RESUMEN
Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response. To model the clinical scenario of multiple tumor sites, we used MMTV-PyMT transgenic mice, which spontaneously develop heterogeneous breast tumors throughout their 10 mammary glands. We found that i.v. administration of a tumor-targeting TLR9 agonist, referred to as PIP-CpG, induced a systemic T cell-mediated immune response that not only promoted regression of existing mammary tumors, but also elicited immune memory capable of delaying growth of independent newly arising tumors. Within the tumor microenvironment, PIP-CpG therapy initiated an inflammatory cascade that dramatically amplified chemokine and cytokine production, prompted robust infiltration and expansion of innate and adaptive immune cells, and led to diverse and unexpected changes in immune phenotypes. This study demonstrates that effective systemic treatment of an autochthonous multisite tumor model can be achieved using a tumor-targeted immunostimulant and provides immunological insights that will inform future therapeutic strategies.
Asunto(s)
Neoplasias de la Mama , Neoplasias Mamarias Animales , Ratones , Animales , Humanos , Femenino , Receptor Toll-Like 9 , Ratones Transgénicos , Adyuvantes Inmunológicos/farmacología , Neoplasias Mamarias Animales/terapia , Neoplasias de la Mama/terapia , Microambiente Tumoral , Línea Celular TumoralRESUMEN
The regulatory mechanisms of circadian rhythms have been studied primarily at the level of the transcription-translation feedback loops of protein-coding genes. Regulatory modules involving noncoding RNAs are less thoroughly understood. In particular, emerging evidence has revealed the important role of microRNAs (miRNAs) in maintaining the robustness of the circadian system. To identify miRNAs that have the potential to modulate circadian rhythms, we conducted a genome-wide miRNA screen using U2OS luciferase reporter cells. Among 989 miRNAs in the library, 120 changed the period length in a dose-dependent manner. We further validated the circadian regulatory function of an miRNA cluster, miR-183/96/182, both in vitro and in vivo. We found that all three members of this miRNA cluster can modulate circadian rhythms. Particularly, miR-96 directly targeted a core circadian clock gene, PER2. The knockout of the miR-183/96/182 cluster in mice showed tissue-specific effects on circadian parameters and altered circadian rhythms at the behavioral level. This study identified a large number of miRNAs, including the miR-183/96/182 cluster, as circadian modulators. We provide a resource for further understanding the role of miRNAs in the circadian network and highlight the importance of miRNAs as a genome-wide layer of circadian clock regulation.
Asunto(s)
Ritmo Circadiano/genética , Regulación de la Expresión Génica/genética , MicroARNs/metabolismo , Proteínas Circadianas Period/metabolismo , Animales , Línea Celular Tumoral , Ritmo Circadiano/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Genómica , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Pulmón/metabolismo , Pulmón/efectos de la radiación , Ratones , MicroARNs/genética , Familia de Multigenes , Especificidad de Órganos , Proteínas Circadianas Period/genética , Retina/metabolismo , Retina/efectos de la radiación , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/efectos de la radiación , Factores de TiempoRESUMEN
Tetraspanins are an evolutionary conserved family of proteins involved in multiple aspects of cell physiology, including proliferation, migration and invasion, protein trafficking, and signal transduction; yet their detailed mechanism of action is unknown. Tetraspanins have no known natural ligands, but their engagement by antibodies has begun to reveal their role in cell biology. Studies of tetraspanin knockout mice and of germline mutations in humans have highlighted their role under normal and pathological conditions. Previously, we have shown that mice deficient in the tetraspanin CD81 developed fewer breast cancer metastases compared to their wild-type (WT) counterparts. Here, we show that a unique anti-human CD81 antibody (5A6) effectively halts invasion of triple-negative breast cancer (TNBC) cell lines. We demonstrate that 5A6 induces CD81 clustering at the cell membrane and we implicate JAM-A protein in the ability of this antibody to inhibit tumor cell invasion and migration. Furthermore, in a series of in vivo studies we demonstrate that this antibody inhibits metastases in xenograft models, as well as in syngeneic mice bearing a mouse tumor into which we knocked in the human CD81 epitope recognized by the 5A6 antibody.
