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Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in â¼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.
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Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/enzimología , Neoplasias/genética , Proteínas Tirosina Quinasas Receptoras/genética , Iniciación de la Transcripción Genética , Alelos , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Femenino , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Intrones/genética , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Lisina/metabolismo , Metilación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Células 3T3 NIH , Neoplasias/tratamiento farmacológico , Oncogenes/genética , Estructura Terciaria de Proteína/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/química , Transducción de SeñalRESUMEN
BACKGROUND: The phosphatidyl 3-inositol kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway frequently is activated in patients with urothelial carcinoma (UC). In the current study, the authors performed a phase 2 study evaluating the efficacy of the pan-isoform class I PI3K inhibitor buparlisib in patients with platinum-refractory metastatic UC. METHODS: Two cohorts were recruited: an initial genetically unselected cohort and a subsequent expansion cohort of patients with PI3K/Akt/mTOR pathway-altered tumors. The primary endpoint was the 2-month progression-free survival rate. A rate of ≥80% was considered promising using a Simon 2-stage minimax design. Secondary endpoints included safety and correlation of markers of PI3K pathway activation with outcome. RESULTS: Six of 13 evaluable patients within the initial cohort demonstrated stable disease and 1 demonstrated a partial response, which was below the cutoff of 9 patients required to proceed to stage 2. Three of the patients with stable disease and the patient with a partial response harbored somatic TSC1 alterations. Four patients subsequently were recruited onto an expansion cohort: 3 patients with TSC1 alterations and 1 patient with a PIK3CA-activating mutation. No patient achieved disease control at 8 weeks and accrual was halted. Of the 19 patients evaluable for toxicity, 17 demonstrated treatment-related toxicities, 2 of whom had to discontinue therapy. CONCLUSIONS: Buparlisib was found to demonstrate modest activity in patients with metastatic UC whose tumors harbored TSC1 loss of function alterations; however, this was not a robust predictor of response to buparlisib. The pattern of genetic coalterations likely influences drug sensitivity. Given the modest clinical activity and substantial toxicity of buparlisib, future trials of PI3K inhibitors in patients with UC should focus on isoform-selective PI3K inhibitors in genomically selected patients. LAY SUMMARY: The phosphatidyl 3-inositol kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway frequently is upregulated in patients with metastatic urothelial carcinoma (UC). This trial explored buparlisib, an inhibitor of the pathway, in patients with heavily pretreated metastatic UC. Although the drug was found to have modest efficacy, with 6 patients experiencing stable disease and 1 patient achieving a partial response at 8 weeks on therapy, significant side effects also were observed. Patients with specific genetic alterations responded to treatment. Further studies of PI3K pathway inhibition are warranted using newer agents that have superior toxicity profiles and are more selective inhibitors of the pathway.
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Aminopiridinas/uso terapéutico , Morfolinas/uso terapéutico , Fosfatidilinositol 3-Quinasa/uso terapéutico , Neoplasias Urológicas/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Aminopiridinas/farmacología , Línea Celular Tumoral , Femenino , Humanos , Masculino , Persona de Mediana Edad , Morfolinas/farmacología , Metástasis de la Neoplasia , Fosfatidilinositol 3-Quinasa/farmacologíaRESUMEN
Clear cell carcinoma of the endometrium is a rare type of endometrial cancer that is generally associated with an aggressive clinical behaviour. Here, we sought to define the repertoire of somatic genetic alterations in endometrial clear cell carcinomas (ECCs), and whether ECCs could be classified into the molecular subtypes described for endometrial endometrioid and serous carcinomas. We performed a rigorous histopathological review, immunohistochemical analysis and massively parallel sequencing targeting 300 cancer-related genes of 32 pure ECCs. Eleven (34%), seven (22%) and six (19%) ECCs showed abnormal expression patterns for p53, ARID1A, and at least one DNA mismatch repair (MMR) protein, respectively. Targeted sequencing data were obtained from 30 of the 32 ECCs included in this study, and these revealed that two ECCs (7%) were ultramutated and harboured mutations affecting the exonuclease domain of POLE. In POLE wild-type ECCs, TP53 (46%), PIK3CA (36%), PPP2R1A (36%), FBXW7 (25%), ARID1A (21%), PIK3R1 (18%) and SPOP (18%) were the genes most commonly affected by mutations; 18% and 11% harboured CCNE1 and ERBB2 amplifications, respectively, and 11% showed DAXX homozygous deletions. ECCs less frequently harboured mutations affecting CTNNB1 and PTEN but more frequently harboured PPP2R1A and TP53 mutations than non-POLE