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Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.
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Polaridad Celular , Quimiocina CXCL9 , Neoplasias de Cabeza y Cuello , Macrófagos , Osteopontina , Carcinoma de Células Escamosas de Cabeza y Cuello , Microambiente Tumoral , Humanos , Quimiocina CXCL9/análisis , Quimiocina CXCL9/metabolismo , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/patología , Macrófagos/inmunología , Osteopontina/análisis , Osteopontina/metabolismo , Pronóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Polaridad Celular/inmunologíaRESUMEN
PURPOSE: The efficiency of the Myriad Homologous Recombination Deficiency (HRD) test to guide the use of poly (ADP-ribose) polymerase (PARP) inhibitors has been demonstrated in several phase III trials. However, a need exists for alternative clinically validated tests. METHODS: A novel biomarker for HRD was developed using The Cancer Genome Atlas database and, as part of the ENGOT HRD European Initiative, applied to 469 samples from the PAOLA-1/ENGOT-ov25 trial. Results were compared with the Myriad myChoice Genomic Instability Score (GIS) with respect to the progression-free survival in the olaparib + bevacizumab and placebo + bevacizumab arms. RESULTS: Analysis of the TCGA cohort revealed that a normalization of the number of large-scale state transitions by the number of whole-genome doubling events allows a better separation and classification of HRD samples than the GIS. Analysis of the PAOLA-1 samples, using the Geneva test (OncoScan + nLST), yielded a lower failure rate (27 of 469 v 59 of 469) and a hazard ratio of 0.40 (95% CI, 0.28 to 0.57) compared with 0.37 for Myriad myChoice (BRCAm or GIS+) in the nLST-positive samples. In patients with BRCAwt, the Geneva test identified a novel subpopulation of patients, with a favorable 1-year PFS (85%) but a poor 2-year PFS (30%) on olaparib + bevacizumab treatment. CONCLUSION: The proposed test efficiently separates HRD-positive from HRD-negative patients, predicts response to PARP inhibition, and can be easily deployed in a clinical laboratory for routine practice. The performance is similar to the available commercial test, but its lower failure rate allows an increase in the number of patients who will receive a conclusive laboratory result.
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Carcinoma , Neoplasias Ováricas , Femenino , Humanos , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Biomarcadores , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Recombinación HomólogaRESUMEN
FOLFOXIRI, i.e., the combination of folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan, is a first-line treatment for colorectal carcinoma (CRC), yet non-personalized and aggressive. In this study, to mimic the clinical situation of patients diagnosed with advanced CRC and exposed to a chronic treatment with FOLFOXIRI, we have generated the CRC cell clones chronically treated with FOLFOXIRI. A significant loss in sensitivity to FOLFOXIRI was obtained in all four cell lines, compared to their treatment-naïve calls, as shown in 2D cultures and heterotypic 3D co-cultures. Acquired drug resistance induction was observed through morphometric changes in terms of the organization of the actin filament. Bulk RNA sequencing revealed important upregulation of glucose transporter family 5 (GLUT5) in SW620 resistant cell line, while in the LS174T-resistant cell line, a significant downregulation of protein tyrosine phosphatase receptor S (PTPRS) and oxoglutarate dehydrogenase-like gene (OGDHL). This acquired resistance to FOLFOXIRI was overcome with optimized low-dose synergistic drug combinations (ODCs) acting via the Ras-Raf-MEK-ERK pathway. The ODCs inhibited the cell metabolic activity in SW620 and LS174T 3Dcc, respectively by up to 82%.
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The role of the proangiogenic factor olfactomedin-like 3 (OLFML3) in cancer is unclear. To characterize OLFML3 expression in human cancer and its role during tumor development, we undertook tissue expression studies, gene expression analyses of patient tumor samples, in vivo studies in mouse cancer models, and in vitro coculture experiments. OLFML3 was expressed at high levels, mainly in blood vessels, in multiple human cancers. We focused on colorectal cancer (CRC), as elevated expression of OLFML3 mRNA correlated with shorter relapse-free survival, higher tumor grade, and angiogenic microsatellite stable consensus molecular subtype 4 (CMS4). Treatment of multiple in vivo tumor models with OLFML3-blocking antibodies and deletion of the Olfml3 gene from mice decreased lymphangiogenesis, pericyte coverage, and tumor growth. Antibody-mediated blockade of OLFML3 and deletion of host Olfml3 decreased the recruitment of tumor-promoting tumor-associated macrophages and increased infiltration of the tumor microenvironment by NKT cells. Importantly, targeting OLFML3 increased the antitumor efficacy of anti-PD-1 checkpoint inhibitor therapy. Taken together, the results demonstrate that OLFML3 is a promising candidate therapeutic target for CRC.
