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To prepare timely motor actions, we constantly predict future events. Regularly repeating events are often perceived as a rhythm to which we can readily synchronize our movements, just as in dancing to music. However, the neuronal mechanisms underlying the capacity to encode and maintain rhythms are not understood. We trained nonhuman primates to maintain the rhythm of a visual metronome of diverse tempos and recorded neural activity in the supplementary motor area (SMA). SMA exhibited rhythmic bursts of gamma band (30–40 Hz) reflecting an internal tempo that matched the extinguished visual metronome. Moreover, gamma amplitude increased throughout the trial, providing an estimate of total elapsed time. Notably, the timing of gamma bursts and firing rate modulations allowed predicting whether monkeys were ahead or behind the correct tempo. Our results indicate that SMA uses dynamic motor plans to encode a metronome for rhythms and a stopwatch for total elapsed time.

Objetivo: describir las complicaciones intraquirúrgicas y posquirúrgicas inmediatas de la histerectomía radical total abdominal más linfadenectomía pélvica y los resultados anatomo-patológicos en pacientes con cáncer de cérvix en estadio temprano del Instituto de Cancerología - Clínica Las Américas en un periodo de ocho años. Materiales y métodos: cohorte histórica descriptiva de mujeres con diagnóstico de cáncer de cérvix confirmado histológicamente, y que al momento de la cirugía se encontraban en estadios entre IA2 a IIA1 según la clasificación FIGO, y sometidas a histerectomía radical tipo II o III entre agosto de 2003 y julio de 2011 en el Instituto de Cancerología - Clínica Las Américas, institución de salud de carácter privado, centro de referencia de alta complejidad en Medellín (Colombia), donde se atienden pacientes del régimen contributivo y subsidiado. Se hizo muestreo consecutivo. Las variables evaluadas fueron edad, índice de masa corporal, tiempo quirúrgico, estancia hospitalaria, sangrado intraoperatorio, estadio clínico, histología, infiltración estromal, compromiso linfovascular, compromiso parametrial, márgenes, conteo y compromiso ganglionar, transfusión sanguínea, complicaciones intraoperatorias, complicaciones posoperatorias, terapia adyuvante. La información se resumió por medio de medidas de tendencia central y dispersión para variables continuas y proporciones para variables categóricas u ordinales. Resultados: se incluyeron 199 pacientes. La mediana de edad fue de 46 años (28-75), 183 pacientes (92%) fueron diagnosticadas en estadio IB1. El diagnóstico histológico más frecuente fue el escamocelular en 125 casos (62,8%), el adenocarcinoma se presentó en 66 casos (33,1%). El tiempo quirúrgico promedio fue de 188 min (90-315); el sangrado estimado en promedio fue 316 cc (30-2000), 19 pacientes (9,5%) requirieron transfusión sanguínea; el promedio de ganglios extraídos fue 22 (9-61); la estancia hospitalaria fue de 2,44 días (1-31) en promedio. La tasa de complicaciones intraoperatorias fue de 9,5%, todas relacionadas con sangrado intraoperatorio y lesiones vasculares. Se presentaron 73 complicaciones posoperatorias (36,7%). La complicación posoperatoria más frecuente fue la fístula urinaria (6,5 %). Un total de 97 (48,7%) pacientes requirieron terapia adyuvante. Conclusiones: la histerectomía radical abdominal realizada en la población de estudio es un procedimiento seguro, reproducible, con una frecuencia de complicaciones intra y posoperatorias dentro de lo esperado para dicha cirugía.Objective: To describe intra-operative and immediate post-operative complications of total radical abdominal hysterectomy with pelvic lymph node dissection, and the pathology results in patients with early-stage cervical cancer at the Instituto de Cancerología - Clínica de Las Américas, over a 8-year period. Materials and methods: Descriptive historical cohort of women with a diagnosis of histologically confirmed cervical cancer, who were diagnosed as stages IA2 to IIA1 according to the FIGO classification at the time of surgery, undergoing type II or III radical hysterectomy between August 2003 and July 2011 at the Instituto de Cancerología - Clínica Las Américas, a private high-complexity referral center in Medellin, Colombia, that provides care to patients of the contributive and subsidized health insurance regimes. A consecutive sample was used. Assessment variables included body mass index, duration of surgery, hospital stay, intraoperative bleeding, clinical stage, histology, stromal infiltration, lymphovascular involvement, parametrial involvement, margins, node count and lymph node involvement, blood transfusion, intra-operative complications, post-operative complications, adjuvant therapy. Information was summarized on the basis of central trend and scatter measurements for continuous variables, and proportions for categorical or ordinal variables. Results: Overall, 199 patients were included with a median age of 46 years (28-75). Of them, 183 (92%) were diagnosed as stage IB1. The most frequent histological diagnosis was squamous cell carcinoma in 125 cases (62.8%), while adenocarcinoma occurred in 66 cases (33.1%). The mean duration of the surgical procedure was 188 min (90-315); average estimated blood loss was 316 cc (30-2000), and 19 patients (9.5%) required a blood transfusion; in average, 22 lymph nodes (9-61) were removed; median hospital stay was 2.44 days (1-31). The rate of intra-operative complications was 9%, all of them associated with vascular lesions and intraoperative bleeding. There were 73 post-operative complications (36.7%), the most frequent of which was urinary fistula (6.5%). Overall, 97 patients (48.7%) required adjuvant therapy. Conclusions: Abdominal radical hysterectomy performed in the study population is a safe, feasible and reproducible, with a frequency of operative and posoperative complications as is expected for that surgery.

