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AIMS: A robust immunohistochemistry (IHC) assay was developed to detect lymphocyte-activation gene 3 (LAG-3) expression by immune cells (ICs) in tumour tissues. LAG-3 is an immuno-oncology target with demonstrable clinical benefit, and there is a need for a standardised, well-characterised assay to measure its expression. This study aims to describe LAG-3 scoring criteria and present the specificity, sensitivity, analytical precision and reproducibility of this assay. METHODS: The specificity of the assay was investigated by antigen competition and with LAG3 knockout cell lines. A melanin pigment removal procedure was implemented to prevent melanin interference in IHC interpretation. Formalin-fixed paraffin-embedded (FFPE) human melanoma samples with a range of LAG-3 expression levels were used to assess the sensitivity and analytical precision of the assay with a ≥1% cut-off to determine LAG-3 positivity. Interobserver and intraobserver reproducibility were evaluated with 60 samples in intralaboratory studies and 70 samples in interlaboratory studies. RESULTS: The LAG-3 IHC method demonstrated performance suitable for analysis of LAG-3 IC expression in clinical melanoma samples. The pretreatment step effectively removed melanin pigment that could interfere with interpretation. LAG-3 antigen competition and analysis of LAG3 knockout cell lines indicated that the 17B4 antibody clone binds specifically to LAG-3. The intrarun repeatability, interday, interinstrument, interoperator and inter-reagent lot reproducibility demonstrated a high scoring concordance (>95%). The interobserver and intraobserver reproducibility and overall interlaboratory and intralaboratory reproducibility also showed high scoring concordance (>90%). CONCLUSIONS: We have demonstrated that the assay reliably assesses LAG-3 expression in FFPE human melanoma samples by IHC.
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Melaninas , Melanoma , Humanos , Inmunohistoquímica , Reproducibilidad de los Resultados , Melanoma/diagnóstico , Melanoma/genética , Melanoma/patologíaRESUMEN
OBJECTIVES: The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment. METHODS: The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms. RESULTS: Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed. CONCLUSIONS: mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force.
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Técnica del Anticuerpo Fluorescente/métodos , Inmunohistoquímica/métodos , Inmunoterapia/métodos , Coloración y Etiquetado/métodos , Microambiente Tumoral/fisiología , HumanosRESUMEN
The premise of this book is the importance of the tumor microenvironment (TME). Until recently, most research on and clinical attention to cancer biology, diagnosis, and prognosis were focused on the malignant (or premalignant) cellular compartment that could be readily appreciated using standard morphology-based imaging.
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Neoplasias/diagnóstico por imagen , Microambiente Tumoral , HumanosRESUMEN
The potential clinical impact of KRAS and epidermal growth factor receptor (EGFR) mutations has been investigated in lung adenocarcinomas; however, their prognostic value remains controversial. In our study, we sought to investigate the prognostic significance of driver mutations using a large cohort of early-stage lung adenocarcinomas. We reviewed patients with pathologic early-stage, lymph node-negative, solitary lung adenocarcinoma who had undergone surgical resection (1995 to 2005; stage I/II=463/19). Tumors were classified according to the IASLC/ATS/ERS classification and genotyped by Sequenom MassARRAY system and polymerase chain reaction-based assays. In stage I disease, the Kaplan-Meier method and cumulative incidence of recurrence analyses were used to estimate the probability of overall survival (OS) and recurrence, respectively. Of all, 129 (27%) patients had mutations in KRAS, 86 (18%) in EGFR, 8 (2%) in BRAF, 8 (2%) in PIK3CA, 4 (1%) in NRAS, and 1 (0.2%) in AKT1. EGFR L858R mutation correlated with lepidic predominant histology (P=0.006), whereas exon 19 deletion correlated with acinar predominant histology (P<0.001). EGFR mutations were not detected in invasive mucinous adenocarcinomas (P=0.033). The 5-year OS of patients with KRAS-mutant tumors was significantly worse (n=124; 5-year OS, 63%) than those with KRAS wild-type (n=339; 77%; P<0.001). In solid predominant tumors, KRAS mutations correlated with worse OS (P=0.008) and increased risk of recurrence (P=0.005). On multivariate analysis, KRAS mutation was an independent prognosticator of OS in all patients (hazard ratio, 1.87; P<0.001) and recurrence in solid predominant tumors (hazard ratio, 4.73; P=0.012). In patients with resected stage I lung adenocarcinomas, KRAS mutation was an independent prognostic factor for OS and recurrence, especially in solid predominant tumors.