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Epithelial ovarian cancer (EOC) is usually diagnosed at an advanced stage and significantly contributes to cancer mortality in women. Despite multimodal treatment associating chemotherapy and surgery, most patients ultimately progress and require palliative systemic therapy. In EOC, the efficacy of anti-HER2 agents is minimal even after selecting patients for HER2 expression. ERBB2 gene amplification is observed in 3-10% of patients, depending on the specific method of detection and cutoffs. We report the case of a young woman with a FIGO stage IV high-grade serous ovarian cancer with an amplification of ERBB2. She was treated with the association of trastuzumab - pertuzumab after two lines of standard treatment and presented an excellent long-lasting partial response after 36 months of treatment. The association of trastuzumab and pertuzumab, without chemotherapy, has not been previously tested in this context and could be more efficacious than monotherapy with either agent. In addition, the significant benefit observed in this case could be attributed to the presence of a high-level focal amplification that is relatively rare and probably more specific than an increase in HER2 expression. In conclusion, prospective trials of the trastuzumab and pertuzumab combination should be considered in an appropriately selected EOC patient population.
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BACKGROUND: Metastatic carcinoma of a renal allograft is a rare but life threatening event with a difficult clinical management. Recent reports suggested a potential role of BK polyomavirus (BKPyV) in the development of urologic tract malignancies in kidney transplant recipients. METHODS: We investigated a kidney-pancreas female recipient with an history of BKPyV nephritis who developed a rapidly progressive and widely metastatic donor-derived renal carcinoma 9 years after transplantation. RESULTS: Histology and fluorescence in situ hybridization analysis revealed a donor-derived (XY tumor cells) collecting (Bellini) duct carcinoma. The presence of BKPyV oncogenic large tumor antigen was identified in large amount within the kidney tumor and the bowel metastases. Whole genome sequencing of the tumor confirmed multiple genome BKPyV integrations. The transplanted kidney was removed, immunosuppression was withdrawn, and recombinant interleukin-2 (IL-2) was administered for 3 months, inducing a complete tumor clearance, with no evidence of disease at 6-year follow-up. The immunological profiling during IL-2 therapy revealed the presence of donor-specific T cells and expanded cytokine-producing bright natural killer cells but no donor-specific antibodies. Finally, we found persistently elevated anti-BK virus IgG titers and a specific anti-BKPyV T cell response. CONCLUSIONS: This investigation showed evidence for the potential oncogenic role of BKPyV in collecting duct carcinoma in renal allografts and demonstrated that immunosuppression withdrawal and IL-2 therapy can lead to an efficient antitumor cellular mediated rejection possibly via 3 distinct mechanisms including (1) host-versus-graft, (2) host-versus-tumor, and (3) anti-BKPyV responses.
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Antineoplásicos/uso terapéutico , Virus BK/patogenicidad , Carcinoma de Células Renales/terapia , Interleucina-2/uso terapéutico , Neoplasias Renales/terapia , Trasplante de Riñón/efectos adversos , Nefrectomía , Trasplante de Páncreas/efectos adversos , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología , Adulto , Virus BK/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/secundario , Carcinoma de Células Renales/virología , Quimioterapia Adyuvante , Femenino , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/efectos adversos , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Neoplasias Renales/virología , Infecciones por Polyomavirus/complicaciones , Infecciones por Polyomavirus/inmunología , Inducción de Remisión , Resultado del Tratamiento , Infecciones Tumorales por Virus/complicaciones , Infecciones Tumorales por Virus/inmunologíaRESUMEN
The current standard of care for colorectal cancer (CRC) is a combination of chemotherapeutics, often supplemented with targeted biological drugs. An urgent need exists for improved drug efficacy and minimized side effects, especially at late-stage disease. We employed the phenotypically driven therapeutically guided multidrug optimization (TGMO) technology to identify optimized drug combinations (ODCs) in CRC. We identified low-dose synergistic and selective ODCs for a panel of six human CRC cell lines also active in heterotypic 3D co-culture models. Transcriptome sequencing and phosphoproteome analyses showed that the mechanisms of action of these ODCs converged toward MAP kinase signaling and cell cycle inhibition. Two cell-specific ODCs were translated to in vivo mouse models. The ODCs reduced tumor