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1.
Thorac Cancer ; 13(15): 2180-2191, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35708207

RESUMEN

BACKGROUND: CT scans are used in routine clinical practice for the diagnosis and treatment surveillance of non-small cell lung cancer (NSCLC). However, more sensitive methods are desirable. Liquid biopsy analyses of RNA and DNA can offer more sensitive diagnostic approaches. Cell-free RNA (cfRNA) has been described in several malignancies, but its clinical utility has not previously been explored. METHODS: We evaluated the clinical utility of cfRNA for early detection and surveillance of tumor disease in a proof-of-concept study. Using real-time-droplet digital polymerase chain reaction we characterized a candidate transcript (MORF4L2) in plasma samples from 41 advanced stage, 38 early stage NSCLC and 39 healthy samples. We compared its diagnostic performance with tumor markers and evaluated its utility for disease monitoring. RESULTS: MORF4L2 cfRNA was more abundant in patients than in healthy donors (p < 0.0001). Using the Youden index approach (cutoff value of 537 copies/ml was established) with a sensitivity of 0.73 (95% CI: 0.61-0.82) and a specificity of 0.87 (95% CI: 0.73-0.96). Positive and negative predictive values of 0.92 (95% CI: 0.83-0.95) and 0.59 (95% CI: 0.47-0.83) were achieved. Combination of cfRNA and Cyfra21-1 improved its predictive value from 89.5% to 94.7%. Low baseline MORF4L2 levels were associated with better overall survival (HR:0.25, 95% CI: 0.09-0.7, p = 0.009) and progression-free survival for patients treated with tyrosine kinase inhibitors (p = 0.011) and chemotherapy (p = 0.019). MORF4L2 profile between baseline and follow-up mirrored radiological response and tumor dynamics better than tumor markers. cfRNA transcripts allowed monitoring tumor dynamics in patients without tumor-reported genetic alterations. CONCLUSION: Our data support clinical utility of cfRNA for detection and surveillance of NSCLC. Further studies with larger cohorts are required.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Antígenos de Neoplasias , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Ácidos Nucleicos Libres de Células/genética , Receptores ErbB/genética , Humanos , Queratina-19 , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Prueba de Estudio Conceptual , Factores de Transcripción/genética
2.
Transl Oncol ; 15(1): 101279, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-34800919

RESUMEN

BACKGROUND: Radiology is the current standard for monitoring treatment responses in lung cancer. Limited sensitivity, exposure to ionizing radiations and related sequelae constitute some of its major limitation. Non-invasive and highly sensitive methods for early detection of treatment failures and resistance-associated disease progression would have additional clinical utility. METHODS: We analyzed serially collected plasma and paired tumor samples from lung cancer patients (61 with stage IV, 48 with stages I-III disease) and 61 healthy samples by means of next-generation sequencing, radiological imaging and droplet digital polymerase chain reaction (ddPCR) mutation and methylation assays. RESULTS: A 62% variant concordance between tumor-reported and circulating-free DNA (cfDNA) sequencing was observed between baseline liquid and tissue biopsies in stage IV patients. Interestingly, ctDNA sequencing allowed for the identification of resistance-mediating p.T790M mutations in baseline plasma samples for which no such mutation was observed in the corresponding tissue. Serial circulating tumor DNA (ctDNA) mutation analysis by means of ddPCR revealed a general decrease in ctDNA loads between baseline and first reassessment. Additionally, serial ctDNA analyses only recapitulated computed tomography (CT) -monitored tumor dynamics of some, but not all lesions within the same patient. To complement ctDNA variant analysis we devised a ctDNA methylation assay (methcfDNA) based on methylation-sensitive restriction enzymes. cfDNA methylation showed and area under the curve (AUC) of > 0.90 in early and late stage cases. A decrease in methcfDNA between baseline and first reassessment was reflected by a decrease in CT-derive tumor surface area, irrespective of tumor mutational status. CONCLUSION: Taken together, our data support the use of cfDNA sequencing for unbiased characterization of the molecular tumor architecture, highlights the impact of tumor architectural heterogeneity on ctDNA-based tumor surveillance and the added value of complementary approaches such as cfDNA methylation for early detection and monitoring.