endometrioid carcinomas from The Cancer Genome Atlas (TCGA). Compared to endometrial serous carcinomas (TCGA), ECCs less frequently harboured TP53 mutations. When a surrogate model for the molecular-based TCGA classification was used, all molecular subtypes previously identified in endometrial endometrioid and serous carcinomas were present in the ECCs studied, including POLE, MMR-deficient, copy-number high (serous-like)/p53 abnormal, and copy-number low (endometrioid)/p53 wild-type, which were significantly associated with disease-free survival in univariate analysis. These findings demonstrate that ECCs constitute a histologically and genetically heterogeneous group of tumours with varying outcomes. Furthermore, our data suggest that the classification of ECCs as being generally 'high-grade' or 'type II' tumours may not be warranted. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Adenocarcinoma de Células Claras/genética , Neoplasias Endometriales/genética , Adenocarcinoma de Células Claras/patología , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Endometriales/patología , Femenino , Dosificación de Gen/genética , Humanos , Persona de Mediana Edad , Mutación/genética , Proteínas de Neoplasias/genética , PronósticoRESUMEN
Leiomyosarcoma (LMS) belongs to the class of genetically complex sarcomas and shows numerous, often non-recurrent chromosomal imbalances and aberrations. We investigated a group of LMS using NGS platform to identify recurrent genetic abnormalities and possible therapeutic targets. Targeted exome sequencing of 230 cancer-associated genes was performed on 35 primary soft tissue and visceral (extra-uterine) LMS. Sequence data were analyzed to identify single nucleotide variants, small insertions/deletions (indels), and copy number alterations. Key alterations were further investigated using FISH assay. The study group included patients with median age of 64 years and median tumor size of 7 cm. The primary sites included retroperitoneal/intra-abdominal, extremity, truncal, and visceral. Thirty-one tumors were high grade LMS, while four were low grade. Losses of chromosomal regions involving key tumor suppressor genes PTEN (10q), RB1 (13q), CDH1 (16q), and TP53 (17p) were the most frequent genetic events. Gains mainly involved chromosome regions 17p11.2 (MYOCD) and 15q25-26 (IGF1R). The most frequent mutations were identified in the TP53 gene in 13 of 35 (37%) cases. FISH analysis showed amplification of the myocardin (MYOCD) gene in 5 of 25 (20%) cases analyzed. None of the four low grade LMS showed losses or mutations of PTEN or TP53 genes. Genetic complexity is the hallmark of LMS with losses of important tumor suppressor genes being a common feature. MYOCD, a key gene associated with smooth muscle differentiation, is amplified in a subset of both retroperitoneal and extremity LMS. Further studies are necessary to investigate the significance of gains/amplifications in the development of these tumors.
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Predisposición Genética a la Enfermedad , Variación Genética , Leiomiosarcoma/genética , Leiomiosarcoma/patología , Análisis de Secuencia de ADN/métodos , Adulto , Anciano , Anciano de 80 o más Años , Exoma , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: The authors characterized the genetic landscape of chemoradiation-treated urothelial carcinoma of the bladder (UCB) with the objective of identifying potential correlates of response. METHODS: Primary tumors with (n = 8) or without (n = 40) matched recurrent tumors from 48 patients who had non-metastatic, high-grade UCB and received treatment primarily with chemoradiation were analyzed using a next-generation sequencing assay enriched for cancer-related and canonical DNA damage response (DDR) genes. Protein expression of meiotic recombination 11 homolog (MRE11), a previously suggested biomarker, was assessed in 44 patients. Recurrent tumors were compared with primary tumors, and the clinical impact of likely deleterious DDR gene alterations was evaluated. RESULTS: The profile of alterations approximated that of prior UCB cohorts. Within 5 pairs of matched primary-recurrent tumors, a median of 92% of somatic mutations were shared. A median 33% of mutations were shared between 3 matched bladder-metastasis pairs. Of 26 patients (54%) who had DDR gene alterations, 12 (25%) harbored likely deleterious alterations. In multivariable analysis, these patients displayed a trend toward reduced bladder recurrence (hazard ratio, 0.32; P = .070) or any recurrence (hazard ratio, 0.37; P = .070). The most common of these alterations, ERCC2 (excision repair cross-complementation group 2) mutations, were associated with significantly lower 2-year metastatic recurrence (0% vs 43%; log-rank P = .044). No impact of MRE11 protein expression on outcome was detected. CONCLUSIONS: A similar mutation profile between primary and recurrent tumors suggests that pre-existing, resistant clonal populations represent the primary mechanism of chemoradiation treatment failure. Deleterious DDR genetic alterations, particularly recurrent alterations in ERCC2, may be associated with improved chemoradiation outcomes in patients with UCB. A small sample size necessitates independent validation of these observations. Cancer 2016;122:3715-23. © 2016 American Cancer Society.