Asunto(s)
Neoplasias de la Mama/patología , Tetraspanina 28/metabolismo , Animales , Anticuerpos/farmacología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Epítopos/metabolismo , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Invasividad Neoplásica , Metástasis de la Neoplasia , Receptores de Superficie Celular/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation.
Asunto(s)
Receptor de Asialoglicoproteína/metabolismo , Lisosomas/metabolismo , Acetilgalactosamina/metabolismo , Humanos , Células Tumorales CultivadasRESUMEN
Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.
Asunto(s)
Inmunoterapia/métodos , Melanoma Experimental/terapia , Neuraminidasa/inmunología , Polisacáridos/química , Receptor ErbB-2/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Aloinjertos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Humanos , Hidrólisis , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Inmunoconjugados/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Terapia Molecular Dirigida , Neuraminidasa/química , Neuraminidasa/genética , Polisacáridos/inmunología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Análisis de Supervivencia , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Despite prognostic grading and staging systems, it is a challenge to predict outcomes for patients with pancreatic neuroendocrine tumors (PanNETs). Sequencing studies of PanNETs have identified alterations in death domain-associated protein (DAXX) and alpha-thalassemia/mental retardation X-linked chromatin remodeler (ATRX). In tumors, mutations in DAXX or ATRX and corresponding loss of protein expression correlate with shorter times of disease-free survival and disease-specific survival of patients. However, DAXX or ATRX proteins were lost in only 50% of distant metastases analyzed. We performed whole-exome sequencing analyses of 20 distant metastases from 20 patients with a single nonsyndrome, nonfunctional PanNET. We found distant metastases contained alterations in multiple endocrine neoplasia type 1 (MEN1) (n = 8), ATRX (n = 5), DAXX (n = 5), TSC2 (n = 3), and DEP domain containing 5 (DEPDC5) (n = 3). We found copy number loss of cyclin dependent kinase inhibitor 2A (CDKN2A) in 15 metastases (75%) and alterations in genes that regulate chromatin remodeling, including set domain containing 2 (SETD2) (n = 4), AT-rich interaction domain 1A (ARID1A) (n = 2), chromodomain helicase DNA binding protein 8 (CHD8) (n = 2), and DNA methyl transferase 1 (DNMT1) (n = 2). In a separate analysis of 347 primary PanNETs, we found loss or deletion of DAXX and ATRX, disruption of SETD2 function (based on loss of H3 lysine 36 trimethylation), loss of ARID1A expression or deletions in CDKN2A in 81% of primary PanNETs with distant metastases. Among patients with loss or deletion of at least 1 of these proteins or genes, 39% survived disease-free for 5 years and 44% had disease-specific survival times of 10 years. Among patients without any of these alterations, 98% survived disease-free for 5 years and 95% had disease-specific survival times of 10 years. Therefore, primary PanNETs with loss of DAXX, ATRX, H3 lysine 36 trimethylation, ARID1A, and/or CDKN2A associate with shorter survival times of patients. Our findings indicate that alterations in chromatin-remodeling genes and CDKN2A contribute to metastasis of PanNETs.