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Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Adenocarcinoma del Pulmón , Adulto , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Receptores ErbB/genética , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/mortalidad , Estadificación de Neoplasias , Neumonectomía , Pronóstico , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
PURPOSE: In patients with chronic myelogenous leukemia (CML), point mutations in the BCR-ABL1 kinase domain are the most common cause of treatment failure with a tyrosine kinase inhibitor (TKI). It is not clear whether the splice variant BCR-ABL1(35INS) is also associated with treatment failure. PATIENTS AND METHODS: We reviewed all CML patients who had BCR-ABL1 kinase mutation analysis performed between August 1, 2007, and January 15, 2014. Patients who had BCR-ABL1(35INS) detected had their medical records reviewed to determine response to TKI therapy. RESULTS: Two hundred and eighty four patients had kinase mutation testing performed; of these, 64 patients (23%) had BCR-ABL1(35INS) detected. Forty-five patients were in chronic phase (70%), 10 were in accelerated phase (16%), 6 were in blastic phase (9%), and 3 were in other settings (5%). Of the 34 chronic phase patients who began therapy with imatinib, 23 patients (68%) failed therapy: 8 patients (24%) had primary refractory disease, 11 patients (32%) progressed, and 4 patients (12%) had disease progression after dose interruption. In contrast to the patients with disease progression or lack of response, none of 23 patients who were responding to imatinib had BCR-ABL1(35INS) detected. DNA sequencing of commonly mutated spliceosomal genes SF3B1, U2AF1, SRSF2, ZRSR2, SFA31, PRPF408, U2A565, and SF1 did not reveal mutations in seven BCR-ABL1(35INS) -positive patients tested. CONCLUSIONS: The splice variant BCR-ABL1(35INS) is frequently found in patients who are resistant to imatinib. Mutations in the commonly mutated spliceosomal proteins do not contribute to this association.
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Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Empalme del ARN/genética , Adolescente , Adulto , Anciano , Femenino , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Masculino , Persona de Mediana Edad , Mutagénesis Insercional , Estudios Retrospectivos , Análisis de Secuencia de ADN , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
Amplicon-based methods for targeted resequencing of cancer genes have gained traction in the clinic as a strategy for molecular diagnostic testing. An 847-amplicon panel was designed with the RainDance DeepSeq system, covering most exons of 28 genes relevant to acute myeloid leukemia and myeloproliferative neoplasms. We developed a paired-sample analysis pipeline for variant calling and sought to assess its sensitivity and specificity relative to a set of samples with previously identified mutations. Thirty samples with known mutations in JAK2, NPM1, DNMT3A, MPL, IDH1, IDH2, CEBPA, and FLT3, were profiled and sequenced to high depth. Variant calling using an unmatched Hapmap DNA control removed a substantial number of artifactual calls regardless of algorithm used or variant class. The removed calls were nonunique, had lower variant frequencies, and tended to recur in multiple unrelated samples. Analysis of sample replicates revealed that reproducible calls had distinctly higher variant allele depths and frequencies compared to nonreproducible calls. On the basis of these differences, filters on variant frequency were chosen to select for reproducible calls. The analysis pipeline successfully retrieved the associated known variant in all tested samples and uncovered additional mutations in some samples corresponding to well-characterized hotspot mutations in acute myeloid leukemia. We have developed a paired-sample analysis pipeline capable of robust identification of mutations from microdroplet-PCR sequencing data with high sensitivity and specificity.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Mutación , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/genética , Reacción en Cadena de la Polimerasa/métodos , Células de la Médula Ósea/metabolismo , Análisis Mutacional de ADN/métodos , Exones , Pruebas Genéticas/métodos , Humanos , Nucleofosmina , Polimorfismo de Nucleótido Simple , Curva ROC , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
IMPORTANCE: Uveal melanoma is characterized by mutations in GNAQ and GNA11, resulting in mitogen-activated protein kinase pathway activation. OBJECTIVE: To assess the efficacy of selumetinib, a selective, non-adenosine triphosphate competitive inhibitor of MEK1 and MEK2, in uveal melanoma. DESIGN, SETTING, AND PARTICIPANTS: Randomized, open-label, phase 2 clinical trial comparing selumetinib vs chemotherapy conducted from August 2010 through December 2013 among 120 patients with metastatic uveal melanoma at 15 academic oncology centers in the United States and Canada. INTERVENTIONS: One hundred one patients were randomized in a 1:1 ratio to receive selumetinib, 75 mg orally twice daily on a continual basis (n = 50), or chemotherapy (temozolomide, 150 mg/m2 orally daily for 5 of every 28 days, or