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Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/terapia , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Quimioradioterapia/métodos , Proteínas de Unión al ADN/genética , Femenino , Genómica/métodos , Humanos , Proteína Homóloga de MRE11 , Masculino , Persona de Mediana Edad , Mutación/genética , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/terapia , Proteína de la Xerodermia Pigmentosa del Grupo D/genéticaRESUMEN
In 2010, the World Health Organization reclassified the entity originally described as intraductal oncocytic papillary neoplasm as the 'oncocytic subtype' of intraductal papillary mucinous neoplasm. Although several key molecular alterations of other intraductal papillary mucinous neoplasm subtypes have been discovered, including common mutations in KRAS, GNAS, and RNF3, those of oncocytic subtype have not been well characterized. We analyzed 11 pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms. Nine pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms uniformly exhibited typical entity-defining morphology of arborizing papillae lined by layers of cells with oncocytic cytoplasm, prominent, nucleoli, and intraepithelial lumina. The remaining two were atypical. One lacked the arborizing papilla and had flat oncocytic epithelium only; the other one had focal oncocytic epithelium in a background of predominantly intestinal subtype intraductal papillary mucinous neoplasm. Different components of this case were analyzed separately. Formalin-fixed, paraffin-embedded specimens of all cases were microdissected and subjected to high-depth-targeted next-generation sequencing for a panel of 300 key cancer-associated genes in a platform that enabled the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Fresh frozen specimens of two cases were also subjected to whole-genome sequencing. For the nine typical pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms, the number of mutations per case, identified by next-generation sequencing, ranged from 1 to 10 (median=4). None of these cases had KRAS or GNAS mutations and only one had both RNF43 and PIK3R1 mutations. ARHGAP26, ASXL1, EPHA8, and ERBB4 genes were somatically altered in more than one of these typical 'oncocytic subtype' of intraductal papillary mucinous neoplasms but not in the other two atypical ones. In the neoplasm with flat oncocytic epithelium, the only mutated gene was KRAS. All components of the intestinal subtype intraductal papillary mucinous neoplasms with focal oncocytic epithelium manifested TP53, GNAS, and RNF43 mutations. In conclusion, this study elucidates that 'oncocytic subtype' of intraductal papillary mucinous neoplasm is not only morphologically distinct but also genetically distinct from other intraductal papillary mucinous neoplasm subtypes. Considering that now its biologic behavior is also being found to be different than other intraductal papillary mucinous neoplasm subtypes, 'oncocytic subtype' of intraductal papillary mucinous neoplasm warrants being recognized separately.
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Biomarcadores de Tumor/genética , Mutación , Neoplasias Quísticas, Mucinosas y Serosas/genética , Células Oxífilas , Neoplasias Pancreáticas/genética , Cromograninas/genética , Análisis Mutacional de ADN/métodos , Proteínas de Unión al ADN/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Perfilación de la Expresión Génica/métodos , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Quísticas, Mucinosas y Serosas/clasificación , Neoplasias Quísticas, Mucinosas y Serosas/patología , Proteínas Oncogénicas/genética , Células Oxífilas/clasificación , Células Oxífilas/patología , Neoplasias Pancreáticas/clasificación , Neoplasias Pancreáticas/patología , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas , Secuenciación Completa del GenomaRESUMEN
PURPOSE: Genomic characterization of radical nephroureterectomy specimens in patients with upper tract urothelial carcinoma may allow for thoughtful integration of systemic and targeted therapies. We sought to determine whether genomic alterations in upper tract urothelial carcinoma are associated with adverse pathological and clinical outcomes. MATERIALS AND METHODS: Next generation exon capture sequencing of 300 cancer associated genes was performed in 83 patients with upper tract urothelial carcinoma. Genomic alterations were assessed individually and also grouped into core signal transduction pathways or canonical cell functions for association with clinicopathological outcomes. Binary outcomes, including grade (high vs low), T stage (pTa/T1/T2 vs pT3/T4) and organ confined status (pT2 or less and N0/Nx vs greater than pT2 or N+) were assessed with the Kruskal-Wallis and Fisher exact tests as appropriate. Associations between alterations and survival were estimated using the Kaplan-Meier method and Cox regression. RESULTS: Of the 24 most commonly altered genes in 9 pathways TP53/MDM2 alterations and FGFR3 mutations were the only 2 alterations uniformly associated with high grade, advanced stage, nonorgan confined disease, and recurrence-free and cancer specific survival. TP53/MDM2 alterations were associated with adverse clinicopathological outcomes whereas FGFR3 mutations were associated with favorable outcomes. We created a risk score using TP53/MDM2 and FGFR3 status that was able to discriminate between adverse pathological and clinical outcomes, including in the subset of patients with high grade disease. The study is limited by small numbers and lack of validation. CONCLUSIONS: Our data indicate that specific genomic alterations in radical nephroureterectomy specimens correlate with tumor grade, stage and cancer specific survival outcomes.