Asunto(s)
Biomarcadores de Tumor/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Tumores Neuroendocrinos/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Adulto , Anciano , Anciano de 80 o más Años , Ensamble y Desensamble de Cromatina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Variaciones en el Número de Copia de ADN , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Mutación , Tumores Neuroendocrinos/mortalidad , Tumores Neuroendocrinos/patología , Tumores Neuroendocrinos/cirugía , Pancreatectomía , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Pronóstico , Secuenciación del ExomaRESUMEN
DNA mismatch repair protein deficient colon cancer frequently displays reduced CDX2 expression, and recent literature has suggested that negative CDX2 expression is a poor prognostic biomarker in colon cancer. We have recently demonstrated that SATB2 is an immunohistochemical marker that is complementary to CDX2. Using a tissue microarray approach, we evaluated SATB2 and CDX2 immunohistochemical expression in 514 patients with colonic adenocarcinoma including 146 with mismatch repair protein deficient tumors and correlated expression with histopathologic variables, molecular alterations, and survival. Overall, SATB2-negative and/or CDX2-negative expression was identified in 33% of mismatch repair protein deficient tumors compared with only 15% of mismatch repair protein proficient tumors (p < 0.001) and in 36% of BRAF V600E mutated compared with only 13% of BRAF wild-type tumors (p < 0.001). Both SATB2-negative and CDX2-negative colonic adenocarcinomas more often displayed lymphatic invasion, venous invasion, and perineural invasion (all with p < 0.05). SATB2-negative expression was also more frequently identified in tumors with mucinous or signet ring cell differentiation (p < 0.01 for both). In a multivariable analysis of survival in patients with mismatch repair protein deficient tumors (n = 131), only tumor stage (p = 0.01) and SATB2-negative and/or CDX2-negative expression (p = 0.009) independently predicted disease-specific survival. Of the 99 patients with stage II or III mismatch repair protein deficient tumors, death from disease only occurred in patients with either SATB2-negative or CDX2-negative tumors, and no patients with SATB2-positive/CDX2-positive tumors developed recurrence or died of disease. SATB2 and CDX2 expression had no effect on patient survival in mismatch repair protein proficient, BRAF-mutated, or KRAS-mutated tumors. In summary, our results suggest that SATB2 and CDX2 are prognostic biomarkers in patients with mismatch repair protein deficient colon cancer and that inclusion of SATB2 and CDX2 immunohistochemistry may be helpful as part of a comprehensive pathologic risk assessment in mismatch repair protein deficient colon cancer.
Asunto(s)
Adenocarcinoma/química , Biomarcadores de Tumor/análisis , Factor de Transcripción CDX2/análisis , Neoplasias del Colon/química , Reparación de la Incompatibilidad de ADN , Proteínas de Unión a la Región de Fijación a la Matriz/análisis , Factores de Transcripción/análisis , Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , California , Neoplasias del Colon/genética , Neoplasias del Colon/mortalidad , Neoplasias del Colon/cirugía , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Pennsylvania , Valor Predictivo de las Pruebas , Supervivencia sin Progresión , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Medición de Riesgo , Factores de Riesgo , Análisis de Matrices Tisulares , Adulto JovenRESUMEN
Gastric and esophageal cancers frequently show genomic instability and aneuploidy. Chromosomal copy number instability (CIN) is a form of genomic instability that exerts pleiotropic effects on cellular biology and is a source of genetic heterogeneity in a population of cells. CIN results in cell-to-cell variation in chromosome copy number which can be detected and quantified by fluorescence in situ hybridization (FISH). CIN is a biomarker associated with differential response to a number of chemotherapy compounds. We quantified chromosome 17 copy number instability (CIN-17) in 348 gastroesophageal adenocarcinomas by centromeric FISH in cases that were tested for HER2 amplification. We evaluated the association between CIN-17 and clinical outcome after surgical and nonsurgical treatment. CIN-17 was detected in 45.4% (158/348) and extreme CIN-17 in 28.4% (99/348). Extreme CIN-17 had no association with outcome in surgically treated patients. However, in patients treated with conventional radiation and/or chemotherapy, extreme CIN-17 was associated with 55% reduction in overall mortality (hazard ratio, 0.448; 95% confidence interval, 0.263-0.763) after adjusting for age and clinical stage at diagnosis. Extreme CIN-17 is detected in over a quarter of gastroesophageal adenocarcinomas and is a favorable prognostic marker in patients treated nonoperatively.
