Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

Impact of travel distance on receipt of indicated adjuvant therapy in resected non-small cell lung cancer.

OBJECTIVE: We have previously demonstrated the negative impact of travel distance on adherence to surveillance imaging guidelines for resected non-small cell lung cancer (NSCLC). The influence of patient residential location on adherence to recommended postoperative treatment plans remains unclear. We sought to characterize the impact of travel distance on receipt of indicated adjuvant therapy in resected NSCLC. METHODS: We performed a single-institution, retrospective review of patients with stage II-III NSCLC who underwent upfront pulmonary resection, 2012-2016. Clinicopathologic and operative/perioperative details of treatment were collected. Travel distance was measured from patients' homes to the operative hospital. Our primary outcome was receipt of adjuvant systemic or radiotherapy. Travel distance was stratified as <100 or >100 miles. Multivariable logistic regression was performed. RESULTS: In total, 391 patients met inclusion criteria, with mean age of 65.9 years and fairly even sex distribution (182 women, 49.2%). Most patients were Non-Hispanic White (n = 309, 83.5%), and most frequent clinical stage was II (n = 254, 64.9%). Indicated adjuvant therapy was received by 266 (71.9%), and median distance traveled was 209 miles (interquartile range, 50.7-617). Multivariate analysis revealed that longer travel distance was inversely associated with receipt of indicated adjuvant therapy (odds ratio, 0.13; 95% confidence interval, 0.06-0.26; P < .001). In addition, Black patients were less likely to receive appropriate treatment (odds ratio, 0.05; 95% confidence interval, 0.02-0.15; P < .001). CONCLUSIONS: Travel distance >100 miles negatively impacts the likelihood of receiving indicated adjuvant therapy in NSCLC. Indications for systemic therapy in earlier staged disease are rapidly expanding, and these findings bear heightened relevance as we aim to provide equitable access to all patients.

Technical Aspects of Robotic First Rib Resection.

Robot-assisted thoracoscopic surgery for the treatment of thoracic outlet syndrome is a novel approach that continues to increase in popularity due to advantages compared with traditional open first rib resection. Following publication of the Society of Vascular Surgeons expert statement in 2016, the diagnosis and management of thoracic outlet syndrome is favorably evolving. Technical mastery of the operation requires precise knowledge of anatomy, comfort with robotic surgical platforms, and understanding of the disease.

Robotic First Rib Resection and Robotic Chest Wall Resection.

Robot-assisted thoracoscopic surgery for the treatment of thoracic outlet syndrome and chest wall lesions are burgeoning topics on thoracic surgery. Following publication of the Society of Vascular Surgeons expert statement in 2016, the diagnosis and management of thoracic outlet syndrome is favorably evolving. Robot-assisted first rib resection is a novel approach to the surgical management of thoracic outlet syndrome that may have advantages compared with traditional open surgical approaches. Robot-assisted chest wall resection is technically feasible for a variety of chest wall conditions and may also have advantages compared with thoracotomy approaches.

Use of weighted reference panels based on empirical estimates of ancestry for capturing untyped variation.

Many association methods use a subset of genotyped single nucleotide polymorphisms (SNPs) to capture or infer genotypes at other untyped SNPs. We and others previously showed that tag SNPs selected to capture common variation using data from The International HapMap Consortium (Nature 437:1299-1320, 2005), The International HapMap Consortium (Nature 449:851-861, 2007) could also capture variation in populations of similar ancestry to HapMap reference populations (de Bakker et al. in Nat Genet 38:1298-1303, 2006; González-Neira et al. in Genome Res 16:323-330, 2006; Montpetit et al. in PLoS Genet 2:282-290, 2006; Mueller et al. in Am J Hum Genet 76:387-398, 2005). To capture variation in admixed populations or populations less similar to HapMap panels, a "cosmopolitan approach," in which all samples from HapMap are used as a single reference panel, was proposed. Here we refine this suggestion and show that use of a "weighted reference panel," constructed based on empirical estimates of ancestry in the target population (relative to available reference panels), is more efficient than the cosmopolitan approach. Weighted reference panels capture, on average, only slightly fewer common variants (minor allele frequency > 5%) than the cosmopolitan approach (mean r (2) = 0.977 vs. 0.989, 94.5% variation captured vs. 96.8% at r (2) > 0.8), across the five populations of the Multiethnic Cohort, but entail approximately 25% fewer tag SNPs per panel (average 538 vs. 718). These results extend a recent study in two Indian populations (Pemberton et al. in Ann Hum Genet 72:535-546, 2008). Weighted reference panels are potentially useful for both the selection of tag SNPs in diverse populations and perhaps in the design of reference panels for imputation of untyped genotypes in genome-wide association studies in admixed populations.

Multidisciplinary Therapy of Esophageal Cancer.