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Biomarcadores de Tumor/genética , Neoplasias Urológicas/genética , Adulto , Anciano , Femenino , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Riñón/patología , Riñón/cirugía , Masculino , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Nefroureterectomía , Pronóstico , Medición de Riesgo/métodos , Análisis de Supervivencia , Uréter/patología , Uréter/cirugía , Neoplasias Urológicas/mortalidad , Neoplasias Urológicas/patologíaRESUMEN
Cutaneous neuroendocrine (Merkel cell) carcinoma most often arises de novo in the background of a clonally integrated virus, the Merkel cell polyomavirus, and is notable for positive expression of retinoblastoma 1 (RB1) protein and low expression of p53 compared with the rare Merkel cell polyomavirus-negative Merkel cell carcinomas. Combined squamous and Merkel cell tumors are consistently negative for Merkel cell polyomavirus. Little is known about their immunophenotypic or molecular profile. Herein, we studied 10 combined cutaneous squamous cell and neuroendocrine carcinomas for immunohistochemical expression of p53, retinoblastoma 1 protein, neurofilament, p63, and cytokeratin 20 (CK20). We compared mutation profiles of five combined Merkel cell carcinomas and seven 'pure' Merkel cell carcinomas using targeted next-generation sequencing. Combined tumors were from the head, trunk, and leg of Caucasian males and one female aged 52-89. All cases were highly p53- and p63-positive and neurofilament-negative in the squamous component, whereas RB1-negative in both components. Eight out of 10 were p53-positive, 3/10 p63-positive, and 3/10 focally neurofilament-positive in the neuroendocrine component. Six out of 10 were CK20-positive in any part. By next-generation sequencing, combined tumors were highly mutated, with an average of 48 mutations per megabase compared with pure tumors, which showed 1.25 mutations per megabase. RB1 and p53 mutations were identified in all five combined tumors. Combined tumors represent an immunophenotypically and genetically distinct variant of primary cutaneous neuroendocrine carcinomas, notable for a highly mutated genetic profile, significant p53 expression and/or mutation, absent RB1 expression in the context of increased RB1 mutation, and minimal neurofilament expression.
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Biomarcadores de Tumor , Carcinoma de Células de Merkel/química , Carcinoma de Células de Merkel/genética , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/genética , Inmunohistoquímica , Neoplasias Complejas y Mixtas/química , Neoplasias Complejas y Mixtas/genética , Neoplasias Cutáneas/química , Neoplasias Cutáneas/genética , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Carcinoma de Células de Merkel/etnología , Carcinoma de Células de Merkel/patología , Carcinoma de Células Escamosas/etnología , Carcinoma de Células Escamosas/patología , Análisis Mutacional de ADN , Femenino , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Queratina-20/análisis , Masculino , Persona de Mediana Edad , Mutación , Neoplasias Complejas y Mixtas/etnología , Neoplasias Complejas y Mixtas/patología , Proteínas de Neurofilamentos/análisis , Fenotipo , Valor Predictivo de las Pruebas , Proteína de Retinoblastoma/análisis , Proteína de Retinoblastoma/genética , Neoplasias Cutáneas/etnología , Neoplasias Cutáneas/patología , Factores de Transcripción/análisis , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/análisis , Población Blanca/genéticaRESUMEN
Context: Androgen levels are generally measured in serum samples, but urine may be a more feasible option, especially in children, as it is a noninvasive alternative. Objective: To assess the correlations of 10 urinary androgen metabolites with 4 serum androgens [dehydroepiandrosterone-sulfate (DHEA-S), androstenedione, and total and free testosterone] and assess if their correlations differ by participant characteristics. Methods: Our study consisted of 44 girls, ages 6-13, who participated in the New York site of the LEGACY Girls Study and had both serum and urine samples collected at the same visit. We performed Pearson's correlation coefficient tests between 4 serum and 10 individual urinary metabolite measures and their sum. We examined the influence of participant characteristics on the magnitude and direction of the correlations. Results: The summed urinary metabolite measures had the highest correlation with free testosterone in serum (global sum, r = 0.83) and correlated least with DHEA-S in serum (global sum, r = 0.64). The correlation between individual urinary metabolites and serum androgens ranged from 0.08 to 0.84.Two 11-oxygenated urinary metabolites (5α-androstane-3α-ol-11,17-dione5ß-androstane-3α,11ß-diol-17-one) were weakly correlated with all serum androgens. Participant age, weight, height, waist:hip ratio, and pubic hair growth stage changed the correlations between urinary and serum androgens measures between 10% and 213%. Conclusion: The sum of urinary androgen metabolites was a good marker of circulating androstenedione, testosterone, and free testosterone. Individual urinary metabolites provide additional information about the metabolic processes of disease development compared to the antecedent serum androgens.