dacarbazine, 1000 mg/m2 intravenously every 21 days [investigator choice]; n = 51) until disease progression, death, intolerable adverse effects, or withdrawal of consent. After primary outcome analysis, 19 patients were registered and 18 treated with selumetinib without randomization to complete the planned 120-patient enrollment. Patients in the chemotherapy group could receive selumetinib at the time of radiographic progression. MAIN OUTCOMES AND MEASURES: Progression-free survival, the primary end point, was assessed as of April 22, 2013. Additional end points, including overall survival, response rate, and safety/toxicity, were assessed as of December 31, 2013. RESULTS: Median progression-free survival among patients randomized to chemotherapy was 7 weeks (95% CI, 4.3-8.4 weeks; median treatment duration, 8 weeks; interquartile range [IQR], 4.3-16 weeks) and among those randomized to selumetinib was 15.9 weeks (95% CI, 8.4-21.1 weeks; median treatment duration, 16.1 weeks; IQR, 8.1-25.3 weeks) (hazard ratio, 0.46; 95% CI, 0.30-0.71; P < .001). Median overall survival time was 9.1 months (95% CI, 6.1-11.1 months) with chemotherapy and 11.8 months (95% CI, 9.8-15.7 months) with selumetinib (hazard ratio, 0.66; 95% CI, 0.41-1.06; P = .09). No objective responses were observed with chemotherapy. Forty-nine percent of patients treated with selumetinib achieved tumor regression, with 14% achieving an objective radiographic response to therapy. Treatment-related adverse events were observed in 97% of patients treated with selumetinib, with 37% requiring at least 1 dose reduction. CONCLUSIONS AND RELEVANCE: In this hypothesis-generating study of patients with advanced uveal melanoma, selumetinib compared with chemotherapy resulted in a modestly improved progression-free survival and response rate; however, no improvement in overall survival was observed. Improvement in clinical outcomes was accompanied by a high rate of adverse events. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01143402.
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Antineoplásicos Alquilantes/uso terapéutico , Bencimidazoles/uso terapéutico , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Melanoma/tratamiento farmacológico , Neoplasias de la Úvea/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Alquilantes/efectos adversos , Bencimidazoles/efectos adversos , Dacarbazina/efectos adversos , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de Supervivencia , Temozolomida , Resultado del TratamientoRESUMEN
During recent years, it has become increasingly evident that donor leukemia following allogeneic transplant may be more common then realized in the past. We identified five cases of potential donor leukemia cases during past five years. The precise mechanism of the origin of such leukemias, however, remains poorly defined. In this short communication, we report a well documented case of donor-derived de novo acute myeloid leukemia (AML) that developed fourteen years after allogeneic stem cell transplantation for treatment induced AML for his primary malignancy Immunoblastic lymphoma. This case allows us to postulate a possible mechanism of the origin of donor leukemia. The de novo AML clone contained a distinct cytogenetic abnormality, trisomy 11, which was simultaneously detected in preserved peripheral blood obtained at the time of transplantation as well as in the current bone marrow from an otherwise clinically and phenotypically normal donor. The findings from this unique case, provides insight into the process of leukemogenesis, and suggests that the sequence of events leading to leukemogenesis in this patient involved the senescence/apoptosis of normal donor hematopoietic cells due to telomere shortening resulting in the selective proliferation and transformation of this clone with MLL (mixed-lineage leukemia) gene amplification.
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The role of the proliferation index (PI) as an outcome predictor in follicular lymphoma (FL) isn't clear. We have previously demonstrated that quantitative image analysis (QIA) is a robust tool for PI determination and the present study aimed to determine the significance of the PI for outcome in low-grade FL. One hundred and twenty-nine patients with grade 1-2 FL were retrospectively analysed. Slides were scanned digitally and follicle/tumour-involved areas were annotated. The intrafollicular PI was estimated by analysing a median of 10 follicles per case. Patients were divided into two groups: PI < 30%, PI ≥ 30% and clinical outcome was analysed. Among the 129 patients analysed, intrafollicular PI ranged from 0·6 to 63·2% with a median of 23·3%. Overall survival was not influenced by PI group. Among those patients initially observed, intrafollicular PI < 30% was associated with longer time to first therapy compared to patients with a PI ≥ 30%. In the group of patients that were treated at diagnosis, PI was not predictive of time to treatment failure (TTTF). Intrafollicular PI is an important predicator of TTFT for patients who are candidates for observation. Further confirmation in an independent cohort of patients is necessary to determine the clinical validity of the results.
