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BACKGROUND: Metaplastic breast carcinoma encompasses a heterogeneous group of tumours with differentiation into squamous and/or spindle, chondroid, osseous or rhabdoid mesenchymal-looking elements. Emerging immunotherapies targeting Programmed Death Ligand 1 (PD-L1) and immune-suppressing T cells (Tregs) may benefit metaplastic breast cancer patients, which are typically chemo-resistant and do not express hormone therapy targets. METHODS: We evaluated the immunohistochemical expression of PD-L1 and FOXP3, and the extent of tumour infiltrating lymphocytes (TILs) in a large cohort of metaplastic breast cancers, with survival data. RESULTS: Metaplastic breast cancers were significantly enriched for PD-L1 positive tumour cells, compared to triple-negative ductal breast cancers (P < 0.0001), while there was no significant difference in PD-L1 positive TILs. Metaplastic breast cancers were also significantly enriched for TILs expressing FOXP3, with FOXP3 positive intra-tumoural TILs (iTILs) associated with an adverse prognostic outcome (P = 0.0226). Multivariate analysis identified FOXP3 iTILs expression status as an important independent prognostic factor for patient survival. CONCLUSIONS: Our findings indicate the clinical significance and prognostic value of FOXP3, PD-1/PD-L1 checkpoint and TILs in metaplastic breast cancer and confirm that a subset of metaplastics may benefit from immune-based therapies.
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Antígeno B7-H1/biosíntesis , Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/patología , Factores de Transcripción Forkhead/biosíntesis , Adulto , Anciano , Neoplasias de la Mama/inmunología , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico , Linfocitos Infiltrantes de Tumor/inmunología , Metaplasia , Persona de Mediana EdadRESUMEN
Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5-year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology - the biological basis and clinical relevance of its seven sub-categories are currently unclear. By establishing the Asia-Pacific MBC (AP-MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan-cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for 'mixed' MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis-1 (NF1) were also overrepresented [16.7% MBCs compared to â¼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed-type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan-cytokeratin expression are important prognostic markers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Mutación , Neoplasias Complejas y Mixtas/genética , Antígenos CD/análisis , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/patología , Cadherinas/análisis , Fosfatidilinositol 3-Quinasa Clase I/genética , Estudios Transversales , Transición Epitelial-Mesenquimal , Receptores ErbB/análisis , Femenino , Predisposición Genética a la Enfermedad , Humanos , Queratinas/análisis , Metaplasia , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias Complejas y Mixtas/química , Neoplasias Complejas y Mixtas/clasificación , Neoplasias Complejas y Mixtas/patología , Neurofibromina 1/genética , Fosfohidrolasa PTEN/genética , Fenotipo , Carga Tumoral , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Schmid metaphyseal chondrodysplasia (MCDS) involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER) in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR) in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K) were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2), generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent) or pathologically redundant (XBP1-dependent). XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-ß, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-ß transcriptional co-factors, GADD45-ß and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-ß-mediated chondrocyte differentiation. Our data suggest that modulation of C/EBP-ß activity in MCDS chondrocytes may offer therapeutic opportunities.
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Enfermedades Óseas/patología , Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Diferenciación Celular/fisiología , Condrocitos/patología , Proteínas de Unión al ADN/fisiología , Estrés del Retículo Endoplásmico/fisiología , Factores de Transcripción/fisiología , Respuesta de Proteína Desplegada/fisiología , Animales , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Ratones , Ratones Transgénicos , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Proteína 1 de Unión a la X-BoxRESUMEN
BACKGROUND: Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. METHODS: As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated '3D on-top cellular assay' to identify novel growth suppressive mechanisms. RESULTS: A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p < 0.05). Consistent with this, depletion of COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis. CONCLUSION: Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells.
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Cobre , Neoplasias , Animales , Ratones , Diferenciación Celular/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Transducción de SeñalRESUMEN
Intratumoral heterogeneity is caused by genomic instability and phenotypic plasticity, but how these features co-evolve remains unclear. SOX10 is a neural crest stem cell (NCSC) specifier and candidate mediator of phenotypic plasticity in cancer. We investigated its relevance in breast cancer by immunophenotyping 21 normal breast and 1860 tumour samples. Nuclear SOX10 was detected in normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In tumours, nuclear SOX10 was almost exclusive to TNBC, and predicted poorer outcome amongst cross-sectional (p = 0.0015, hazard ratio 2.02, n = 224) and metaplastic (p = 0.04, n = 66) cases. To understand SOX10's influence over the transcriptome during the transition from normal to malignant states, we performed a systems-level analysis of co-expression data, de-noising the networks with an eigen-decomposition method. This identified a core module in SOX10's normal mammary epithelial network that becomes rewired to NCSC genes in TNBC. Crucially, this reprogramming was proportional to genome-wide promoter methylation loss, particularly at lineage-specifying CpG-island shores. We propose that the progressive, genome-wide methylation loss in TNBC simulates more primitive epigenome architecture, making cells vulnerable to SOX10-driven reprogramming. This study demonstrates potential utility for SOX10 as a prognostic biomarker in TNBC and provides new insights about developmental phenotypic mimicry-a major contributor to intratumoral heterogeneity.