RESUMEN
Salivary duct carcinoma (SDC) is a rare, aggressive malignancy with limited treatment options and poor outcome. Twenty-nine primary resected SDC, including 15 SDC de novo (SDCDN), and 14 SDC ex pleomorphic adenoma (SDCXPA) were subjected to the massive parallel sequencing assay (MSK-IMPACT) targeting 287 to 468 cancer-related genes. TP53 was the most frequently altered gene (69%). TP53 mutations and ERBB2 amplification were more frequent in SDCXPA than in SDCDN (Pâ¯=â¯.0007 and Pâ¯=â¯.01, respectively). Potentially targetable mutations were detected in 79% (23/29) of SDC involving ERBB2 (31%), PIK3CA (28%), HRAS (21%), ALK (7%) and BRAF (3%), and 22% (5/23) of those cases harbored possible primary resistance mutations involving CCNE1, NF1 and PTEN. A novel HNRNPH3-ALK rearrangement was found in one SDCDN. In another case, EML4-ALK fusion detected in the primary tumor was associated with ALK G1202R secondary resistance mutation in the post-treatment metastasis. A germline analysis of the DNA repair genes revealed a case with a pathogenic BRCA1 E23fs germline variant. SDCDN and SDCXPA are genetically distinct. Although the majority of SDC may be amenable to molecular targeted therapy, concurrent possible resistance mutations may be found in a significant minority of cases. A broad genomic profiling is necessary to ensure detection of rare but clinically actionable somatic alterations in SDC.
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Quinasa de Linfoma Anaplásico/genética , Reordenamiento Génico/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Neoplasias de las Glándulas Salivales/genética , Adenoma Pleomórfico/genética , Adenoma Pleomórfico/patología , Anciano , Anciano de 80 o más Años , Femenino , Mutación de Línea Germinal , Humanos , Masculino , Persona de Mediana Edad , Mutación , Neoplasias de la Parótida/genética , Neoplasias de la Parótida/patología , Receptor ErbB-2/genética , Neoplasias de las Glándulas Salivales/patología , Neoplasias de la Glándula Submandibular/genética , Neoplasias de la Glándula Submandibular/patología , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Biopsies from patients with high-risk (HR) non-muscle-invasive urothelial carcinoma (NMIUC), especially flat urothelial carcinoma in situ, frequently contain scant diagnostic material or denuded mucosa only, and this precludes further extensive genomic analysis. This study evaluated the use of next-generation sequencing (NGS) analysis of urine cytology material from patients with HR NMIUC in an attempt to identify genetic alterations that might correlate with clinical features and responses to bacille Calmette-Guérin (BCG) treatment. METHODS: Forty-one cytology slides from patients with HR NMIUC treated with intravesical BCG were selected for this study. Histological confirmation was available for all cases. The specimens were subjected to NGS analysis with a customized targeted exome capture assay composed of 341 genes. RESULTS: In this cohort, genomic alterations were successfully identified in all cytology samples. Mutations were detected down to a 2% allele frequency and chromosomal rearrangements including copy number alterations and gene fusions were identified. The most frequently altered genes included telomerase reverse transcriptase (TERT), tumor protein 53 (TP53), Erb-B2 receptor tyrosine kinase 2 (ERBB2), and chromatin remodeling genes such as lysine demethylase 6A (KDM6A) and AT-rich interaction domain 1A (ARID1A). For patients with matched tumor tissue, cytology specimens revealed all mutations detected in tissue as well as additional mutations, and this suggested that urine might more effectively capture the full genetic heterogeneity of disease than an individual cystectomy. Alterations in multiple genes correlated with clinical and histopathological features, including responses to BCG treatment, flat architecture versus papillary architecture, and smoking history. CONCLUSIONS: Urine specimens can replace tissue as a substrate for NGS analysis of HR NMIUC. Several genomic alterations identified in urine specimens might be associated with histological features and clinical characteristics. Cancer Cytopathol 2017;125:416-26. © 2017 American Cancer Society.