Asunto(s)
Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Inestabilidad Cromosómica , Cromosomas Humanos Par 17/genética , Neoplasias Esofágicas/genética , Neoplasias Gástricas/genética , Adenocarcinoma/patología , Anciano , Variaciones en el Número de Copia de ADN , Neoplasias Esofágicas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptor ErbB-2/genética , Neoplasias Gástricas/patologíaRESUMEN
AIMS: Pulmonary sarcomatoid carcinoma (PSC) is a poorly differentiated non-small-cell lung carcinoma (NSCLC) with aggressive behaviour. This study aimed to evaluate the prognostic clinicopathological and genetic characteristics of PSCs. METHODS AND RESULTS: Fifty-three cases of surgically treated PSCs were selected, 23 of which were subjected to mutation and copy number variation analysis using the 50-gene Ion AmpliSeq Cancer Panel. The majority of the patients were male (32 of 53, 60.3%) and smokers (51 of 53, 96.2%). Overall, 25 (47.1%) patients died within 2-105 months (mean = 22.7 months, median = 15 months) after diagnosis, and 28 were alive 3-141 months (mean = 38.7 months, median = 21.5 months) after diagnosis. Five-year overall survival was 12.5%. KRAS codon 12/13 mutation in adenocarcinomas (P = 0.01), age more than 70 years (P = 0.008) and tumour size ≥4.0 cm (P = 0.02) were associated strongly with worse outcome. TP53 (17 of 23, 74.0%) and KRAS codon 12 of 13 mutations (10 of 23, 43.4%) were the most common genetic alterations. Potentially actionable variants were identified including ATM (four of 23, 17.3%), MET, FBXW7 and EGFR (two of 23, 8.7%), AKT1, KIT, PDGFRA, HRAS, JAK3 and SMAD4 (one of 23, 4.3%). MET exon 14 skipping and missense mutations were identified in two (11.1%) cases with adenocarcinoma histology. Copy number analysis showed loss of RB1 (three of 23, 13%) and ATM (two of 23, 8.7%). Copy number gains were seen in EGFR (two of 23, 13.0%) and in one (4.3%) of each PIK3CA, KRAS, MET and STK11. CONCLUSIONS: Potentially targetable mutations can be identified in a subset of PSC, although most tumours harbour currently untargetable prognostically adverse TP53 and KRAS mutations.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
Sclerosing mucoepidermoid carcinoma with eosinophilia is a rare thyroid neoplasm of uncertain pathogenesis that resembles salivary gland mucoepidermoid carcinoma. This multi-institutional study characterizes the clinicopathologic and molecular features of this tumor by utilizing next-generation sequencing to assess common mutations and gene fusions involved in thyroid carcinogenesis as well as fluorescence in-situ hybridization for MAML2 translocations typical of salivary gland mucoepidermoid carcinoma. Nine cases (6 females and 3 males, mean age: 59 years, range 30-77 years) were identified. All cases were comprised of nests and strands of tumor cells with both squamous and mucinous differentiation embedded in a fibrohyaline stroma with an inflammatory infiltrate replete with eosinophils. All cases were p63 positive, thyroglobulin negative and showed variable expression of TTF-1. All nine cases were negative for MAML2 rearrangements. Five cases successfully tested by next-generation sequencing (ThyroSeq v.2 assay) were negative for mutations and translocations commonly involved in thyroid carcinogenesis. NTRK1 showed overexpression but no evidence of translocation. On follow-up, one patient died of persistent disease, whereas one of four remaining patients with available follow-up (mean: 7.3 years, range 4-11 years) demonstrated recurrence at 4 years. Thus, we show that sclerosing mucoepidermoid carcinoma with eosinophilia appears molecularly and morphologically distinct from follicular and C-cell-derived thyroid tumors as well as from salivary gland mucoepidermoid carcinoma. The overall and recurrence-free survival for these patients may be lower than for other well-differentiated thyroid cancers.