3.
Clin Chem ; 66(12): 1510-1520, 2020 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-33257977

RESUMEN

BACKGROUND: We assessed the usefulness of circulating tumor DNA (ctDNA) pre- or post-treatment initiation for outcome prediction and treatment monitoring in metastatic colorectal cancer (mCRC). METHODS: Droplet digital PCR was used to measure absolute mutant V-Ki-ras2 Kirsten rat sarcoma viral oncogene ((mut)KRAS) ctDNA concentrations in 214 healthy controls (plasma and sera) and in 151 tissue-based mutKRAS positive patients with mCRC from the prospective multicenter phase 3 trial AIO KRK0207. Serial mutKRAS ctDNA was analyzed prior to and 2-3 weeks after first-line chemotherapy initiation with fluoropyrimidine, oxaliplatin, and bevacizumab in patients with mCRC and correlated with clinical parameters. RESULTS: mut KRAS ctDNA was detected in 74.8% (113/151) of patients at baseline and in 59.6% (90/151) at follow-up. mutKRAS ctDNA at baseline and follow-up was associated with poor overall survival (OS) (hazard ratio [HR] =1.88, 95% confidence interval [CI] 1.20-2.95; HR = 2.15, 95% CI 1.47-3.15) and progression-free survival (PFS) (HR = 2.53, 95% CI 1.44-4.46; HR = 1.90, 95% CI 1.23-2.95), respectively. mutKRAS ctDNA clearance at follow-up conferred better disease control (P = 0.0075), better OS (log-rank P = 0.0018), and PFS (log-rank P = 0.0018). Measurable positive mutKRAS ctDNA at follow-up was the strongest and most significant independent prognostic factor on OS in multivariable analysis (HR = 2.31, 95% CI 1.40-3.25). CONCLUSIONS: Serial analysis of circulating mutKRAS concentrations in mCRC has prognostic value. Post treatment mutKRAS concentrations 2 weeks after treatment initiation were associated with therapeutic response in multivariable analysis and may be an early response predictor in patients receiving first-line combination chemotherapy. CLINICALTRIALSGOV IDENTIFIER: NCT00973609.


Asunto(s)
ADN Tumoral Circulante , Neoplasias del Colon , Neoplasias Colorrectales , Biomarcadores de Tumor , ADN Tumoral Circulante/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Mutación , Pronóstico , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/genética
4.
Cancers (Basel) ; 12(2)2020 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-32033141

RESUMEN

Early detection of cancer holds high promise for reducing cancer-related mortality. Detection of circulating tumor-specific nucleic acids holds promise, but sensitivity and specificity issues remain with current technology. We studied cell-free RNA (cfRNA) in patients with non-small cell lung cancer (NSCLC; n = 56 stage IV, n = 39 stages I-III), pancreatic cancer (PDAC, n = 20 stage III), malignant melanoma (MM, n = 12 stage III-IV), urothelial bladder cancer (UBC, n = 22 stage II and IV), and 65 healthy controls by means of next generation sequencing (NGS) and real-time droplet digital PCR (RT-ddPCR). We identified 192 overlapping upregulated transcripts in NSCLC and PDAC by NGS, more than 90% of which were noncoding. Previously reported transcripts (e.g., HOTAIRM1) were identified. Plasma cfRNA transcript levels of POU6F2-AS2 discriminated NSCLC from healthy donors (AUC = 0.82 and 0.76 for stages IV and I-III, respectively) and significantly associated (p = 0.017) with the established tumor marker Cyfra 21-1. cfRNA yield and POU6F2-AS transcript abundance discriminated PDAC patients from healthy donors (AUC = 1.0). POU6F2-AS2 transcript was significantly higher in MM (p = 0.044). In summary, our findings support further validation of cfRNA detection by RT-ddPCR as a biomarker for early detection of solid cancers.

5.
Nat Commun ; 8: 15440, 2017 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-28561041

RESUMEN

Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8+ T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.