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Carcinoma de Células Transicionales/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias de la Vejiga Urinaria/genética , Orina/citología , Adyuvantes Inmunológicos/uso terapéutico , Administración Intravesical , Anciano , Anciano de 80 o más Años , Vacuna BCG/uso terapéutico , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/patología , Carcinoma de Células Transicionales/orina , Proteínas de Unión al ADN , Femenino , Histona Demetilasas/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Invasividad Neoplásica , Estadificación de Neoplasias , Proteínas Nucleares/genética , Receptor ErbB-2/genética , Análisis de Secuencia de ADN , Telomerasa/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/orinaRESUMEN
Plasmacytoid bladder cancer is an aggressive histologic variant with a high risk of disease-specific mortality. Using whole-exome and targeted sequencing, we find that truncating somatic alterations in the CDH1 gene occur in 84% of plasmacytoid carcinomas and are specific to this histologic variant. Consistent with the aggressive clinical behavior of plasmacytoid carcinomas, which frequently recur locally, CRISPR/Cas9-mediated knockout of CDH1 in bladder cancer cells enhanced cell migration.
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Cadherinas/genética , Plasmacitoma/genética , Neoplasias de la Vejiga Urinaria/genética , Antígenos CD , Línea Celular Tumoral , Análisis Mutacional de ADN , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Análisis Multivariante , Mutación , Modelos de Riesgos Proporcionales , Neoplasias de la Vejiga Urinaria/mortalidadRESUMEN
Desmoplastic melanoma (DM) is a rare variant of melanoma with distinct clinical, histopathologic, and immunohistochemical features. Clinically, DM differs from conventional melanoma by a higher propensity for local recurrence and less frequent metastatic spread to regional lymph nodes. In its pure form, DM has a distinct appearance displaying a low density of fusiform melanocytes in a collagen-rich matrix. Whereas a number of mutations have been identified in primary melanoma, including BRAF, NRAS, GNAQ, GNA11, and KIT, and the occurrence of these mutations has been found to correlate to some extent with the histopathologic features, anatomic site, and/or mode of sun exposure, no distinct set of mutations has so far been reported for DM. To study the potential association of neurofibromin (NF1) mutations with DM, we examined 15 desmoplastic and 20 non-DMs by next-generation sequencing. Mutations of the NF1 gene were found in 14 of 15 (93%) DMs and 4 of 20 (20%) non-DMs. The high frequency of NF1 mutations in DMs suggests an important role for NF1 in the biology of this type of melanoma.
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Biomarcadores de Tumor/genética , Genes de Neurofibromatosis 1 , Melanoma/genética , Mutación , Neoplasias Cutáneas/genética , Adulto , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN/métodos , Femenino , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Fenotipo , Neoplasias Cutáneas/patología , Adulto JovenRESUMEN
BACKGROUND: Mutation analysis for personalized treatment has become increasingly important in the management of different types of cancer. The advent of new DNA extraction protocols and sequencing platforms with reduced DNA input requirements might allow the use of cytology specimens for high-throughput mutation analysis. In this study, the authors evaluated the use of effusion fluid for next-generation sequencing-based, multigene mutation profiling. METHODS: Four specimens from each of 5 patients who had at least stage III, high-grade serous ovarian carcinoma were selected: effusion fluid; frozen tumor; formalin-fixed, paraffin embedded tumor; and matched normal blood. Frozen tumors from each patient were previously characterized by The Cancer Genomic Atlas (TCGA). DNA was extracted from all specimens and was sequenced using a custom hybridization capture-based assay. Genomic alterations were compared among all specimens from each patient as well as with mutations reported in TCGA for the same tumors. RESULTS: In total, 17 distinct somatic mutations were identified in the cohort. Ten of 17 mutations were reported in TCGA and were called in all 3 malignant specimens procured from the patients. Of the remaining 7 mutations, 2 were called in all 3 specimens, and the other 5 were sample-specific. Two mutations were detected only in the cytology specimens. Copy number profiles were concordant among the tumors analyzed. CONCLUSIONS: Cytology specimens represent suitable material for high-throughput sequencing, because all mutations described by TCGA were independently identified in the effusion fluid. Differences in mutations detected in samples procured from the same patient may reflect tumor heterogeneity.