Objetivo: presentar dos casos de pacientes con cáncer de cérvix estadios IA2, con deseos de paridad, que fueron sometidas a traquelectomía radical vaginal como cirugía conservadora de la fertilidad, y realizar una revisión de la literatura científica disponible acerca de las complicaciones, tasas de recurrencia, calidad de vida y fertilidad posterior. Materiales y métodos: se presentan dos casos de pacientes con cáncer de cérvix estadios IA2 con deseo de fertilidad futura, que fueron sometidas a traquelectomía radical vaginal con linfadenectomía pélvica laparoscópica, en el Hospital San José, institución privada de referencia ubicada en Bogotá, Colombia, en el año 2011. La búsqueda de la literatura se hizo a través de Medline en idioma inglés y español. Se buscaron reportes y series de casos, ensayos clínicos controlados y revisiones de tema en el periodo comprendido entre 1980 al 2011. Conclusión: la traquelectomía radical vaginal y la linfadenectomía pélvica laparoscópica son una alternativa que se debe considerar para el manejo conservador de la fertilidad en pacientes con estadios tempranos de cáncer invasivo de cérvix.

The present report is the northernmost capture of the piscicolid leech Stibarobdella moorei in the western South Atlantic Ocean. This is also the first time S. moorei is found associated to a batoid fish in the Brazilian coast, the eyespot skate Atlantoraja cyclophora. Stibarobdella moorei was found fixed in the dorsal side of a male eyespot skate, caught by bottom trawl around of the São Paulo coast, southeastern Brazil. A brief description of the morphology of the parasite and a discussion on the taxonomic status of the S. moorei are presented.

El cáncer es una enfermedad caracterizada por la proliferación anormal de células neoplásicas, dada en esencia por alteraciones genéticas y epigenéticas. El control de las diferentes funciones celulares está dado por los genes codificados en el ADN, por lo tanto algunas alteraciones en genes que codifican para las proteínas involucradas en el ciclo de proliferación celular pue­ den inducir una cascada de eventos que llevan a la producción del fenotipo cancerígeno. La transformación maligna requiere que ocurran alteraciones en genes específicos que controlan la proliferación celular, la apoptosis y el mantenimiento de la integridad del ADN en la misma célula. Las mutaciones tienen la posibilidad de aparecer de manera esporádica o de heredarse, pueden ser sustituciones de bases, adiciones, deleciones o cambios epigenéticos. El presente artículo revisa conceptos moleculares involucrados en la génesis del cáncer.