growth by ~80%, outperforming standard chemotherapy (FOLFOX). No toxicity was observed for the ODCs, while significant side effects were induced in the group treated with FOLFOX therapy. Identified ODCs demonstrated significantly enhanced bioavailability of the individual components. Finally, ODCs were also active in primary cells from CRC patient tumor tissues. Taken together, we show that the TGMO technology efficiently identifies selective and potent low-dose drug combinations, optimized regardless of tumor mutation status, outperforming conventional chemotherapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Relación Dosis-Respuesta a Droga , Femenino , Células HCT116 , Humanos , Masculino , Ratones , Transcriptoma/efectos de los fármacos , Resultado del TratamientoAsunto(s)
COVID-19 , Colecistitis Aguda , Vesícula Biliar , SARS-CoV-2/aislamiento & purificación , Anciano de 80 o más Años , COVID-19/diagnóstico , COVID-19/epidemiología , COVID-19/fisiopatología , COVID-19/terapia , Colecistectomía/métodos , Colecistitis Aguda/sangre , Colecistitis Aguda/fisiopatología , Colecistitis Aguda/terapia , Colecistitis Aguda/virología , Deterioro Clínico , Comorbilidad , Diagnóstico Diferencial , Resultado Fatal , Femenino , Vesícula Biliar/diagnóstico por imagen , Vesícula Biliar/cirugía , Vesícula Biliar/virología , Humanos , Pruebas de Función Hepática/métodos , Masculino , Enfermedades no Transmisibles/epidemiología , Manejo de Atención al Paciente/métodos , ARN Viral/aislamiento & purificación , Ultrasonografía/métodosRESUMEN
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, characterized by a high degree of intertumoral heterogeneity. However, a common feature of the GBM microenvironment is hypoxia, which can promote radio- and chemotherapy resistance, immunosuppression, angiogenesis, and stemness. We experimentally defined common GBM adaptations to physiologically relevant oxygen gradients, and we assessed their modulation by the metabolic drug metformin. We directly exposed human GBM cell lines to hypoxia (1% O2) and to physioxia (5% O2). We then performed transcriptional profiling and compared our in vitro findings to predicted hypoxic areas in vivo using in silico analyses. We observed a heterogenous hypoxia response, but also a common gene signature that was induced by a physiologically relevant change in oxygenation from 5% O2 to 1% O2. In silico analyses showed that this hypoxia signature was highly correlated with a perinecrotic localization in GBM tumors, expression of certain glycolytic and immune-related genes, and poor prognosis of GBM patients. Metformin treatment of GBM cell lines under hypoxia and physioxia reduced viable cell number, oxygen consumption rate, and partially reversed the hypoxia gene signature, supporting further exploration of targeting tumor metabolism as a treatment component for hypoxic GBM.
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Liquid biopsy, the analysis of circulating tumor DNA (ctDNA), is a promising tool in oncology, especially in personalized medicine. Although its main applications currently focus on selection and adjustment of therapy, ctDNA may also be used to monitor residual disease, establish prognosis, detect relapses, and possibly screen at-risk individuals. CtDNA represents a small and variable proportion of circulating cell-free DNA (ccfDNA) which is itself present at a low concentration in normal individuals and so analyzing ctDNA is technically challenging. Various commercial systems have recently appeared on the market, but it remains difficult for practitioners to compare their performance and to determine whether they yield comparable results. As a first step toward establishing national guidelines for ctDNA analyses, four laboratories in Switzerland joined a comparative exercise to assess ccfDNA extraction and ctDNA analysis by sequencing. Extraction was performed using six distinct methods and yielded ccfDNA of equally high quality, suitable for sequencing. Sequencing of synthetic samples containing predefined amounts of eight mutations was performed on three different systems, with similar results. In all four laboratories, mutations were easily identified down to 1% allele frequency, whereas detection at 0.1% proved challenging. Linearity was excellent in all cases and while molecular yield was superior with one system this did not impact on sensitivity. This study also led to several additional conclusions: First, national guidelines should concentrate on principles of good laboratory practice rather than recommend a particular system. Second, it is essential that laboratories thoroughly validate every aspect of extraction and sequencing, in particular with respect to initial amount of DNA and average sequencing depth. Finally, as software proved critical for mutation detection, laboratories should validate the performance of variant callers and underlying algorithms with respect to various types of mutations.