Asunto(s)
Carcinoma Mucoepidermoide/patología , Eosinofilia/patología , Fusión Génica , Glándula Tiroides/patología , Neoplasias de la Tiroides/patología , Adulto , Anciano , Carcinoma Mucoepidermoide/genética , Carcinoma Mucoepidermoide/metabolismo , Supervivencia sin Enfermedad , Eosinofilia/genética , Eosinofilia/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Translocación GenéticaRESUMEN
AIMS: The 2013 College of American Pathologists, the Association for Molecular Pathology and the International Association for the Study of Lung Cancer guideline for EGFR and ALK testing in lung carcinoma indicates that either the primary tumour or the metastasis is suitable for testing. The heterogeneity of gene mutations has been studied extensively, while similar reports on gene rearrangements are limited. The aim of this study was to determine if ALK status between primary tumour and matched metastasis differs. METHODS AND RESULTS: Fifteen ALK fluorescence in-situ hybridization (FISH) rearranged and 19 non-ALK FISH rearranged adenocarcinomas were collected retrospectively based on availability of tissue from a matched metastatic site. Sixty-eight samples were tested by ALK FISH (Vysis ALK break-apart FISH kit) and ALK immunohistochemistry (IHC) (Ventana ALK D5F3 CDx assay). Overall agreement of FISH and IHC was 88%, with IHC showing 100% specificity and 71% sensitivity. Concordance between primary site and metastasis by ALK FISH was seen in 30 cases (88%), and in 32 cases (94%) by ALK IHC. Five discordant cases were found (15%). Three ALK FISH discordant cases had low percentage of ALK FISH-positive tumour cells (average 23%, range: 18-31%) and all were negative by ALK IHC. One IHC discordant case had a high percentage of ALK FISH-positive tumour cells (67%), and was ALK IHC-negative. One FISH discordant case showed ALK FISH- and ALK IHC-positive primary tumour, but ALK FISH- and ALK IHC-negative metastasis. CONCLUSIONS: ALK FISH results show more frequent discordances between primary tumour and matched metastases than ALK IHC, due probably to technical challenges and sample quality. This observation indicates that the quality of sample and technical expertise of the laboratory should guide the decision about ALK testing in clinical practice.
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Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Técnicas de Diagnóstico Molecular/métodos , Metástasis de la Neoplasia/genética , Proteínas Tirosina Quinasas Receptoras/genética , Adulto , Anciano , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico , Femenino , Reordenamiento Génico , Humanos , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Masculino , Persona de Mediana Edad , Proteínas Tirosina Quinasas Receptoras/análisis , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: The authors hypothesized that histogenetic classification of salivary duct carcinoma (SDC) could account for de novo tumors and those with morphologic or molecular evidence (pleomorphic adenoma gene 1 [PLAG1], high-mobility group AT hook 2 [HMGA2] rearrangement, amplification) of pleomorphic adenoma (PA). METHODS: SDCs (n = 66) were reviewed for morphologic evidence of PA. PLAG1 and HMGA2 alterations were detected by fluorescence in situ hybridization (FISH). PLAG1-positive tumors were tested by FISH for fibroblast growth factor receptor 1 (FGFR1) rearrangement. Thirty-nine tumors were analyzed using a commercial panel for mutations and copy number variations in 50 cancer-related genes. RESULTS: On the basis of combined morphologic and molecular evidence of PA, 4 subsets of SDC emerged: 1) carcinomas with morphologic evidence of PA but intact PLAG1 and HMGA2 (n = 22); 2) carcinomas with PLAG1 alteration (n = 18) or 3) HMGA2 alteration (n = 12); and 4) de novo carcinomas, without morphologic or molecular evidence of PA (n = 14). The median disease-free survival was 37 months (95% confidence interval, 28.4-45.6 months). Disease-free survival and other clinicopathologic parameters did not differ for the subsets defined above. Combined Harvey rat sarcoma viral oncogene homolog/phosphatidylinositol-4,5-biphosphate 3-kinase, catalytic subunit α (HRAS/PIK3CA) mutations were observed predominantly in de novo carcinomas (5 of 8 vs 2 of 31 tumors; P = .035). Erb-B2 receptor tyrosine kinase 2 (ERBB2) copy number gain was not observed in de novo carcinomas (0 of 8 vs 12 of 31 tumors; P = .08). Tumor protein 53 (TP53) mutations were more common in SDC ex pleomorphic adenomas than in de novo carcinomas (17 of 31 vs 1 of 8 tumors; P = .033). CONCLUSIONS: The genetic profile of SDC varies with the absence or presence of pre-existing PA and its cytogenetic signature. Most de novo SDCs harbor combined HRAS/PIK3CA mutations and no ERBB2 amplification. Cancer 2016;122:3136-44. © 2016 American Cancer Society.