Abstract Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP). The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE) samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Large scale comprehensive genomic profiling (CGP) has led to an improved understanding of oncogenic mutations in acute myeloid leukemia (AML), as well as identification of alterations that can serve as targets for potential therapeutic intervention. We sought to gain insight into age-associated variants in AML through comparison of extensive DNA and RNA-based GP results from pediatric and adult AML. Sequencing of 932 AML specimens (179 pediatric (age 0-18), 753 adult (age ≥ 19)) from diagnostic, relapsed, and refractory times points was performed. Comprehensive DNA (405 genes) and RNA (265) sequencing to identify a variety of structural and short variants was performed. We found that structural variants were highly prevalent in the pediatric cohort compared to the adult cohort (57% vs. 30%; p < 0.001), with certain structural variants detected only in the pediatric cohort. Fusions were the most common structural variant and were highly prevalent in AML in very young children occurring in 68% of children < 2 years of age. We observed an inverse trend in the prevalence of fusions compared to the average number of mutations per patient. In contrast to pediatric AML, adult AML was marked by short variants and multiple mutations per patient. Mutations that were common in adult AML were much less common in the adolescent and young adult cohort and were rare or absent in the pediatric cohort. Clinical CGP demonstrates the biologic differences in pediatric vs. adult AML that have significant therapeutic impacts on prognosis, therapeutic allocation, disease monitoring, and the use of more targeted therapies., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest in this article.

Background: High tumor mutational burden (TMB) is an emerging biomarker of sensitivity to immune checkpoint inhibitors and has been shown to be more significantly associated with response to PD-1 and PD-L1 blockade immunotherapy than PD-1 or PD-L1 expression, as measured by immunohistochemistry (IHC). The distribution of TMB and the subset of patients with high TMB has not been well characterized in the majority of cancer types., Methods: In this study, we compare TMB measured by a targeted comprehensive genomic profiling (CGP) assay to TMB measured by exome sequencing and simulate the expected variance in TMB when sequencing less than the whole exome. We then describe the distribution of TMB across a diverse cohort of 100,000 cancer cases and test for association between somatic alterations and TMB in over 100 tumor types., Results: We demonstrate that measurements of TMB from comprehensive genomic profiling are strongly reflective of measurements from whole exome sequencing and model that below 0.5 Mb the variance in measurement increases significantly. We find that a subset of patients exhibits high TMB across almost all types of cancer, including many rare tumor types, and characterize the relationship between high TMB and microsatellite instability status. We find that TMB increases significantly with age, showing a 2.4-fold difference between age 10 and age 90 years. Finally, we investigate the molecular basis of TMB and identify genes and mutations associated with TMB level. We identify a cluster of somatic mutations in the promoter of the gene PMS2, which occur in 10% of skin cancers and are highly associated with increased TMB., Conclusions: These results show that a CGP assay targeting ~1.1 Mb of coding genome can accurately assess TMB compared with sequencing the whole exome. Using this method, we find that many disease types have a substantial portion of patients with high TMB who might benefit from immunotherapy. Finally, we identify novel, recurrent promoter mutations in PMS2, which may be another example of regulatory mutations contributing to tumorigenesis.

Purpose: The clinical utility of next-generation sequencing (NGS) in breast cancer has not been demonstrated. We hypothesized that we could perform NGS of a new biopsy from patients with metastatic triple-negative breast cancer (TNBC) in a clinically actionable timeframe., Experimental Design: We planned to enroll 40 patients onto a prospective study, Individualized Molecular Analyses Guide Efforts (IMAGE), to evaluate the feasibility of obtaining a new biopsy of a metastatic site, perform NGS (FoundationOne), and convene a molecular tumor board to formulate treatment recommendations within 28 days. We collected blood at baseline and at time of restaging to assess cell-free circulating plasma tumor DNA (ptDNA)., Results: We enrolled 26 women with metastatic TNBC who had received ≥1 line of prior chemotherapy, and 20 (77%) underwent NGS of a metastatic site biopsy. Twelve (60%) evaluable patients received treatment recommendations within 28 days of consent. The study closed after 20 patients underwent NGS, based on protocol-specified interim futility analysis. Three patients went on to receive genomically directed therapies. Twenty-four of 26 patients had genetic alterations successfully detected in ptDNA. Among 5 patients, 4 mutations found in tumor tissues were not identified in blood, and 4 mutations found in blood were not found in corresponding tumors. In 9 patients, NGS of follow-up blood samples showed 100% concordance with baseline blood samples., Conclusions: This study demonstrates challenges of performing NGS on prospective tissue biopsies in patients with metastatic TNBC within 28 days, while also highlighting the potential use of blood as a more time-efficient and less invasive method of mutational assessment. Clin Cancer Res; 23(2); 379-86. ©2016 AACR., (©2016 American Association for Cancer Research.)