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ADN Tumoral Circulante/aislamiento & purificación , Análisis Mutacional de ADN , Biopsia Líquida/estadística & datos numéricos , Humanos , Laboratorios/estadística & datos numéricosRESUMEN
Alterations of the Retinoblastoma (Rb) pathway are frequent in ovarian cancer, typically resulting from CDKN2A down-regulation, CCNE1 amplification, CCND1/2 amplification, and RB1 loss. However, bi-allelic CDKN2A mutation or homozygous deletion is a very rare event, concerning less than 5% of patients.Initial trials with palbociclib in serous ovarian cancer have shown very modest benefit in unselected patient populations, thus underlining the need for a biomarker predicting response. We report the case of a heavily pre-treated patient with a serous ovarian tumor harboring a homozygous deletion of the CDKN2A gene that derived significant, prolonged clinical benefit from palbociclib, a CDK4/6 oral inhibitor, with letrozole. Treatment with palbociclib and letrozole started on February 2018, with an ongoing response after 12 months.In conclusion, homozygous CDKN2A deletion is rare and could be used to predict response to CDK4/6 inhibitors in association with other genomic features. We encourage further trials in this direction.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Cistadenocarcinoma Seroso/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Ciclina E/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Femenino , Amplificación de Genes , Humanos , Letrozol/administración & dosificación , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Proteínas Oncogénicas/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Piperazinas/administración & dosificación , Pronóstico , Piridinas/administración & dosificación , Proteínas de Unión a Retinoblastoma/genética , Eliminación de Secuencia , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Pleomorphic hyalinizing angiectatic tumor (PHAT) is a rare mesenchymal tumor of intermediate malignancy. PHAT, and the related hemosiderotic fibrolipomatous tumor, show a recurrent t(1;10)(p22;q24). Fluorescence in situ hybridization (FISH) and BAC (bacterial artificial chromosome) clones have previously identified TGFBR3 and MGEA5 as fusion partners. However, targeted RNA-sequencing allowed for the correct identification of FBXW4 and not MGEA5 as the fusion partner of TGFBR3 in a subcutaneous PHAT, a finding further confirmed by RT-PCR. FBXW4 and MGEA5 share a common cytogenetic location at 10q24.32, thereby suggesting that the use of less precise technology may have led to inaccurate gene identification. The study of additional cases is however required.
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Antígenos de Neoplasias/genética , Histona Acetiltransferasas/genética , Hialuronoglucosaminidasa/genética , Proteoglicanos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Sarcoma/genética , Análisis de Secuencia de ARN/métodos , Neoplasias de los Tejidos Blandos/genética , Proteínas F-Box/genética , Humanos , Masculino , Persona de Mediana Edad , Fusión de Oncogenes , Sarcoma/patología , Neoplasias de los Tejidos Blandos/patologíaRESUMEN
Metastases and tumor recurrence have a major prognostic impact in head and neck squamous cell carcinoma (HNSCC); however, cellular models that comprehensively characterize metastatic and recurrent HNSCC are lacking. To this end, we obtained genomic, transcriptomic, and copy number profiles of the UM-SCC cell line panel, encompassing patient-matched metastatic and recurrent cells. UM-SCC cells recapitulate the most prevalent genomic alterations described in HNSCC, featuring common TP53, PI3K, NOTCH, and Hippo pathway mutations. This analysis identified a novel F977Y kinase domain PIK3CA mutation exclusively present in a recurrent cell line (UM-SCC14B), potentially conferring resistance to PI3K inhibitors. Small proline-rich protein 2A (SPRR2A), a protein involved in epithelial homeostasis and invasion, was one of the most consistently downregulated transcripts in metastatic and recurrent UM-SCC cells. Assessment of SPRR2A protein expression in a clinical cohort of patients with HNSCC confirmed common SPRR2A downregulation in primary tumors (61.9% of cases) and lymph node metastases (31.3%), but not in normal tissue. High expression of SPRR2A in lymph node metastases was, along with nonoropharyngeal location of the primary tumor, an independent prognostic factor for regional disease recurrence after surgery and radiotherapy (HR 2.81; 95% CI, 1.16-6.79; P = 0.02). These results suggest that SPRR2A plays a dual role in invasion and therapeutic resistance in HNSCC, respectively through its downregulation and overexpression. IMPLICATIONS: The current study reveals translationally relevant mechanisms underlying metastasis and recurrence in HNSCC and represents an adjuvant tool for preclinical research in this disease setting. Underlining its discovery potential this approach identified a PIK3CA-resistant mutation as well as SPRR2A as possible theragnostic markers.
