identified in 30.5% of BTCs including 39.1% of IHC. Integrative cluster analysis revealed four distinct molecular clusters, with cluster 4 predominately associated with FGFR2 rearrangements and BAP1 mutations in IHC. Immune-related biomarkers indicative of an inflamed tumor-immune microenvironment were elevated in gallbladder cancers and in cluster 1, which was enriched for TP53, KRAS, and ATM mutations. Multiple common driver genes, including TP53, FGFR2, IDH1, TERT, BRAF, and BAP1, were individually associated with unique BTC immune microenvironments. CONCLUSION: BTC subtypes exhibit diverse DNA alterations, RNA inflammatory signatures, and immune biomarkers. The association between specific BTC anatomical subsets, molecular alterations, and immunophenotypes highlights new opportunities for therapeutic development.
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Neoplasias dos Ductos Biliares , Neoplasias do Sistema Biliar , Colangiocarcinoma , Neoplasias da Vesícula Biliar , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias do Sistema Biliar/genética , Colangiocarcinoma/genética , Neoplasias da Vesícula Biliar/genética , Genômica , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Transcriptoma/genética , Microambiente Tumoral/genéticaRESUMO
Aging is a universal biological process that increases the risk of multiple diseases including cancer. Growing evidence shows that alterations in the genome and epigenome, driven by similar mechanisms, are found in both aged cells and cancer cells. In this review, we detail the genetic and epigenetic changes associated with normal aging and the mechanisms responsible for these changes. By highlighting genetic and epigenetic alterations in the context of tumorigenesis, cancer progression, and the aging tumor microenvironment, we examine the possible impacts of the normal aging process on malignant transformation. Finally, we examine the implications of age-related genetic and epigenetic alterations in both tumors and patients for the treatment of cancer.
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Metilação de DNA , Neoplasias , Idoso , Envelhecimento/genética , Envelhecimento/patologia , Epigênese Genética/genética , Epigenoma , Humanos , Neoplasias/genética , Neoplasias/terapia , Microambiente Tumoral/genéticaRESUMO
Activating variants in the PEST region of NOTCH1 have been associated with aggressive phenotypes in human cancers, including triple-negative breast cancer (TNBC). Previous studies suggested that PEST domain variants in TNBC patients resulted in increased cell proliferation, invasiveness, and decreased overall survival. In this study, we assess the phenotypic transformation of activating NOTCH1 variants and their response to standard of care therapies. AAV-mediated gene targeting was used to isogenically incorporate 3 NOTCH1 variants, including a novel TNBC frameshift variant, in two non-tumorigenic breast epithelial cell lines, MCF10A and hTERT-IMEC. Two different variants at the NOTCH1 A2241 site (A2441fs and A2441T) both demonstrated increased transformative properties when compared to a non-transformative PEST domain variant (S2523L). These phenotypic changes include proliferation, migration, anchorage-independent growth, and MAPK pathway activation. In contrast to previous studies, activating NOTCH1 variants did not display sensitivity to a gamma secretase inhibitor (GSI) or resistance to chemotherapies. This study demonstrates distinct transformative phenotypes are specific to a given variant within NOTCH1 and these phenotypes do not correlate with sensitivities or resistance to chemotherapies or GSIs. Although previous studies have suggested NOTCH1 variants may be prognostic for TNBC, our study does not demonstrate prognostic ability of these variants and suggests further characterization would be required for clinical applications.
Assuntos
Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Proliferação de Células/genética , Inibidores e Moduladores de Secretases gama , Humanos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais , Padrão de Cuidado , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/terapiaRESUMO
Tumor involvement of major vascular structures limits surgical options in pancreatic adenocarcinoma (PDAC), which in turn limits opportunities for cure. Despite advances in locoregional approaches, there is currently no role for incomplete resection. This study evaluated a gelatinized neoantigen-targeted vaccine applied to a grossly positive resection margin in preventing local recurrence. Incomplete surgical resection was performed in mice bearing syngeneic flank Panc02 tumors, leaving a 1 mm rim adherent to the muscle bed. A previously validated vaccine consisting of neoantigen peptides, a stimulator of interferon genes (STING) agonist and AddaVaxTM (termed PancVax) was embedded in a hyaluronic acid hydrogel and applied to the tumor bed. Tumor remnants, regional lymph nodes, and spleens were analyzed using histology, flow cytometry, gene expression profiling, and ELISPOT assays. The immune microenvironment at the tumor margin after surgery alone was characterized by a transient influx of myeloid-derived suppressor cells (MDSCs), prolonged neutrophil influx, and near complete loss of cytotoxic T cells. Application of PancVax gel was associated with enhanced T cell activation in the draining lymph node and expansion of neoantigen-specific T cells in the spleen. Mice implanted with PancVax gel demonstrated no evidence of residual tumor at two weeks postoperatively and healed incisions at two months postoperatively without local recurrence. In summary, application of PancVax gel at a grossly positive tumor margin led to systemic expansion of neoantigen-specific T cells and effectively prevented local recurrence. These findings support further work into locoregional adjuncts to immune modulation in PDAC.