Asunto(s)
Resistencia a Antineoplásicos/genética , Interferón gamma/inmunología , Melanoma/genética , Neoplasias Cutáneas/genética , Linfocitos T/inmunología , Escape del Tumor/genética , Presentación de Antígeno/genética , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Biopsia , Línea Celular Tumoral , Análisis Mutacional de ADN , Conjuntos de Datos como Asunto , Resistencia a Antineoplásicos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoterapia/métodos , Interferón gamma/metabolismo , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Melanoma/patología , Mutación , Tasa de Mutación , Modelación Específica para el Paciente , Medicina de Precisión/métodos , Transducción de Señal/genética , Transducción de Señal/inmunología , Piel/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T/metabolismo , Resultado del Tratamiento , Secuenciación Completa del Genoma
6.
Cancer Res ; 76(15): 4347-58, 2016 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-27261508

RESUMEN

Melanoma often recurs after a latency period of several years, presenting a T cell-edited phenotype that reflects a role for CD8(+) T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen-specific CD8(+) T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8(+) T cell-resistant, HLA class I-negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8(+) T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Jun(high)/MITF(low) phenotype, possibly associated with immunosuppression, which contrasted with a c-Jun(low)/MITF(high) phenotype of T cell-edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347-58. ©2016 AACR.


Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Melanoma/genética , Proteínas de Neoplasias/inmunología , Humanos , Melanoma/patología , Metástasis de la Neoplasia
7.
Clin Cancer Res ; 20(24): 6593-604, 2014 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-25294904

RESUMEN

PURPOSE: CD8(+) T lymphocytes can kill autologous melanoma cells, but their activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here, we analyzed three consecutive melanoma lesions obtained within one year of developing stage IV disease for their recognition by autologous T cells. EXPERIMENTAL DESIGN: One skin (Ma-Mel-48a) and two lymph node (Ma-Mel-48b, Ma-Mel-48c) metastases were analyzed for T-cell infiltration. Melanoma cell lines established from the respective lesions were characterized, determining the T-cell-stimulatory capacity, expression of surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor-T-cell interaction. RESULTS: Metastases Ma-Mel-48a and Ma-Mel-48b, in contrast with Ma-Mel-48c, were infiltrated by T cells. The T-cell-stimulatory capacity was found to be strong for Ma-Mel-48a, lower for Ma-Mel-48b, and completely abrogated for Ma-Mel-48c cells. The latter proved to be HLA class I-negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. The same deletion was already present in Ma-Mel-48a and Ma-Mel-48b cells, pointing to an early acquired genetic event predisposing to development of ß2m deficiency. Notably, the same chronology of genetic alterations was also observed in a second ß2m-deficient melanoma model. CONCLUSION: Our study reveals a progressive loss in melanoma immunogenicity during the course of metastatic disease. The genetic evolvement of T-cell resistance suggests screening tumors for genetic alterations affecting immunogenicity could be clinically relevant in terms of predicting patient responses to T-cell-based immunotherapy.


Asunto(s)
Evolución Molecular , Melanoma/genética , Melanoma/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Alelos , Antígenos B7/genética , Antígenos B7/inmunología , Butorfanol , Línea Celular Tumoral , Análisis por Conglomerados , Citocinas/metabolismo , Progresión de la Enfermedad , Expresión Génica , Predisposición Genética a la Enfermedad , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Melanoma/terapia , Mutación , Metástasis de la Neoplasia , Estadificación de Neoplasias , Fenotipo , Polimorfismo de Nucleótido Simple , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genética
8.
Mod Pathol ; 27(4): 502-8, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24030750

