Effect of Antigen Retrieval on Genomic DNA From Immunodissected Samples.

Immunohistochemical (IHC) staining is an established technique for visualizing proteins in tissue sections for research studies and clinical applications. IHC is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immunodissection, computer-aided laser dissection (CALD), expression microdissection (xMD), and other techniques. The initial antigen retrieval (AR) process increases epitope availability and improves staining characteristics; however, the procedure can damage DNA. To better understand the effects of AR on DNA quality and quantity in immunodissected samples, both clinical specimens (KRAS gene mutation positive cases) and model system samples (lung cancer patient-derived xenograft tissue) were subjected to commonly employed AR methods (heat induced epitope retrieval [HIER], protease digestion) and the effects on DNA were assessed by Qubit, fragment analysis, quantitative PCR, digital droplet PCR (ddPCR), library preparation, and targeted sequencing. The data showed that HIER resulted in optimal IHC staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased overall yields. However, neither of the AR methods combined with IHC prevented ddPCR amplification of small amplicons and gene mutations were successfully identified from immunodissected clinical samples. The results indicate for the first time that DNA recovered from immunostained slides after standard AR and IHC processing can be successfully employed for genomic mutation analysis via ddPCR and next-generation sequencing (NGS) short-read methods.

Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine.

Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.

Preoperative nutritional counselling in patients undergoing oesophagectomy.

BACKGROUND: Patients undergoing surgery for oesophageal cancer are at high risk of malnutrition due to pathology and neoadjuvent therapy. This study sought to determine if oesophageal cancer patients undergoing oesophagectomy achieve superior clinical outcomes when preoperative nutritional counselling is performed. METHODS: Oesophageal cancer patients undergoing oesophagectomy were retrospectively divided into cohorts based on those who received (n = 48) and did not receive (n = 58) preoperative nutritional counselling. We compared weight loss, length of stay, 30-day readmission related to nutrition or feeding tube problems, and 90-day mortality. RESULTS: Per cent weight loss was less in patients who received preoperative nutritional counselling. There was a trend toward decreased mean length of stay and there were fewer readmissions for feeding tube-related complications in patients who received counselling. CONCLUSIONS: Nutritional counselling before surgery may lead to decreased weight loss and reduced readmissions for feeding tube-related complications in patients with oesophageal cancer undergoing oesophagectomy.

An advanced molecular medicine case report of a rare human tumor using genomics, pathomics, and radiomics.

Background: Pulmonary Sclerosing Pneumocytoma (PSP) is a rare tumor of the lung with a low malignant potential that primarily affects females. Initial studies of PSP focused primarily on analyzing features uncovered using conventional X-ray or CT imaging. In recent years, because of the widespread use of next-generation sequencing (NGS), the study of PSP at the molecular-level has emerged. Methods: Analytical approaches involving genomics, radiomics, and pathomics were performed. Genomics studies involved both DNA and RNA analyses. DNA analyses included the patient's tumor and germline tissues and involved targeted panel sequencing and copy number analyses. RNA analyses included tumor and adjacent normal tissues and involved studies covering expressed mutations, differential gene expression, gene fusions and molecular pathways. Radiomics approaches were utilized on clinical imaging studies and pathomics techniques were applied to tumor whole slide images. Results: A comprehensive molecular profiling endeavor involving over 50 genomic analyses corresponding to 16 sequencing datasets of this rare neoplasm of the lung were generated along with detailed radiomic and pathomic analyses to reveal insights into the etiology and molecular behavior of the patient's tumor. Driving mutations (AKT1) and compromised tumor suppression pathways (TP53) were revealed. To ensure the accuracy and reproducibility of this study, a software infrastructure and methodology known as NPARS, which encapsulates NGS and associated data, open-source software libraries and tools including versions, and reporting features for large and complex genomic studies was used. Conclusion: Moving beyond descriptive analyses towards more functional understandings of tumor etiology, behavior, and improved therapeutic predictability requires a spectrum of quantitative molecular medicine approaches and integrations. To-date this is the most comprehensive study of a patient with PSP, which is a rare tumor of the lung. Detailed radiomic, pathomic and genomic molecular profiling approaches were performed to reveal insights regarding the etiology and molecular behavior. In the event of recurrence, a rational therapy plan is proposed based on the uncovered molecular findings.