Multimodality therapy is the standard of care for locoregional esophageal cancers (greater than clinical T3 or Nþ), including Siewert type 1 and 2 gastroesophageal junction tumors. Induction regimen, chemotherapy only or chemoradiation, is an area of controversy and often institution-specific, as neither has shown to be superior. Response to induction therapy is an important prognostic marker. For esophageal squamous cell carcinoma, it may be acceptable to observe clinical complete responders after chemoradiotherapy and perform salvage esophagectomy for recurrent disease. Clinical T2N0 esophageal cancer presents a unique challenge given its inaccuracy in clinical staging; management of this particular subset is controversial.

Plasma circulating tumor DNA as a potential tool for disease monitoring in head and neck cancer.

BACKGROUND: Recommendations for perioperative therapy in head and neck cancer are not explicit and recurrence occurs frequently. Circulating tumor DNA is an emerging cancer biomarker, but has not been extensively explored for detection of recurrence in head and neck cancer. METHODS: Patients diagnosed with head and neck squamous cell carcinoma were recruited into the study protocol. Tumors were sequenced to identify patient-specific mutations. Mutations were then identified in plasma circulating tumor DNA from pre-treatment blood samples and longitudinally during standard follow-up. Circulating tumor DNA status during follow-up was correlated to disease recurrence. RESULTS: Samples were taken from eight patients. Tumor mutations were verified in seven patients. Baseline circulating tumor DNA was positive in six patients. Recurrence occurred in four patients, two of whom had detectable circulating tumor DNA prior to recurrence. CONCLUSION: Circulating tumor DNA is a potential tool for disease and recurrence monitoring following curative therapy in head and neck cancer, allowing for better prognostication, and/or modification of treatment strategies.

Detection of Circulating Tumor DNA in Plasma: A Potential Biomarker for Esophageal Adenocarcinoma.

BACKGROUND: Recent literature has demonstrated the potential of "liquid biopsy" and detection of circulating tumor (ct)DNA as a cancer biomarker. However, to date there is a lack of data specific to esophageal adenocarcinoma (EAC). This study was conducted to determine how detection and quantification of ctDNA changes with disease burden in patients with EAC and evaluate its potential as a biomarker in this population. METHODS: Blood samples were obtained from patients with stage I to IV EAC. Longitudinal blood samples were collected from a subset of patients. Imaging studies and pathology reports were reviewed to determine disease course. Tumor samples were sequenced to identify mutations. Mutations in plasma DNA were detected using custom, barcoded, patient-specific sequencing libraries. Mutations in plasma were quantified, and associations with disease stage and response to therapy were explored. RESULTS: Plasma samples from a final cohort of 38 patients were evaluated. Baseline plasma samples were ctDNA positive for 18 patients (47%) overall, with tumor allele frequencies ranging from 0.05% to 5.30%. Detection frequency of ctDNA and quantity of ctDNA increased with stage. Data from longitudinal samples indicate that ctDNA levels correlate with and precede evidence of response to therapy or recurrence. CONCLUSIONS: ctDNA can be detected in plasma of EAC patients and correlates with disease burden. Detection of ctDNA in early-stage EAC is challenging and may limit diagnostic applications. However, our data demonstrate the potential of ctDNA as a dynamic biomarker to monitor treatment response and disease recurrence in patients with EAC.

Association studies of common variants in 10 hypogonadotropic hypogonadism genes with age at menarche.

CONTEXT: Although the timing of puberty is a highly heritable trait, little is known about the genes that regulate pubertal timing in the general population. Several genes have been identified that, when mutated, cause disorders of delayed or absent puberty such as hypogonadotropic hypogonadism (HH). OBJECTIVE: Because severe variants in HH-related genes cause a severe puberty phenotype, we hypothesized that common subtle variation in these genes could contribute to the population variation in pubertal timing. DESIGN: We assessed common genetic variation in 10 HH-related genes in 1801 women from the Hawaii and Los Angeles Multiethnic Cohort with either early (age<11 yr) or late (age>14 yr) menarche and in other replication samples. In addition to these common variants, we also studied the most frequently reported HH mutations to assess their role in the population variation in pubertal timing. SETTING AND PATIENTS/OTHER PARTICIPANTS: Within the general community, 1801 women from the Hawaii and Los Angeles Multiethnic Cohort participated. MAIN OUTCOME MEASURES: We assessed the association of genetic variation with age at menarche. RESULTS: We found no significant association between any of the variants tested and age at menarche, although we cannot rule out modest effects of these variants or of other variants at long distances from the coding region. In several self-reported racial/ethnic groups represented in our study, we observed an association between estimated genetic ancestry and age at menarche. CONCLUSIONS: Our results suggest that common variants near 10 HH-related loci do not play a substantial role in the regulation of age at menarche in the general population.

Post-resection complications: abscesses, empyemas, bronchopleural fistulas.

The role of thoracic surgeons in the management of pulmonary infection has evolved over time as the medical treatments have improved. We herein review historical and current management for surgically-treated pulmonary infections-lung abscesses, empyemas, and bronchopleural fistulas. In particular, we review when the surgeons need to be involved for infectious cases, our algorithm/approach to empyemas, and summary of post-operative bronchopleural fistula in tuberculosis cases.

Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing.

Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.