Asunto(s)
Adenoma Pleomórfico/diagnóstico , Biomarcadores de Tumor/genética , Carcinoma Ductal/diagnóstico , Proteínas de Unión al ADN/genética , Reordenamiento Génico , Variación Genética/genética , Proteína HMGA2/genética , Neoplasias de las Glándulas Salivales/diagnóstico , Adenoma Pleomórfico/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Ductal/clasificación , Carcinoma Ductal/genética , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Mutación , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Neoplasias de las Glándulas Salivales/clasificación , Neoplasias de las Glándulas Salivales/genética , Tasa de SupervivenciaRESUMEN
Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.
Asunto(s)
Mucinas , Neoplasias , Animales , Ratones , Mucinas/metabolismo , Péptido Hidrolasas/metabolismo , ProteolisisRESUMEN
Promoting immune activation within the tumor microenvironment (TME) is a promising therapeutic strategy to reverse tumor immunosuppression and elicit anti-tumor immunity. To enable tumor-localized immunotherapy following intravenous administration, we chemically conjugated a polyspecific integrin-binding peptide (PIP) to an immunostimulant (Toll-like receptor 9 [TLR9] agonist: CpG) to generate a tumor-targeted immunomodulatory agent, referred to as PIP-CpG. We demonstrate that systemic delivery of PIP-CpG induces tumor regression and enhances therapeutic efficacy compared with untargeted CpG in aggressive murine breast and pancreatic cancer models. Furthermore, PIP-CpG transforms the immune-suppressive TME dominated by myeloid-derived suppressor cells into a lymphocyte-rich TME infiltrated with activated CD8+ T cells, CD4+ T cells, and B cells. Finally, we show that T cells are required for therapeutic efficacy and that PIP-CpG treatment generates tumor-specific CD8+ T cells. These data demonstrate that conjugation to a synthetic tumor-targeted peptide can improve the efficacy of systemically administered immunostimulants and lead to durable anti-tumor immune responses.
Asunto(s)
Adyuvantes Inmunológicos , Neoplasias , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Linfocitos T CD8-positivos , Línea Celular Tumoral , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Microambiente TumoralRESUMEN
BACKGROUND: Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. METHODS: PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH, and phosphoproteomics. RESULTS: Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. CONCLUSIONS: Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Inmunoconjugados , Preparaciones Farmacéuticas , Anticuerpos Monoclonales Humanizados , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/tratamiento farmacológico , HumanosRESUMEN
V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA. Our anti-VISTA antibody (SG7) inhibits VISTA function and blocks purported interactions with both PSGL-1 and VSIG3 proteins. SG7 binds a unique epitope on the surface of VISTA, which partially overlaps with other clinically relevant antibodies. As a monotherapy, and to a greater extent as a combination with anti-PD1, SG7 slows tumor growth in multiple syngeneic mouse models. SG7 is a promising clinical candidate that can be tested in fully immunocompetent mouse models and its binding epitope can be used for future campaigns to develop species cross-reactive inhibitors of VISTA.