Genomic studies have identified recurrent somatic mutations in acute leukemias. However, current murine models do not sufficiently encompass the genomic complexity of human leukemias. To develop preclinical models, we transplanted 160 samples from patients with acute leukemia (acute myeloid leukemia, mixed lineage leukemia, B-cell acute lymphoblastic leukemia, T-cell ALL) into immunodeficient mice. Of these, 119 engrafted with expected immunophenotype. Targeted sequencing of 374 genes and 265 frequently rearranged RNAs detected recurrent and novel genetic lesions in 48 paired primary tumor (PT) and patient-derived xenotransplant (PDX) samples. Overall, the frequencies of 274 somatic variant alleles correlated between PT and PDX samples, although the data were highly variable for variant alleles present at 0-10%. Seventeen percent of variant alleles were detected in either PT or PDX samples only. Based on variant allele frequency changes, 24 PT-PDX pairs were classified as concordant while the other 24 pairs showed various degree of clonal discordance. There was no correlation of clonal concordance with clinical parameters of diseases. Significantly more bone marrow samples than peripheral blood samples engrafted discordantly. These data demonstrate the utility of developing PDX banks for modeling human leukemia, and emphasize the importance of genomic profiling of PDX and patient samples to ensure concordance before performing mechanistic or therapeutic studies., Competing Interests: KW, MKN, JH, ALD, GMF, DL, SR, PJS, EMS, TB, GAO, RY, VAM are employees and equity holders, RLL is a consultant for Foundation Medicine, Inc.

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance., (© 2016 by The American Society of Hematology.)

Context: Metastatic metaplastic breast carcinoma (MPBC) is an uncommon, but aggressive, tumor resistant to conventional chemotherapy., Objective: To learn whether next-generation sequencing could identify potential targets of therapy for patients with relapsed and metastatic MPBC., Design: Hybridization capture of 3769 exons from 236 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer was applied to a minimum of 50 ng of DNA extracted from 20 MPBC formalin-fixed, paraffin-embedded specimens and sequenced to high uniform coverage., Results: The 20 patients with MPBC had a median age of 62 years (range, 42-86 years). There were 9 squamous (45%), 9 chondroid (45%), and 2 spindle cell (10%) MPBCs, all of which were high grade. Ninety-three genomic alterations were identified, (range, 1-11) with 19 of the 20 cases (95%) harboring an alteration that could potentially lead to a targeted treatment option. The most-common alterations were in TP53 (n = 69; 75%), PIK3CA (n = 37; 40%), MYC (n = 28; 30%), MLL2 (n = 28; 30%), PTEN (n = 23; 25%), CDKN2A/B (n = 19; 20%), CCND3 (n = 14; 15%), CCNE1 (n = 9; 10%), EGFR (n = 9; 10%), and KDM6A (n = 9; 10%); AKT3, CCND1, CCND2, CDK4, FBXW7, FGFR1, HRAS, NF1, PIK3R1, and SRC were each altered in a single case. All 16 MPBCs (100%) that were negative for ERBB2 (HER2) overexpression by immunohistochemistry and/or ERBB2 (HER2) amplification by fluorescence in situ hybridization were also uniformly (100%) negative for ERBB2 amplification by next-generation sequencing-based copy-number assessment., Conclusions: Our results indicate that genomic profiling using next-generation sequencing can identify clinically meaningful alterations that have the potential to guide targeted treatment decisions in most patients with metastatic MPBC.