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Linfoma Folicular/patología , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Linfoma Folicular/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Pronóstico , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95-99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.
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Análisis Mutacional de ADN/métodos , Técnicas de Diagnóstico Molecular/métodos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Variaciones en el Número de Copia de ADN , Frecuencia de los Genes , Humanos , Neoplasias/diagnóstico , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Recurrent mutations in JAK2 and MPL genes are genetic hallmarks of BCR-ABL1-negative myeloproliferative neoplasms. Detection of JAK2 and MPL mutations has been incorporated into routine diagnostic algorithms for these diseases. This Special Article summarizes results from a nationwide laboratory survey of JAK2 and MPL mutation analysis. Based on the current practice pattern and the literature, this Special Article provides recommendations and guidelines for laboratory practice for detection of mutations in the JAK2 and MPL genes, including clinical manifestations for prompting the mutation analysis, current and recommended methodologies for testing the mutations, and standardization for reporting the test results. This Special Article also points to future directions for genomic testing in BCR-ABL1-negative myeloproliferative neoplasms.
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Neoplasias Hematológicas/diagnóstico , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/diagnóstico , Receptores de Trombopoyetina/genética , Análisis Mutacional de ADN , Detección Precoz del Cáncer , Pruebas Genéticas/normas , Neoplasias Hematológicas/genética , Humanos , Técnicas de Diagnóstico Molecular/normas , Mutación , Trastornos Mieloproliferativos/genética , Estándares de ReferenciaRESUMEN
Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD(+) disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD(-) complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell-mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.
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Antígenos CD19/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfocitos T/metabolismo , Linfocitos T/fisiología , Adulto , Anciano , Células Cultivadas , Femenino , Humanos , Inmunoterapia/métodos , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Tumor-infiltrating lymphocyte (TIL) counts in colorectal cancer liver metastases (CRCLM) predict survival following resection. While CD4 and CD8 T cells have been correlated with outcome following CRCLM resection, the role of regulatory T cells (Treg) is not well defined. METHODS: TIL in 188 patients who underwent CRCLM resection between 1998 and 2000 were analyzed by immunohistochemistry using tissue microarrays. Correlation between TIL composition and outcome was determined while controlling for established prognostic factors. Total T cells (CD3), helper T cells (CD4), cytotoxic T cells (CD8), and Treg (FoxP3) were analyzed. RESULTS: Median follow-up time was 40 months for all patients and 95 months for survivors. Overall survival (OS) at 5 and 10 years was 40 and 25%, respectively. The CD4 T cell count correlated with OS (p = .02) and recurrence-free survival (p = .04). A high number of CD8 T cells relative to total T cells (CD8:CD3 ratio) predicted longer OS times (p = .05). Analysis of Treg revealed that high FoxP3:CD4 (p = .03) and FoxP3:CD8 (p = .05) ratios were independent predictors of shorter OS. Patients with a high clinical risk score (CRS) were more likely to have a high number of intratumoral Treg, and patients ≥65 years old had a less robust CRCLM T cell infiltration. CONCLUSIONS: A high number of Treg relative to CD4 or CD8 T cells predicted poor outcome, suggesting an immunosuppressive role for FoxP3 + TIL. The intratumoral immune response was an independent predictor of outcome in patients with colorectal liver metastases.