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Fosfatidilinositol 3-Quinasa Clase I/genética , Proteínas Ricas en Prolina del Estrato Córneo/genética , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Neoplasias de Cabeza y Cuello/genética , Recurrencia Local de Neoplasia/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/química , Regulación hacia Abajo , Resistencia a Antineoplásicos , Femenino , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Masculino , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Dominios Proteicos , Análisis de Secuencia de ARN , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Secuenciación del ExomaRESUMEN
Isocitrate Dehydrogenase-1 (IDH1) is a driver gene in several cancers including brain tumors such as low-grade and high-grade gliomas. Mutations of SMARCB1 were described in atypical teratoid rhabdoid tumors and to date have not been associated with the pathogenesis of medulloblastoma. We report concurrent IDH1 and SMARCB1 mutations in a medulloblastoma patient. We searched the catalog of somatic mutations in cancer (COSMIC) database and other mutation databases and -to our knowledge- this is the first reported case of medulloblastoma harboring both mutations together. Our patient is a 13-year-old male presenting with headache and vomiting at diagnosis. MRI revealed left cerebellar expansive lesion with no evidence of metastasis. A histopathological diagnosis of desmoplastic/nodular medulloblastoma was made after complete resection of the tumor. Immunophenotypic characterization and methylation profiling suggested a medulloblastoma with SHH activation. Next generation sequencing of a panel of 400 genes revealed heterozygous somatic IDH1(p.R132C), SMARCB1(p.R201Q), and CDH11(p.L625T) mutations. The patient was treated according to the HIT-SIOP PNET 4 protocol. He is in complete remission more than 2 years after diagnosis. In conclusion, increasing use of high throughput sequencing will certainly increase the frequency with which rare mutations or mutation combinations are identified. The exact frequency of this mutation combination and whether it has any particular therapeutic implications or prognostic relevance requires further investigation.
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PLEKHA7 is a junctional protein, which participates in a complex that stabilizes E-cadherin at the zonula adhaerens. Since E-cadherin is involved in epithelial morphogenesis, signaling, and tumor progression, we explored PLEKHA7 expression in cancer. PLEKHA7 expression was assessed in invasive ductal and lobular carcinomas of the breast by immunohistochemistry, immunofluorescence and quantitative RT-PCR. PLEKHA7 was detected at epithelial junctions of normal mammary ducts and lobules, and of tubular and micropapillary structures within G1 and G2 ductal carcinomas. At these junctions, the localization of PLEKHA7 was along the circumferential belt (zonula adhaerens), and only partially overlapping with that of E-cadherin, p120ctn and ZO-1, as shown previously in rodent tissues. PLEKHA7 immunolabeling was strongly decreased in G3 ductal carcinomas and undetectable in lobular carcinomas. PLEKHA7 mRNA was detected in both ductal and lobular carcinomas, with no observed correlation between mRNA levels and tumor type or grade. In summary, PLEKHA7 is a junctional marker of epithelial cells within tubular structures both in normal breast tissue and ductal carcinomas, and since PLEKHA7 protein but not mRNA expression is strongly decreased or lost in high grade ductal carcinomas and in lobular carcinomas, loss of PLEKHA7 is a newly characterized feature of these carcinomas.
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Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Proteínas Portadoras/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/patología , Línea Celular Tumoral , Femenino , HumanosRESUMEN
The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.