Importance: For carcinoma of unknown primary site (CUP), determining the primary tumor site may be uninformative and often does not improve outcome., Objective: To discover opportunities for targeted therapies in patients with CUP not currently searched for in routine practice., Design, Setting, and Participants: Comprehensive genomic profiling on 200 CUP formalin-fixed paraffin-embedded specimens (mean, 756× coverage) using the hybrid-capture-based FoundationOne assay at academic and community oncology clinics., Main Outcomes and Measures: Presence of targetable genomic alterations (GAs) in CUP and responses to targeted therapies., Results: There were 125 adenocarcinomas of unknown primary site (ACUPs) and 75 carcinomas of unknown primary site without features of adenocarcinoma (non-ACUPs). At least 1 GA was found in 192 (96%) of CUP specimens, with a mean (SD) of 4.2 (2.8) GAs per tumor. The most frequent GAs were in TP53 (110 [55%]), KRAS (40 [20%]), CDKN2A (37 [19%]), MYC (23 [12%]), ARID1A (21 [11%]), MCL1 (19 [10%]), PIK3CA (17 [9%]), ERBB2 (16 [8%]), PTEN (14 [7%]), EGFR (12 [6%]), SMAD4 (13 [7%]), STK11 (13 [7%]), SMARCA4 (12 [6%]), RB1 (12 [6%]), RICTOR (12 [6%]), MLL2 (12 [6%]), BRAF (11 [6%]), and BRCA2 (11 [6%]). One or more potentially targetable GAs were identified in 169 of 200 (85%) CUP specimens. Mutations or amplifications of ERBB2 were more frequent in ACUPs (13 [10%]) than in non-ACUPs (3 [4%]). Alterations of EGFR (10 [8%] vs 2 [3%]) and BRAF (8 [6%] vs 3 [4%]) were more common in ACUPs than in non-ACUPs. Strikingly, clinically relevant alterations in the receptor tyrosine kinase (RTK)/Ras signaling pathway including alterations in ALK, ARAF, BRAF, EGFR, FGFR1, FGFR2, KIT, KRAS, MAP2K1, MET, NF1, NF2, NRAS, RAF1, RET, and ROS1 were found in 90 (72%) ACUPs but in only 29 (39%) non-ACUPs (P < .001)., Conclusions and Relevance: Almost all CUP samples harbored at least 1 clinically relevant GA with potential to influence and personalize therapy. The ACUP tumors were more frequently driven by GAs in the highly druggable RTK/Ras/mitogen-activated protein kinase (MAPK) signaling pathway than the non-ACUP tumors. Comprehensive genomic profiling can identify novel treatment paradigms to address the limited options and poor prognoses of patients with CUP.

Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-(6-iodobenzo[d][1.3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purine-6-amine (PU-H71). Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML.

Aims: Adrenocortical carcinoma (ACC) carries a poor prognosis and current systemic cytotoxic therapies result in only modest improvement in overall survival. In this retrospective study, we performed a comprehensive genomic profiling of 29 consecutive ACC samples to identify potential targets of therapy not currently searched for in routine clinical practice., Methods: DNA from 29 ACC was sequenced to high, uniform coverage (Illumina HiSeq) and analysed for genomic alterations (GAs)., Results: At least one GA was found in 22 (76%) ACC (mean 2.6 alterations per ACC). The most frequent GAs were in TP53 (34%), NF1 (14%), CDKN2A (14%), MEN1 (14%), CTNNB1 (10%) and ATM (10%). APC, CCND2, CDK4, DAXX, DNMT3A, KDM5C, LRP1B, MSH2 and RB1 were each altered in two cases (7%) and EGFR, ERBB4, KRAS, MDM2, NRAS, PDGFRB, PIK3CA, PTEN and PTCH1 were each altered in a single case (3%). In 17 (59%) of ACC, at least one GA was associated with an available therapeutic or a mechanism-based clinical trial., Conclusions: Next-generation sequencing can discover targets of therapy for relapsed and metastatic ACC and shows promise to improve outcomes for this aggressive form of cancer., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Aims: Small cell lung cancer (SCLC) carries a poor prognosis, and the systemic therapies currently used as treatments are only modestly effective, as demonstrated by a low 5-year survival at only ∼5%. In this retrospective collected from March 2013 to study, we performed comprehensive genomic profiling of 98 small cell undifferentiated lung cancer (SCLC) samples to identify potential targets of therapy not currently searched for in routine clinical practice., Methods: DNA from 98 SCLC was sequenced to high, uniform coverage (Illumina HiSeq 2500) and analysed for all classes of genomic alterations., Results: A total of 386 alterations were identified for an average of 3.9 alterations per tumour (range 1–10). Fifty-two (53%) of cases harboured at least 1 actionable alteration with the potential to personalise therapy including base substitutions, amplifications or homozygous deletions in RICTOR (10%), KIT (7%), PIK3CA (6%), EGFR (5%), PTEN (5%), KRAS (5%), MCL1 (4%), FGFR1 (4%), BRCA2, (4%), TSC1 (3%), NF1 (3%), EPHA3 (3%) and CCND1. The most common non-actionable genomic alterations were alterations in TP53 (86% of SCLC cases), RB1 (54%) and MLL2 (17%)., Conclusions: Greater than 50% of the SCLC cases harboured at least one actionable alteration. Given the limited treatment options and poor prognosis of patients with SCLC, comprehensive genomic profiling has the potential to identify new treatment paradigms and meet an unmet clinical need for this disease.