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Neoplasias Colorrectales/mortalidad , Neoplasias Hepáticas/mortalidad , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Femenino , Estudios de Seguimiento , Factores de Transcripción Forkhead/metabolismo , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios Prospectivos , Tasa de Supervivencia , Adulto JovenRESUMEN
BRAF(V600E) is the most common mutation in cutaneous melanoma and has become the target of treatment for patients with metastatic melanoma. A number of methods are currently available to determine mutation status. Recently, a monoclonal antibody (VE1) against mutant BRAF(V600E) was generated. Its use permits assessment of the mutant protein expression throughout a tumor sample and may allow faster and cheaper determination of the mutation status in selected cases. However, for BRAF(V600E) protein expression analysis to be of clinical value, high sensitivity and specificity of the antibody is a prerequisite. In this study we analyzed 44 metastatic melanoma samples with a known BRAF(V600E) mutation status with immunohistochemical expression of the BRAF(V600E) protein. None of the 22 tumors that lacked the BRAF(V600E) mutation labeled with the antibody VE1. This set of VE1-immunonegative tumors included 4 metastatic lesions with the BRAF(V600E) mutation. All 22 tumor samples that were known to carry the BRAF mutation were immunoreactive with VE1. Sixteen of them stained strongly and homogenously throughout the tumor sample. However, 6 tumor samples contained both BRAF(V600E)-immunopositive and BRAF(V600E)-immunonegative cell populations. When the BRAF status was compared with immunoreactivity for melanocyte differentiation antigens, no significant difference in the expression of melan-A, microphthalmia transcription factor, gp100, or tyrosinase was found between mutant and wild-type tumors. In addition to metastatic lesions, we also examined 20 primary melanomas for the expression of BRAF(V600E). Seven of 10 superficial spreading melanomas were immunoreactive with the antibody VE1. Five tumors were strongly and homogenously immunoreactive. In 2 primary tumors the staining was focal, involving only a subpopulation of the tumor. None of the nonsuperficial spreading melanomas was immunoreactive. In 7 primary tumors the mutation status could be analyzed: only tumors carrying the BRAF(V600E) mutation were immunoreactive with VE1. The high specificity and sensitivity of VE1 for the detection of mutant BRAF(V600E) suggests a valuable reagent for clinical purposes. Heterogeneity in BRAF expression may be relevant for treatment response to BRAF inhibitors.
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Anticuerpos Monoclonales , Biomarcadores de Tumor/análisis , Análisis Mutacional de ADN/métodos , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Femenino , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/metabolismo , Melanoma/patología , Persona de Mediana Edad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The Polycomb group protein Bmi1 is a transcriptional silencer of the Ink4a-Arf locus, which encodes the cell cycle regulator p16(Ink4a) and the tumor suppressor p19(Arf). Bmi1 plays a key role in oncogenesis and stem cell self-renewal. We report that phosphorylation of human Bmi1 at Ser³¹6 by Akt impaired its function by triggering its dissociation from the Ink4a-Arf locus, which resulted in decreased ubiquitylation of histone H2A and the inability of Bmi1 to promote cellular proliferation and tumor growth. Moreover, Akt-mediated phosphorylation of Bmi1 also inhibited its ability to promote self-renewal of hematopoietic stem and progenitor cells. Our study provides a mechanism for the increased abundance of p16(Ink4a) and p19(Arf) seen in cancer cells with an activated phosphoinositide 3-kinase to Akt signaling pathway and identifies crosstalk between phosphorylation events and chromatin structure.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Sitios Genéticos , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Cromatina/genética , Cromatina/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Silenciador del Gen , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Complejo Represivo Polycomb 1/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Represoras/genética , Transducción de Señal/genética , Ubiquitinación/genéticaRESUMEN