Asunto(s)
Anticuerpos/metabolismo , Antígenos B7/antagonistas & inhibidores , Epítopos/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Animales , Reacciones Antígeno-Anticuerpo , Antígenos B7/genética , Antígenos B7/inmunología , Sitios de Unión , Moléculas de Adhesión Celular/metabolismo , Técnicas de Visualización de Superficie Celular , Reacciones Cruzadas , Epítopos/genética , Femenino , Humanos , Inmunoglobulinas/metabolismo , Macaca fascicularis , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Moleculares , Unión Proteica , Ingeniería de ProteínasRESUMEN
SATB2 is a sensitive immunohistochemistry marker of colorectal carcinoma and non-neoplastic colorectal epithelium that is complementary to CDX2. However, its expression is affected by molecular alterations. Inflammatory bowel disease-associated neoplasia demonstrates molecular alterations that are different from those in sporadic colorectal neoplasia. Given these differences, we examined SATB2 expression in 73 cases of inflammatory bowel disease-associated neoplasia including 37 dysplasia cases and 36 carcinomas and compared the expression patterns with 50 cases of nondysplastic colorectal mucosa in patients with active inflammatory bowel disease, 40 sporadic colonic polyps (20 conventional adenomas and 20 sessile serrated lesions/polyps), and 343 sporadic colorectal adenocarcinomas to assess SATB2 immunohistochemistry as a biomarker of inflammatory bowel disease-associated neoplasia. Loss of SATB2 expression was only identified in colorectal dysplasia arising in inflammatory bowel disease (15/37, 41%) and was not seen in nondysplastic colorectal mucosa with active inflammatory bowel disease or sporadic colonic polyps (P<0.001). Loss of SATB2 expression was identified in both endoscopically visible dysplasia (11/28, 39%) and invisible (4/9, 44%) dysplasia. Loss of SATB2 expression was identified in 67% (24/36) of inflammatory bowel disease-associated carcinomas and was significantly more frequent compared with sporadic colorectal carcinomas (47/343, 14%, P<0.001). There was no difference in positive CDX2 expression between inflammatory bowel disease-associated colorectal carcinoma and sporadic colorectal carcinoma (89% vs. 85%, P=1.0). In conclusion, loss of SATB2 expression is common in inflammatory bowel disease-associated colorectal dysplasia and adenocarcinoma and may be a helpful ancillary biomarker when evaluating for inflammatory bowel disease-associated dysplasia.
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Adenocarcinoma/química , Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/química , Enfermedades Inflamatorias del Intestino/complicaciones , Proteínas de Unión a la Región de Fijación a la Matriz/análisis , Factores de Transcripción/análisis , Adenocarcinoma/etiología , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , Regulación hacia Abajo , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Análisis de Matrices TisularesRESUMEN
CONTEXT.: The separation of reactive from malignant mesothelial proliferations is often a difficult morphologic problem. There is contradictory information in the literature on whether methylthioadenosine phosphorylase (MTAP) immunohistochemistry can be used for this purpose. OBJECTIVE.: To determine the utility of MTAP immunohistochemistry in distinguishing reactive from malignant mesothelial proliferations. DESIGN.: We stained a tissue microarray containing 20 epithelioid malignant mesotheliomas and 17 reactive mesothelial proliferations. For the mesotheliomas, comparisons were made between MTAP staining and BRCA-associated nuclear protein 1 (BAP1) immunohistochemistry, cyclin-dependent kinase inhibitor 2A ( CDKN2A) fluorescence in situ hybridization, and neurofibromin 2 ( NF2) fluorescence in situ hybridization, which are established techniques for making this separation. RESULTS.: Loss of MTAP was seen in 0 of 17 reactive mesothelial proliferations and 13/20 (65%) malignant mesotheliomas. Almost all cases with loss showed loss in 100% of mesothelial cells. Background inflammatory and stromal cells served as a positive internal control. CDKN2A fluorescence in situ hybridization on the mesotheliomas showed concordance with MTAP staining in 14 of 17 evaluable cases. BAP1 immunohistochemistry showed loss of nuclear staining in 11 of 20 mesotheliomas (55%). No cases showed loss of NF2. A total of 18 of 20 mesotheliomas (90%) showed loss of either MTAP or BAP1. CONCLUSIONS.: In the context of a mesothelial proliferation, loss of MTAP staining is 100% specific for malignant mesothelioma. In this study the combination of MTAP and BAP1 immunohistochemical staining allowed separation of reactive from epithelial malignant mesothelial proliferations in 90% of cases.