Background: Intrahepatic cholangiocarcinoma (ICC) is a subtype of primary liver cancer that is rarely curable by surgery and is rapidly increasing in incidence. Relapsed ICC has a poor prognosis, and current systemic nontargeted therapies are commonly extrapolated from those used in other gastrointestinal malignancies. We hypothesized that genomic profiling of clinical ICC samples would identify genomic alterations that are linked to targeted therapies and that could facilitate a personalized approach to therapy., Methods: DNA sequencing of hybridization-captured libraries was performed for 3,320 exons of 182 cancer-related genes and 36 introns of 14 genes frequently rearranged in cancer. Sample DNA was isolated from 40 μm of 28 formalin-fixed paraffin-embedded ICC specimens and sequenced to high coverage., Results: The most commonly observed alterations were within ARID1A (36%), IDH1/2 (36%), and TP53 (36%) as well as amplification of MCL1 (21%). Twenty cases (71%) harbored at least one potentially actionable alteration, including FGFR2 (14%), KRAS (11%), PTEN (11%), CDKN2A (7%), CDK6 (7%), ERBB3 (7%), MET (7%), NRAS (7%), BRCA1 (4%), BRCA2 (4%), NF1 (4%), PIK3CA (4%), PTCH1 (4%), and TSC1 (4%). Four (14%) of the ICC cases featured novel gene fusions involving the tyrosine kinases FGFR2 and NTRK1 (FGFR2-KIAA1598, FGFR2-BICC1, FGFR2-TACC3, and RABGAP1L-NTRK1)., Conclusion: Two thirds of patients in this study harbored genomic alterations that are associated with targeted therapies and that have the potential to personalize therapy selection for to individual patients.

Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 μm of formalin-fixed, paraffin-embedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 × and evaluated for all classes of genomic alterations (GAs). Actionable GAs were defined as those impacting the selection of targeted anticancer therapies on the market or in registered clinical trials. A total of 139 GAs were identified, with an average of 4.0 GAs per tumor (range 0-10), of which 78 (56%) were considered actionable, with an average of 2.2 per tumor (range 0-7). Twenty-nine (83%) cases harbored at least one actionable GA including: PIK3CA (9 cases; 26%); CDKN2A/B (8 cases; 23%); CCND1 (5 cases; 14%); FGFR1 (5 cases; 14%); CCND3 (4 cases; 11%); FGFR3 (4 cases; 11%); MCL1 (4 cases; 11%); MDM2 (4 cases; 11%); EGFR (2 cases, 6%); ERBB2 (HER2/neu) (2 cases, 6%); NF1 (2 cases, 6%) and TSC1 (2 cases, 6%). Notable additional alterations included TP53 (19 cases, 54%) and RB1 (6 cases; 17%). Genes involved in chromatin modification were altered by nonsense mutation, splice site mutation or frameshift indel in a mutually exclusive manner in nearly half of all cases including KDM6A (10 cases; 29%) and ARID1A (7 cases; 20%). Comprehensive NGS of 35 UCs of the bladder revealed a diverse spectrum of actionable GAs in 83% of cases, which has the potential to inform treatment decisions for patients with relapsed and metastatic disease.

We hypothesized that next-generation sequencing could reveal actionable genomic alterations (GAs) and potentially expand treatment options for patients with advanced adenoid cystic carcinoma (ACC). Genomic profiling using next-generation sequencing was performed on hybridization-captured, adapter ligation libraries derived from 28 relapsed and metastatic formalin-fixed paraffin-embedded ACC. The 3230 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer were fully sequenced using the Illumina HiSeq 2000. All classes of GAs were evaluated. Actionable GAs were defined as those impacting targeted anticancer therapies on the market or in registered clinical trials. A total of 44 GAs were identified in the 28 ACC tumors, with 12 of 28 (42.9%) of tumors harboring at least 1 potentially actionable GA. The most common nonactionable GAs were identified in KD6MA (5 cases; 18%), ARID1A (4 cases; 14%), RUNX1 (2 cases; 7%), and MYC (2 cases; 7%). Actionable GAs included NOTCH1 (3 cases; 11%), MDM2 (2 cases; 7%), PDGFRA (2 cases; 7%), and CDKN2A/B (p16) (2 cases; 7%). Other potentially actionable GAs identified in a single case included: mutations in AKT1, BAP1, EGFR, and PIK3CA, homozygous deletion of FBXW7, and amplifications of CDK4, FGFR1, IGF1R, KDR, KIT, and MCL1. The frequency of GA in ACC is lower than that seen in the more common solid tumors. Comprehensive genomic profiling of ACC can identify actionable GAs in a subset of patients that could influence therapy for these difficult-to-treat progressive neoplasms.