NPARS-A Novel Approach to Address Accuracy and Reproducibility in Genomic Data Science.

Background: Accuracy and reproducibility are vital in science and presents a significant challenge in the emerging discipline of data science, especially when the data are scientifically complex and massive in size. Further complicating matters, in the field of genomic-based science high-throughput sequencing technologies generate considerable amounts of data that needs to be stored, manipulated, and analyzed using a plethora of software tools. Researchers are rarely able to reproduce published genomic studies. Results: Presented is a novel approach which facilitates accuracy and reproducibility for large genomic research data sets. All data needed is loaded into a portable local database, which serves as an interface for well-known software frameworks. These include python-based Jupyter Notebooks and the use of RStudio projects and R markdown. All software is encapsulated using Docker containers and managed by Git, simplifying software configuration management. Conclusion: Accuracy and reproducibility in science is of a paramount importance. For the biomedical sciences, advances in high throughput technologies, molecular biology and quantitative methods are providing unprecedented insights into disease mechanisms. With these insights come the associated challenge of scientific data that is complex and massive in size. This makes collaboration, verification, validation, and reproducibility of findings difficult. To address these challenges the NGS post-pipeline accuracy and reproducibility system (NPARS) was developed. NPARS is a robust software infrastructure and methodology that can encapsulate data, code, and reporting for large genomic studies. This paper demonstrates the successful use of NPARS on large and complex genomic data sets across different computational platforms.

A rare case of chordoma presenting as a Pancoast tumor.

The notochord is the defining structure of all chordate embryos. It is a midline structure ventral to the ectoderm, neural plates, and neural arch. Remnants of the notochord ultimately give rise to the nucleus pulposus. The function of the notochord is to organize the surrounding structures. Chordoma is a rare malignant bone tumor arising from remnants of the notochord. These tumors are indolent and can present as incidental or locally advanced involving adjacent structures. These tumors typically present at the skull base and sacral spine but more rarely can be seen on the cervical and thoracic spine. Rare cases of chordoma invading the brachial plexus have been recorded. Surgical resection is the mainstay of treatment for chordomas. We would like to discuss a novel presentation of a chordoma as a Pancoast tumor, and aim to highlight the clinical importance of accurate diagnosis and planning therapy along with poor prognosis of incomplete surgical resection.

Treatment of sternoclavicular joint osteomyelitis with debridement and delayed resection with muscle flap coverage improves outcomes.

The objective of this study was to evaluate the efficacy of various treatment options for sternoclavicular joint osteomyelitis. We evaluated patients with a diagnosis of sternoclavicular joint osteomyelitis, treated at our hospital from 2002 to 2012. Four treatment options were compared. Three out of twelve patients were successfully cured with antibiotics alone (25%). Debridement with or without negative pressure therapy was successful for one of three patients (33%). Simultaneous debridement, bone resection, and muscle flap coverage of the acquired defect successfully treated one of two patients (50%). Debridement with delayed bone resection and muscle flap coverage was successful in five of five patients (100%). Osteomyelitis of the sternoclavicular joint is a rare disease that has become more prevalent in recent years and can be associated with increasing use of long-term indwelling catheters. Initial debridement with delayed bone resection and pectoralis major muscle flap coverage can effectively treat sternoclavicular joint osteomyelitis.

Acute shingles after resection of thoracic schwannoma.

Herpes zoster is relatively uncommon after surgery in immunocompetent patients. To our knowledge, there have been no reports of herpes zoster after the resection of a thoracic schwannoma. We report the case of a 48-year-old woman in whom acute shingles developed after the video-assisted thoracic surgical resection of a posterior mediastinal schwannoma adjacent to the 4th thoracic vertebral body. The patient recovered after receiving timely antiviral therapy. Rash and pain are common in patients who have wound infections and contact dermatitis after surgery, so the possible reactivation of varicella virus might not be prominent in the surgeon's mind. This case serves as a reminder that viral infections such as shingles should be considered in the differential diagnosis of postoperative erythema and pain.