RESUMEN

Activating mutations in the TERT promoter leading to increased telomerase expression were recently identified in cutaneous melanoma and subsequently in many other types of cancer. These mutations lead to increased telomerase expression, allowing cells to proliferate continuously without entering apoptosis or senescence. Atypical fibroxanthomas and pleomorphic dermal sarcomas are genetically poorly understood tumors developing in the skin of older patients. Known genetic events in these tumors are mutations in TP53 (atypical fibroxanthoma and pleomorphic dermal sarcoma) and RAS (pleomorphic dermal sarcoma) genes, often having a UV signature. We analyzed a cohort of 27 atypical fibroxanthomas and 34 pleomorphic dermal sarcomas for the presence of TERT promoter mutations by conventional Sanger sequencing. TERT promoter mutations were identified in 25 (93%) atypical fibroxanthomas and in 26 (76%) pleomorphic dermal sarcomas. Mutations were found to have a UV signature (C>T or CC>TT) and were largely identical to those detected in cutaneous melanoma. Our data show that TERT promoter mutations are the most frequent mutations in atypical fibroxanthomas and pleomorphic dermal sarcomas reported to date. The identified mutations confirm the pathogenetic role of UV exposure in both atypical fibroxanthomas and pleomorphic dermal sarcomas and suggest that telomere maintenance through increased expression of telomerase plays an important role in the pathogenesis of these tumors.


Asunto(s)
Mutación , Regiones Promotoras Genéticas , Sarcoma/genética , Neoplasias Cutáneas/genética , Telomerasa/genética , Xantomatosis/genética , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sarcoma/enzimología , Sarcoma/patología , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/patología , Xantomatosis/enzimología , Xantomatosis/patología
9.
Diagn Pathol ; 8: 87, 2013 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-23694694

RESUMEN

BACKGROUND: Melanoma is a deadly disease affecting people worldwide. Genetic studies have identified different melanoma subtypes characterized by specific recurrently mutated genes and led to the successful clinical introduction of targeted therapies. Hotspot mutations in SF3B1 were recently reported in uveal melanoma. Our aim was to see if these mutations also occur in cutaneous melanoma. FINDINGS: We analyzed a cohort of 85 cutaneous melanoma including 22 superficial spreading, 24 acral-lentiginous, 36 nodular, and 3 lentigo-maligna melanomas. Exon 14 of SF3B1, containing the site of recurrent mutations described in uveal melanoma, was sequenced in all samples. Additionally, NRAS exon 1 and 2 and BRAF exon 15 were sequenced in all, KIT exons 9, 11, 13, 17, and 18 in 30 samples. High numbers of BRAF and NRAS mutations were identified with frequencies varying according to melanoma subtype. None of the samples were found to harbor a SF3B1 mutation. CONCLUSIONS: We conclude that recurrent mutations in codon 625 of SF3B1 as reported in uveal melanoma are not present in most types of cutaneous melanoma. This highlights the genetic differences between cutaneous and uveal melanoma and the need for subtype specific therapeutic approaches.


Asunto(s)
Melanoma/genética , Mutación/genética , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Neoplasias Cutáneas/genética , Neoplasias de la Úvea/genética , Secuencia de Bases/genética , Exones/genética , Femenino , Genes ras , Humanos , Masculino , Melanoma/diagnóstico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Empalme de ARN , Piel/patología , Neoplasias Cutáneas/diagnóstico , Neoplasias de la Úvea/diagnóstico
10.
Am J Dermatopathol ; 34(3): 266-9, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22197861

RESUMEN

According to the seventh edition of the American Joint Committee on Cancer guidelines, the TNM staging category in thin cutaneous melanomas depends on the mitotic rate (MR). In this study, we analyze the interobserver agreement of the MR in a series of 92 thin cutaneous melanomas. Serial sections of the tumors were either stained with hematoxylin and eosin or immunohistochemically stained with pHH3, an antibody for phosphohistone H3, and analyzed by 4 observers. Determination of MR with pHH3 immunostaining resulted in higher sensitivity in counting mitosis for all observers. Moreover, interobserver agreement was higher with pHH3. Immunostaining with pHH3 is a sensitive method to detect mitosis in thin cutaneous melanomas, with good reproducibility of MR between independent observers. Further studies are needed to find out if higher sensitivity in the detection of mitosis by pHH3 immunostaining has additional prognostic relevance.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Histonas/metabolismo , Melanoma/diagnóstico , Índice Mitótico , Neoplasias Cutáneas/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Histonas/inmunología , Humanos , Inmunohistoquímica/métodos , Masculino , Melanoma/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Variaciones Dependientes del Observador , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Fosforilación , Sensibilidad y Especificidad , Neoplasias Cutáneas/metabolismo , Adulto Joven
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