Purpose: Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options., Experimental Design: DNA was extracted from 40 μm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors. Sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries to high coverage for 3,230 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alteration., Results: Mutations in the extracellular domain of ERBB2 were identified in 6 of 15 (40%) of MPUC: S310F (four cases), S310Y (one case), and R157W (one case). All six cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification and Erbb2 overexpression. In contrast, 6 of 64 (9.4%) non-MPUC harbored an ERBB2 alteration, including base substitution (three cases), amplification (two cases), and gene fusion (one case), which is higher than the 2 of 159 (1.3%) protein-changing ERBB2 mutations reported for urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared with non-MPUC is significant both between this series (P < 0.0084) and for all types of urinary tract cancer in COSMIC (P < 0.001)., Conclusions: NGS of MPUC revealed a high incidence of mutation in the extracellular domain of ERBB2, a gene for which there are five approved targeted therapies. NGS can identify genomic alteration, which inform treatment options for the majority of MPUC patients.

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.

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.

Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of approximately 30% were obtained for a range of ZMW diameters (70-100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.

The HCV internal ribosome entry site (IRES) directly regulates the assembly of translation initiation complexes on viral mRNA by a sequential pathway that is distinct from canonical eukaryotic initiation. The HCV IRES can form a binary complex with an eIF-free 40S ribosomal subunit. Next, a 48S-like complex assembles at the AUG initiation codon upon association of eIF3 and ternary complex. 80S complex formation is rate limiting and follows the GTP-dependent association of the 60S subunit. Efficient assembly of the 48S-like and 80S complexes on the IRES mRNA is dependent upon maintenance of the highly conserved HCV IRES structure. This revised model of HCV IRES translation initiation provides a context to understand the function of different HCV IRES domains during translation initiation.

Complex RNA structures regulate many biological processes, but are often too large for structure determination by NMR methods. The 5' untranslated region (5' UTR) of the hepatitis C viral (HCV) RNA genome contains an internal ribosome entry site (IRES) that binds to 40S ribosomal subunits with high affinity and specificity to control translation. Domain II of the HCV IRES forms a 25-kDa folded subdomain that may alter ribosome conformation. We report here the structure of domain II as determined using an NMR approach that combines short- and long-range structural data. Domain II adopts a distorted L-shape structure, and its overall shape in the free form is markedly similar to its 40S subunit-bound form; this suggests how domain II may modulate 40S subunit conformation. The results show how NMR can be used for structural analysis of large biological RNAs.

Translation of the hepatitis C virus genomic RNA is mediated by an internal ribosome entry site (IRES). The 330-nt IRES RNA forms a binary complex with the small 40S ribosomal subunit as a first step in translation initiation. Here chemical probing and 4-thiouridine-mediated crosslinking are used to characterize the interaction of the HCV IRES with the HeLa 40S subunit. No IRES-18S rRNA contacts were detected, but several specific crosslinks to 40S ribosomal proteins were observed. The identity of the crosslinked proteins agrees well with available structural information and provides new insights into HCV IRES function. The protein-rich surface of the 40S subunit thus mediates the IRES-ribosome interaction.

Translation of the hepatitis C virus (HCV) polyprotein is initiated at an internal ribosome entry site (IRES) element in the 5' untranslated region of HCV RNA. The HCV IRES element interacts directly with the 40S subunit, and biochemical experiments have implicated RNA elements near the AUG start codon as required for IRES-40S subunit complex formation. The data we present here show that two RNA stem loops, domains IIId and IIIe, are involved in IRES-40S subunit interaction. The structures of the two RNA domains were solved by NMR spectroscopy and reveal structural features that may explain their role in IRES function.