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AIMS: BRAF mutation has been shown in a large meta-analysis to be an independent prognostic marker for papillary thyroid carcinoma (PTC) with poorer survival and higher recurrence rates. METHODS: We studied prevalence of BRAF mutation in 77 patients with PTC from an Australian cohort using competitive polymerase chain reaction (C-PCR) and immunohistochemistry (IHC) with BRAF-specific antibody, VE1. Clinicopathological parameters, recurrence and mortality were analysed according to BRAF mutation status. RESULTS: Median follow-up was 84.5 months. BRAF mutation was demonstrated in 65% of cases combining both C-PCR and IHC; in 71% (37/77) of tumours >1 cm and 52% (13/25) of microcarcinomas (<1 cm). IHC was positive in 69% (49/71) and C-PCR in 53% (41/77); 87% (67/77) of our patients were treated with total thyroidectomy and 65% (50/77) also had radioactive ablation. BRAF positive tumours had a significantly higher rate of subsequent lymph node metastases (p=0.035). Significant association was found between BRAF mutation and male sex (p=0.034), but not between age >45 years at diagnosis, size of primary tumour, extrathyroidal extension, lymph node or distant metastases or clinical stage at diagnosis. CONCLUSIONS: BRAF mutation in PTC determined by IHC is associated with significantly increased risk of lymph node recurrence.
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Carcinoma Papilar/genética , Mutación , Recurrencia Local de Neoplasia/genética , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/genética , Adulto , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Papilar/metabolismo , Carcinoma Papilar/mortalidad , Carcinoma Papilar/patología , Femenino , Humanos , Inmunohistoquímica/métodos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Pronóstico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/mortalidad , Neoplasias de la Tiroides/patologíaRESUMEN
PURPOSE: Amplification of cyclin E1 (CCNE1) is associated with poor outcome in breast, lung, and other solid cancers, and is the most prominent structural variant associated with primary treatment failure in high-grade serous ovarian cancer (HGSC). We have previously shown that CCNE1-amplified tumors show amplicon-dependent sensitivity to CCNE1 suppression. Here, we explore targeting CDK2 as a novel therapeutic strategy in CCNE1-amplified cancers and mechanisms of resistance. EXPERIMENTAL DESIGN: We examined the effect of CDK2 suppression using RNA interference and small-molecule inhibitors in SK-OV-3, OVCAR-4, and OVCAR-3 ovarian cancer cell lines. To identify mechanisms of resistance, we derived multiple, independent resistant sublines of OVCAR-3 to CDK2 inhibitors. Resistant cells were extensively characterized by gene expression and copy number analysis, fluorescence-activated cell sorting profiling and conventional karyotyping. In addition, we explored the relationship between CCNE1 amplification and polyploidy using data from primary tumors. RESULTS: We validate CDK2 as a therapeutic target in CCNE1-amplified cells by showing selective sensitivity to suppression, either by gene knockdown or using small-molecule inhibitors. In addition, we identified two resistance mechanisms, one involving upregulation of CDK2 and another novel mechanism involving selection of polyploid cells from the pretreatment tumor population. Our analysis of genomic data shows that polyploidy is a feature of cancer genomes with CCNE1 amplification. CONCLUSIONS: These findings suggest that cyclinE1/CDK2 is an important therapeutic target in HGSC, but that resistance to CDK2 inhibitors may emerge due to upregulation of CDK2 target protein and through preexisting cellular polyploidy.
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Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Amplificación de Genes , Proteínas Oncogénicas/genética , Neoplasias Ováricas/genética , Poliploidía , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Supervivencia Celular/genética , Análisis por Conglomerados , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/metabolismo , Pirazoles/farmacología , Pirrolidinonas/farmacologíaRESUMEN
Familial digital arthropathy-brachydactyly (FDAB) is a dominantly inherited condition that is characterized by aggressive osteoarthropathy of the fingers and toes and consequent shortening of the middle and distal phalanges. Here we show in three unrelated families that FDAB is caused by mutations encoding p.Gly270Val, p.Arg271Pro and p.Phe273Leu substitutions in the intracellular ankyrin-repeat domain of the cation channel TRPV4. Functional testing of mutant TRPV4 in HEK-293 cells showed that the mutant proteins have poor cell-surface localization. Calcium influx in response to the synthetic TRPV4 agonists GSK1016790A and 4αPDD was significantly reduced, and mutant channels did not respond to hypotonic stress. Others have shown that gain-of-function TRPV4 mutations cause skeletal dysplasias and peripheral neuropathies. Our data indicate that TRPV4 mutations that reduce channel activity cause a third phenotype, inherited osteoarthropathy, and show the importance of TRPV4 activity in articular cartilage homeostasis. Our data raise the possibility that TRPV4 may also have a role in age- or injury-related osteoarthritis.