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Cistadenocarcinoma Seroso/genética , ADN de Neoplasias/genética , Perfilación de la Expresión Génica/métodos , Predisposición Genética a la Enfermedad , Neoplasias Ováricas/genética , Anciano , Líquido Ascítico/patología , Estudios de Casos y Controles , Cistadenocarcinoma Seroso/patología , Análisis Mutacional de ADN , Femenino , Secciones por Congelación/métodos , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Persona de Mediana Edad , Mutación , Invasividad Neoplásica/patología , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Valores de Referencia , Muestreo , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Adhesión del Tejido/métodosRESUMEN
BACKGROUND: Intraductal papillary mucinous neoplasms (IPMN) are being increasingly recognized as important precursors to pancreatic adenocarcinoma. Elucidation of the genetic changes underlying IPMN carcinogenesis may improve the diagnosis and management of IPMN. We sought to determine whether different histologic subtypes of IPMN would exhibit different frequencies of specific genetic mutations. STUDY DESIGN: Patients with resected IPMN-associated invasive carcinoma (IPMN-INV) between 1997 and 2012 were reviewed. Areas of carcinoma, high-grade dysplasia, and low-grade dysplasia were micro-dissected from each pathologic specimen. Targeted, massively parallel sequencing was then performed on a panel of 275 genes (including KRAS, GNAS, and RNF43). RESULTS: Thirty-eight patients with resected IPMN-INV and sufficient tissue for micro-dissection were identified. Median follow-up was 2.6 years. Mutations in GNAS were more prevalent in colloid-type IPMN-INV than tubular-type IPMN-INV (89% vs 32% respectively; p = 0.0003). Conversely, KRAS mutations were more prevalent in tubular-type than colloid-type IPMN-INV (89% vs 52%, respectively; p = 0.01). For noninvasive IPMN subtypes, GNAS mutations were more prevalent in intestinal (74%) compared with pancreatobiliary (31%) and gastric (50%) subtypes (p = 0.02). The presence of these mutations did not vary according to the degree of dysplasia (GNAS: invasive 61%, high-grade 59%, low-grade 53%; KRAS: invasive 71%, high-grade 62%, low-grade 74%), suggesting that mutations in these genes occur early in IPMN carcinogenesis. CONCLUSIONS: Colloid carcinoma associated with IPMN and its intestinal-type preinvasive precursor are associated with high frequencies of GNAS mutations. The mutation profile of tubular carcinoma resembles that of conventional pancreatic adenocarcinoma. Preoperative determination of mutational status may assist with clinical treatment decisions.
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Adenocarcinoma Mucinoso/genética , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Papilar/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Adenocarcinoma Mucinoso/mortalidad , Adenocarcinoma Mucinoso/patología , Adenocarcinoma Mucinoso/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/cirugía , Carcinoma Papilar/mortalidad , Carcinoma Papilar/patología , Carcinoma Papilar/cirugía , Cromograninas , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Mutación , Proteínas Oncogénicas/genética , Pancreatectomía , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Pronóstico , Proteínas Proto-Oncogénicas p21(ras) , Estudios Retrospectivos , Ubiquitina-Proteína Ligasas , Neoplasias PancreáticasRESUMEN
The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.
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Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , ADN/genética , Genotipo , Humanos , Mutación , Adhesión en Parafina , Reproducibilidad de los ResultadosRESUMEN
UNLABELLED: Urothelial carcinoma of the bladder (UCB) is genomically heterogeneous, with frequent alterations in genes regulating chromatin state, cell cycle control, and receptor kinase signaling. To identify prognostic genomic markers in high-grade UCB, we used capture-based massively parallel sequencing to analyze 109 tumors. Mutations were detected in 240 genes, with 23 genes mutated in ≥5% of cases. The presence of a recurrent phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutation was associated with improved recurrence-free survival (RFS) (hazard ratio [HR]: 0.35; p=0.014) and improved cancer-specific survival (CSS) (HR: 0.35; p=0.040) in patients treated with radical cystectomy (RC). In multivariable analyses controlling for pT and pN stages, PIK3CA mutation remained associated with RFS (HR: 0.39; p=0.032). The most frequent alteration, TP53 mutation (57%), was more common in extravesical disease (69% vs 32%, p=0.005) and lymph node-positive disease (77% vs 56%, p=0.025). Patients with cyclin-dependent kinase inhibitor 2A (CDKN2A)-altered tumors experienced worse RFS (HR: 5.76; p<0.001) and worse CSS (HR: 2.94; p=0.029) in multivariable analyses. Mutations in chromatin-modifying genes were highly prevalent but not associated with outcomes. In UCB patients treated with RC, PIK3CA mutations are associated with favorable outcomes, whereas TP53 and CDKN2A alterations are associated with poor outcomes. Genomic profiling may aid in the identification of UCB patients at highest risk following RC. PATIENT SUMMARY: Using next-generation sequencing, we identified genomic subsets of high-grade urothelial bladder cancer associated with favorable and unfavorable outcomes. These findings may aid in the selection of patients most likely to benefit from novel combined modality approaches.