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Adenocarcinoma , Vacinas Anticâncer , Neoplasias Pancreáticas , Adenocarcinoma/prevenção & controle , Adenocarcinoma/cirurgia , Animais , Hidrogéis , Imunoterapia , Camundongos , Microambiente TumoralRESUMO
Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity; however, their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to coexist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell contact-mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor-positive and -negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insight into nonrandom subclonal architecture of heterogenous tumors, which may aid the understanding of tumor evolution and inform future strategies for personalized medicine.
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Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Comunicação Celular/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Técnicas de Cocultura , Feminino , Fibronectinas/antagonistas & inibidores , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Técnicas de Inativação de Genes , Humanos , Imuno-Histoquímica , Células MCF-7 , Mutação , Fenótipo , Receptor ErbB-2/genéticaRESUMO
Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers.
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Reprogramação Celular , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias , Fosfoproteínas , Proteoma/metabolismo , Fatores de Processamento de RNA , Serina , Transcriptoma , Animais , Linhagem Celular Tumoral , Metabolismo Energético/genética , Glicina , Humanos , Camundongos , Proteínas de Neoplasias/genética , Neoplasias/dietoterapia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteoma/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Estrogen receptor-alpha (ER) is a therapeutic target of ER-positive (ER+) breast cancers. Although ER signaling is complex, many mediators of this pathway have been identified. Specifically, phosphorylation of ER at serine 118 affects responses to estrogen and therapeutic ligands and has been correlated with clinical outcomes in ER+ breast cancer patients. We hypothesized that a newly described germline variant (S118P) at this residue would drive cellular changes consistent with breast cancer development and/or hormone resistance. METHODS: Isogenic human breast epithelial cell line models harboring ER S118P were developed via genome editing and characterized to determine the functional effects of this variant. We also examined the frequency of ER S118P in a case-control study (N = 536) of women with and without breast cancer with a familial risk. RESULTS: In heterozygous knock-in models, the S118P variant demonstrated no significant change in proliferation, migration, MAP Kinase pathway signaling, or response to the endocrine therapies tamoxifen and fulvestrant. Further, there was no difference in the prevalence of S118P between women with and without cancer relative to population registry databases. CONCLUSIONS: This study suggests that the ER S118P variant does not affect risk for breast cancer or hormone therapy resistance. Germline screening and modification of treatments for patients harboring this variant are likely not warranted.
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Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/epidemiologia , Receptor alfa de Estrogênio/genética , Mutação em Linhagem Germinativa , Adulto , Idoso , Neoplasias da Mama/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Receptor alfa de Estrogênio/metabolismo , Feminino , Fulvestranto/uso terapêutico , Variação Genética , Humanos , Incidência , Células MCF-7 , Pessoa de Meia-Idade , Fosforilação , Análise de Sobrevida , Tamoxifeno/uso terapêutico , Resultado do TratamentoRESUMO
BACKGROUND: Molecular-based diagnostics have great utility for cancer detection. We have used droplet digital PCR (ddPCR) as a platform for identifying mutations in circulating plasma tumor DNA (ptDNA). We present the unexpected finding of a spurious mutant allele fraction that was discovered to be artifactual because of the presence of a single-nucleotide polymorphism (SNP) in a patient sample. DESIGN AND METHODS: Probe and primer combinations for the K700 and V701 loci of the SF3B1 spliceosome gene were designed for ddPCR to identify the percentage of mutant and wild-type alleles. Clinical samples from patients with cancer with known SF3B1 mutations were collected and tested to evaluate the assays' ability to detect SF3B1 mutations in ptDNA. RESULTS: Patient samples showed SF3B1 K700E mutations within the ptDNA of 4 patients with acute leukemia and 3 with myelodysplastic syndrome who were known to harbor this mutation. A blood sample from a patient with lung cancer with a known SF3B1 V701F mutation was also analyzed and this mutation was successfully identified in ptDNA. However, 1 of the patients with a K700E mutation was found to have a mutational burden of 98%. After careful analysis of this locus by Sanger sequencing and ddPCR, this patient was found to have an SNP (R702R), which prevented binding of the ddPCR wild-type probe to its cognate allele. CONCLUSIONS: These results further support that ddPCR-based assays may be valuable companion diagnostics for the identification and monitoring of patients with cancer, but the results also emphasize the need to identify SNPs at loci that are being analyzed.