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Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/diagnóstico , Mesotelioma/diagnóstico , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Células Epitelioides/metabolismo , Epitelio/metabolismo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Mesotelioma Maligno , Neurofibromatosis 2/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Análisis de Matrices Tisulares , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Background: Chronic idiopathic inflammatory bowel disease (IBD) is a significant risk factor for the development of intestinal adenocarcinoma. The underlying molecular alterations in IBD-associated intestinal adenocarcinoma remain largely unknown. Methods: We compared the clinicopathologic and molecular features of 35 patients with 47 IBD-associated intestinal adenocarcinomas with a consecutive series of 451 patients with sporadic colorectal carcinoma identified at our institution and published data on sporadic colorectal carcinoma. Results: c-MYC amplification was the most frequent molecular alteration identified in 33% of IBD-associated intestinal adenocarcinoma that is a significantly higher frequency than in sporadic colorectal carcinoma (8%) (P = 0.0001). Compared to sporadic colorectal carcinoma, IBD-associated intestinal adenocarcinomas more frequently demonstrated mucinous differentiation (60% vs 25%, P < 0.001) and signet ring cell differentiation (28% vs 4%, P < 0.001). Mucinous and signet ring cell differentiation were significantly associated with the presence of c-MYC amplification (both with P < 0.05). HER2 positivity (11%), KRAS exon 2 or 3 mutation (10%), and IDH1 mutation (7%) were less commonly observed in IBD-associated intestinal adenocarcinoma. There was an association between poor survival and HER2 status with 3 of 4 patients having HER2-positive adenocarcinoma dead of disease at last clinical follow-up; however, no statistically significant survival effect was identified for any of the molecular alterations identified. Conclusions: We demonstrate that IBD-associated intestinal adenocarcinomas have a high frequency of c-MYC amplification that is associated with mucinous and signet ring cell differentiation. Many of the identified molecular alterations have potential therapeutic relevance, including HER2 amplification, IDH1 mutation, and low frequency KRAS mutation.
Asunto(s)
Adenocarcinoma Mucinoso/genética , Carcinoma de Células en Anillo de Sello/genética , Neoplasias Colorrectales/genética , Enfermedades Inflamatorias del Intestino/genética , Inestabilidad de Microsatélites , Adenocarcinoma Mucinoso/patología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células en Anillo de Sello/patología , Diferenciación Celular/genética , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/complicaciones , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptor ErbB-2/genéticaRESUMEN
We hypothesized that there is a relationship between the preexisting pleomorphic adenoma [PA]), histologic grade of epithelial-myoepithelial carcinomas (EMCAs), and genetic alterations. EMCAs (n=39) were analyzed for morphologic and molecular evidence of preexisting PA (PLAG1, HMGA2 status by fluorescence in situ hybridization, FISH, and FGFR1-PLAG1 fusion by next-generation sequencing, NGS). Twenty-three EMCAs were further analyzed by NGS for mutations and copy number variation in 50 cancer-related genes. On the basis of combined morphologic and molecular evidence of PA, the following subsets of EMCA emerged: (a) EMCAs with morphologic evidence of preexisting PA, but intact PLAG1 and HMGA2 (12/39, 31%), (b) Carcinomas with PLAG1 alterations (9/39, 23%), or (c) HMGA2 alterations (10/39, 26%), and (d) de novo carcinomas, without morphologic or molecular evidence of PA (8/39, 21%). Twelve high-grade EMCAs (12/39, 31%) occurred across all subsets. The median disease-free survival was 80 months (95% confidence interval, 77-84 mo). Disease-free survival and other clinicopathologic parameters did not differ by the above defined subsets. HRAS mutations were more common in EMCAs with intact PLAG1 and HMGA2 (7/9 vs. 1/14, P<0.001). Other genetic abnormalities (TP53 [n=2], FBXW7 [n=1], SMARCB1 deletion [n=1]) were seen only in high-grade EMCAs with intact PLAG1 and HMGA2. We conclude that most EMCAs arose ex PA (31/39, 80%) and the genetic profile of EMCA varies with the absence or presence of preexisting PA and its cytogenetic signature. Progression to higher grade EMCA with intact PLAG1 and HMGA2 correlates with the presence of TP53, FBXW7 mutations, or SMARCB1 deletion.