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Neoplasias de la Mama/metabolismo , Proteómica/métodos , Receptor ErbB-2/metabolismo , Femenino , Formaldehído , Humanos , Hibridación Fluorescente in Situ , Espectrometría de Masas , Adhesión en Parafina , Péptidos/metabolismo , Fijación del TejidoRESUMEN
BACKGROUND: Atherosclerosis is a chronic inflammatory disease of large- to medium-sized arteries and is the main underlying cause of death worldwide. The lymphatic vasculature is critical for processes that are intimately linked to atherogenesis such as the immune response and cholesterol metabolism. However, whether lymphatic vessels truly contribute to the pathogenesis of atherosclerosis is less clear despite increasing research efforts in this field. DESIGN: PubMed and Ovid MEDLINE databases were searched. In addition, key review articles were screened for relevant original publications. RESULTS: Current knowledge about lymphatic vessels in the arterial wall came from studies that examined the presence and location of such vessels in human atherosclerotic plaque specimens, as well as in a variety of arteries in animal models for atherosclerosis (e.g. rabbits, dogs, rats and mice). Generally, three experimental approaches have been used to investigate the functional role of plaque-associated lymphatic vessels; experimental lymphostasis was used to investigate lymphatic drainage of the arterial wall, and more recently, studies with genetic interventions and/or surgical transplantation have been performed. CONCLUSIONS: Lymphatic vessels seem to be mostly present in the adventitial layer of the arterial walls of animals and humans. They are involved in reverse cholesterol transport from atherosclerotic lesions, and arteries with a dense lymphatic network seem naturally protected against atherosclerosis. Lymphangiogenesis is a process that is an important part of the inflammatory loop in atherosclerosis. However, how augmenting or impeding the distribution of lymphatic vessels impacts disease progression remains to be investigated in future studies.
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Aterosclerosis/etiología , Vasos Linfáticos/fisiología , Placa Aterosclerótica/etiología , Animales , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Progresión de la Enfermedad , Perros , Humanos , Linfangiogénesis/fisiología , Ratones , Microvasos/fisiología , Placa Aterosclerótica/patología , Placa Aterosclerótica/fisiopatología , Conejos , RatasRESUMEN
BACKGROUND: The distinction between adenoid cystic carcinoma (ACC) and pleomorphic adenoma (PA) on fine-needle aspiration biopsy (FNAB) can be challenging. Recently, a specific translocation t(6;9) involving the v-myb avian myeloblastosis viral oncogene homolog (MYB) and nuclear factor I/B (NFIB) genes was identified in ACCs, in which it contributes to MYB overexpression. The authors investigated the use of MYB immunocytochemistry in FNAB specimens as an ancillary test for the cytologic diagnosis of ACC. METHODS: The expression of MYB was assessed in alcohol-fixed cytologic smears of histologically confirmed ACCs (n = 20) and PAs (n = 20) using immunocytochemistry. The corresponding ACCs and PAs from formalin-fixed, paraffin embedded surgical resection specimens also were stained immunohistochemically for MYB. The nuclear expression of MYB was assessed semiquantitatively using a scoring system (from 0 to 6) that combined the proportion and the intensity of staining. A score ≥4 was considered positive, and a score ≤3 was considered negative. RESULTS: On FNAB material, 80% of ACCs (N = 16 of 20) were immunocytochemically positive for MYB. In contrast, all PAs (N = 20 of 20) were negative for MYB (P < .0001). The sensitivity of MYB on FNAB was 80%, and the specificity was 100% relative to PA. Results on corresponding histology were similar to the cytology results; however, there was often a zonal staining pattern with central areas of tumor (range, 20%-90%) that lacked immunoreactivity, thus suggesting that the immunoreactivity was maintained better in alcohol-fixed FNAB material. CONCLUSIONS: In contrast to PAs, a majority of ACCs are immunocytochemically positive for MYB. The test is more effective using alcohol-fixed FNAB material and is potentially useful for the cytologic distinction of ACC and PA.
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Adenoma Pleomórfico/patología , Carcinoma Adenoide Quístico/patología , Regulación Neoplásica de la Expresión Génica , Genes myb/genética , Neoplasias de las Glándulas Salivales/patología , Adenoma Pleomórfico/diagnóstico , Adenoma Pleomórfico/genética , Biopsia con Aguja Fina , Carcinoma Adenoide Quístico/diagnóstico , Carcinoma Adenoide Quístico/genética , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Adhesión en Parafina , Neoplasias de las Glándulas Salivales/diagnóstico , Neoplasias de las Glándulas Salivales/genéticaRESUMEN
Proteomic analysis of tissues has advanced in recent years as instruments and methodologies have evolved. The ability to retrieve peptides from formalin-fixed paraffin-embedded tissues followed by shotgun or targeted proteomic analysis is offering new opportunities in biomedical research. In particular, access to large collections of clinically annotated samples should enable the detailed analysis of pathologically relevant tissues in a manner previously considered unfeasible. In this paper, we review the current status of proteomic analysis of formalin-fixed paraffin-embedded tissues with a particular focus on targeted approaches and the potential for this technique to be used in clinical research and clinical diagnosis. We also discuss the limitations and perspectives of the technique, particularly with regard to application in clinical diagnosis and drug discovery.