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Alelos , DNA de Neoplasias/genética , Fosfoproteínas/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética , Fatores de Processamento de RNA/genética , DNA de Neoplasias/sangue , Reações Falso-Positivas , Humanos , MutaçãoRESUMO
Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2-irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivoConclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123-34. ©2017 AACR.
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Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Terapia de Alvo Molecular , Receptor ErbB-2/genética , Afatinib , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Lapatinib , Camundongos , Mutação , Quinazolinas/administração & dosagem , Quinazolinas/efeitos adversos , Receptor ErbB-2/antagonistas & inibidores , Receptores de Estrogênio/genética , Transdução de Sinais/efeitos dos fármacos , Trastuzumab/administração & dosagem , Trastuzumab/efeitos adversos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: Although most human cancers display a single histology, there are unusual cases where two or more distinct tissue types present within a primary tumor. One such example is metaplastic breast carcinoma, a rare but aggressive cancer with a heterogeneous histology, including squamous, chondroid, and spindle cells. Metaplastic carcinomas often contain an admixed conventional ductal invasive or in situ mammary carcinoma component, and are typically triple-negative for estrogen receptor, progesterone receptor, and HER-2 amplification/overexpression. An unanswered question is the origin of metaplastic breast cancers. While they may arise independently from their ductal components, their close juxtaposition favors a model that postulates a shared origin, either as two derivatives from the same primary cancer or one histology as an outgrowth of the other. Understanding the mechanism of development of these tumors may inform clinical decisions.Experimental Design: We performed exome sequencing for paired metaplastic and adjacent conventional invasive ductal carcinomas in 8 patients and created a pipeline to identify somatic variants and predict their functional impact, without having normal tissue. We then determined the genetic relationships between the histologically distinct compartments.Results: In each case, the tumor components have nearly identical landscapes of somatic mutation, implying that the differing histologies do not derive from genetic clonal divergence.Conclusions: A shared origin for tumors with differing histologies suggests that epigenetic or noncoding changes may mediate the metaplastic phenotype and that alternative therapeutic approaches, including epigenetic therapies, may be required for metaplastic breast cancers. Clin Cancer Res; 23(16); 4875-84. ©2017 AACR.
Assuntos
Neoplasias da Mama/genética , Mama/metabolismo , Carcinoma Ductal de Mama/genética , Sequenciamento do Exoma/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Células Clonais/metabolismo , Células Clonais/patologia , Variações do Número de Cópias de DNA , Feminino , Humanos , Metaplasia/genética , Metaplasia/metabolismo , Pessoa de Meia-Idade , Mutação , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND/PURPOSE: The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies. EXPERIMENTAL DESIGN/METHODS: We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies. RESULTS: Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic. CONCLUSIONS: This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação , Fenótipo , Proteína Supressora de Tumor p53/genética , Animais , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Centrômero/genética , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Amplificação de Genes , Edição de Genes , Técnicas de Inativação de Genes , Instabilidade Genômica , Genótipo , Humanos , Camundongos , Paclitaxel/farmacologiaRESUMO
PURPOSE: The clinical utility of next-generation sequencing (NGS) in breast cancer has not been demonstrated. We hypothesized that we could perform NGS of a new biopsy from patients with metastatic triple-negative breast cancer (TNBC) in a clinically actionable timeframe. EXPERIMENTAL DESIGN: We planned to enroll 40 patients onto a prospective study, Individualized Molecular Analyses Guide Efforts (IMAGE), to evaluate the feasibility of obtaining a new biopsy of a metastatic site, perform NGS (FoundationOne), and convene a molecular tumor board to formulate treatment recommendations within 28 days. We collected blood at baseline and at time of restaging to assess cell-free circulating plasma tumor DNA (ptDNA). RESULTS: We enrolled 26 women with metastatic TNBC who had received ≥1 line of prior chemotherapy, and 20 (77%) underwent NGS of a metastatic site biopsy. Twelve (60%) evaluable patients received treatment recommendations within 28 days of consent. The study closed after 20 patients underwent NGS, based on protocol-specified interim futility analysis. Three patients went on to receive genomically directed therapies. Twenty-four of 26 patients had genetic alterations successfully detected in ptDNA. Among 5 patients, 4 mutations found in tumor tissues were not identified in blood, and 4 mutations found in blood were not found in corresponding tumors. In 9 patients, NGS of follow-up blood samples showed 100% concordance with baseline blood samples. CONCLUSIONS: This study demonstrates challenges of performing NGS on prospective tissue biopsies in patients with metastatic TNBC within 28 days, while also highlighting the potential use of blood as a more time-efficient and less invasive method of mutational assessment. Clin Cancer Res; 23(2); 379-86. ©2016 AACR.