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Biomarcadores de Tumor/genética , Carcinoma/genética , Genómica , Mutación , Neoplasias de la Vejiga Urinaria/genética , Urotelio/patología , Carcinoma/mortalidad , Carcinoma/secundario , Carcinoma/cirugía , Fosfatidilinositol 3-Quinasa Clase I , Cistectomía , Supervivencia sin Enfermedad , Perfilación de la Expresión Génica , Genes p16 , Predisposición Genética a la Enfermedad , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metástasis Linfática , Análisis Multivariante , Clasificación del Tumor , Recurrencia Local de Neoplasia , Fenotipo , Fosfatidilinositol 3-Quinasas/genética , Valor Predictivo de las Pruebas , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/cirugía , Urotelio/cirugíaRESUMEN
BACKGROUND: Despite a similar histologic appearance, upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) tumors have distinct epidemiologic and clinicopathologic differences. OBJECTIVE: To investigate whether the differences between UTUC and UCB result from intrinsic biological diversity. DESIGN, SETTING, AND PARTICIPANTS: Tumor and germline DNA from patients with UTUC (n=83) and UCB (n=102) were analyzed using a custom next-generation sequencing assay to identify somatic mutations and copy number alterations in 300 cancer-associated genes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We described co-mutation patterns and copy number alterations in UTUC. We also compared mutation frequencies in high-grade UTUC (n=59) and high-grade UCB (n=102). RESULTS AND LIMITATIONS: Comparison of high-grade UTUC and UCB revealed significant differences in the prevalence of somatic alterations. Genes altered more commonly in high-grade UTUC included FGFR3 (35.6% vs 21.6%; p=0.065), HRAS (13.6% vs 1.0%; p=0.001), and CDKN2B (15.3% vs 3.9%; p=0.016). Genes less frequently mutated in high-grade UTUC included TP53 (25.4% vs 57.8%; p<0.001), RB1 (0.0% vs 18.6%; p<0.001), and ARID1A (13.6% vs 27.5%; p=0.050). Because our assay was restricted to genomic alterations in a targeted panel, rare mutations and epigenetic changes were not analyzed. CONCLUSIONS: High-grade UTUC tumors display a spectrum of genetic alterations similar to high-grade UCB. However, there were significant differences in the prevalence of several recurrently mutated genes including HRAS, TP53, and RB1. As relevant targeted inhibitors are being developed and tested, these results may have important implications for the site-specific management of patients with urothelial carcinoma. PATIENT SUMMARY: Comparison of next-generation sequencing of upper tract urothelial carcinoma (UTUC) with urothelial bladder cancer identified that similar mutations were present in both cancer types but at different frequencies, indicating a potential need for unique management strategies. UTUC tumors were found to have a high rate of mutations that could be targeted with novel therapies.
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Carcinoma de Células Transicionales/genética , Genómica , Neoplasias Renales/genética , Mutación , Neoplasias Ureterales/genética , Neoplasias de la Vejiga Urinaria/genética , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, highly aggressive form of ovarian cancer primarily diagnosed in young women. We identified inactivating biallelic SMARCA4 mutations in 100% of the 12 SCCOHT tumors examined. Protein studies confirmed loss of SMARCA4 expression, suggesting a key role for the SWI/SNF chromatin-remodeling complex in SCCOHT.
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Carcinoma de Células Pequeñas/genética , ADN Helicasas/genética , Mutación/genética , Proteínas Nucleares/genética , Neoplasias Ováricas/genética , Factores de Transcripción/genética , Secuencia de Bases , Ensamble y Desensamble de Cromatina/genética , Biología Computacional , ADN Complementario/genética , Femenino , Componentes del Gen , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
PURPOSE: Rapalogs are allosteric mTOR inhibitors and approved agents for advanced kidney cancer. Reports of clonal heterogeneity in this disease challenge the concept of targeted monotherapy, yet a small subset of patients derives extended benefit. Our aim was to analyze such outliers and explore the genomic background of extreme rapalog sensitivity in the context of intratumor heterogeneity. EXPERIMENTAL DESIGN: We analyzed archived tumor tissue of 5 patients with renal cell carcinoma, who previously achieved durable disease control with rapalogs (median duration, 28 months). DNA was extracted from spatially separate areas of primary tumors and metastases. Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Whole-exome sequence analysis was added to investigate genes beyond this original target list. RESULTS: Five long-term responders contributed 14 specimens to explore clonal heterogeneity. Genomic alterations with activating effect on mTOR signaling were detected in 11 of 14 specimens, offering plausible explanation for exceptional treatment response through alterations in two genes (TSC1 and MTOR). In two subjects, distinct yet functionally convergent alterations activated the mTOR pathway in spatially separate sites. In 1 patient, concurrent genomic events occurred in two separate pathway components across different tumor regions. CONCLUSIONS: Analysis of outlier cases can facilitate identification of potential biomarkers for targeted agents, and we implicate two genes as candidates for further study in this class of drugs. The previously reported phenomenon of clonal convergence can occur within a targetable pathway which might have implications for biomarker development beyond this disease and this class of agents.