Pathologic grading for prognostic stratification of neuroendocrine tumors (NETs) is critical but presents a challenging interpretive dilemma. Tumor cell proliferative rate is an important factor in the determination of prognosis, and immunohistochemical analysis with Ki67 is becoming more widely used to quantify the proliferative rate. However, Ki67 assessment has limitations due to lack of uniformity and consistency in quantification. These limitations are accentuated in well-differentiated NETs, as differences in the range of 1% to 5% can alter tumor grade, with potential implications for treatment. We therefore performed a concordance study to assess different Ki67 quantification techniques including: (a) digital image analysis (DIA); (b) manual counting (MC) of >2000 cells; and (c) "eyeballed" estimate (EE) of labeling percentage by pathologists (n=18), including individuals experienced in evaluating Ki67 labeling as well as others who had little prior experience assessing Ki67 percentages. Forty-five Ki67 images were selected and analyzed using the 3 methods. On the basis of the recommendations of the World health Organization (WHO) for grading NETs, MC of 2000 cells was used as the "gold standard" reference against which the other techniques were compared. Three images were presented twice, the second being inverted, to assess intraobserver consistency. Statistical analyses were performed to evaluate: (a) the concordance between methods; (b) intraobserver and interobserver consistency; and (c) correlation of NET grades on the basis of Ki67 scores by EE versus the gold standard. Agreement between scores was assessed by intraclass correlation (ICC). DIA and MC were highly concordant (ICC=0.98). The ICC between DIA and the mean EE of all observers was 0.88. However, there was discordance among individual observers on all cases quantified by EE (ICC=0.13). The ICC for intraobserver consistency was 0.39±0.26. With Ki67 in the ranges of <1%, 2% to 3%, and >20%, the mean of Ki67 by EE was, respectively, 93%±2%, 55%±7%, and 55%±15% correct against the gold standard. The κ statistics for EE exhibited low agreement (κ=0.24; 95% confidence interval, 0.23-0.25) for all WHO NET grades. Incorrect assessment by EE resulted in upgrading of all WHO G1 group tumors (n=14); in the WHO G2 group, downgrading of 41% cases occurred (n=11) when Ki67 was <5% (by DIA or MC), and upgrading of 59% cases occurred (n=16) when Ki67 was >5%. We conclude that DIA and MC are the acceptable standards for Ki67 assessment. Given the inherent discordance in determining the grade, the use of an approximate EE of the Ki67-labeling index requires critical reevaluation, especially for NETs with a labeling index straddling the cut-points between grades. Consequently, determination of therapeutic strategies should be guided by an amalgamation of clinicopathologic characteristics, including but not limited to the Ki67 index.
Asunto(s)
Proliferación Celular , Técnicas de Laboratorio Clínico , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Neoplasias Intestinales/química , Antígeno Ki-67/análisis , Tumores Neuroendocrinos/química , Neoplasias Pancreáticas/química , Diferenciación Celular , Competencia Clínica , Técnicas de Laboratorio Clínico/normas , Humanos , Procesamiento de Imagen Asistido por Computador/normas , Inmunohistoquímica/normas , Neoplasias Intestinales/patología , Intestino Delgado/química , Intestino Delgado/patología , Clasificación del Tumor , Tumores Neuroendocrinos/patología , Variaciones Dependientes del Observador , Neoplasias Pancreáticas/patología , Valor Predictivo de las Pruebas , Pronóstico , Neoplasias del Recto/química , Neoplasias del Recto/patología , Reproducibilidad de los ResultadosRESUMEN
Leukemic transformation (LT) of myeloproliferative neoplasms (MPNs) is associated with a poor prognosis and resistance to therapy. Although previous candidate genetic studies have identified mutations in MPN patients who develop acute leukemia, the complement of genetic abnormalities in MPN patients who undergo LT is not known nor have specific molecular abnormalities been shown to have clinical relevance in this setting. We performed high-throughput resequencing of 22 genes in 53 patients with LT after MPN to characterize the frequency of known myeloid mutations in this entity. In addition to JAK2 and TET2 mutations, which occur commonly in LT after MPN, we identified recurrent mutations in the serine/arginine-rich splicing factor 2 (SRSF2) gene (18.9%) in acute myeloid leukemia (AML) transformed from MPNs. SRSF2 mutations are more common in AML derived from MPNs compared with LT after myelodysplasia (4.8%) or de novo AML (5.6%), respectively (P=.05). Importantly, SRSF2 mutations are associated with worsened overall survival in MPN patients who undergo LT in univariate (P=.03; HR, 2.77; 95% CI, 1.10-7.00) and multivariate analysis (P<.05; HR, 2.11; 95% CI, 1.01-4.42). These data suggest that SRSF2 mutations contribute to the pathogenesis of LT and may guide novel therapeutic approaches for MPN patients who undergo LT.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Hematológicas/genética , Leucemia/patología , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/genética , Proteínas Nucleares/genética , Ribonucleoproteínas/genética , Secuencia de Bases , Transformación Celular Neoplásica/patología , Estudios de Cohortes , Análisis Mutacional de ADN , Progresión de la Enfermedad , Frecuencia de los Genes , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/mortalidad , Neoplasias Hematológicas/patología , Humanos , Leucemia/diagnóstico , Leucemia/genética , Leucemia/mortalidad , Mutación/fisiología , Trastornos Mieloproliferativos/mortalidad , Trastornos Mieloproliferativos/patología , Pronóstico , Factores de Empalme Serina-Arginina , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
We evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.