Jennifer K. Sehn

Major Complications and Comparison Between 3-column Osteotomy Techniques in 105 Consecutive Spinal Deformity Procedures

Study Design. A retrospective review. Objective. To characterize the risk factors for the development of major complications in 3-column osteotomies and determine whether the presence of a major complication affects ultimate clinical outcomes. Summary of Background Data. Three-column spinal osteotomies, including pedicle subtraction osteotomy (PSO) and vertebral column resection (VCR), are common techniques to correct severe and/or rigid spinal deformities. Methods. Two hundred forty consecutive PSO (n = 156) and VCR (n = 84) procedures in 237 patients were performed at a single institution between 1995 and 2008. Of these, 105 patients (87 PSOs, 18 VCRs) had complete preoperative and minimum 2-year postoperative clinical outcomes data available for analysis. Using established criteria, we reported complications as major or minor and further stratified complications as surgical versus medical and permanent versus transient. Risk factors for complications and their effect on Scoliosis Research Society (SRS) clinical outcomes at baseline and at 2 years or more were assessed. Results. Major medical and surgical complications occurred at similar rates in both PSOs and VCRs (38%, 33 of 87 vs. 22%, 4 of 18; P = 0.28). Overall, 24.8% (26 of 105) experienced major surgical complications (3 permanent) and 15.2% (16 of 105) experienced major medical complications (4 permanent). Patients with PSO were older (53 vs. 29 yr; P < 0.001), had greater estimated blood loss (1867 vs. 1278 mL; P = 0.02), and showed a trend toward fewer fused levels (10.1 vs. 12.2; P = 0.06). Risk factors for major complications included preoperative sagittal imbalance of 40 mm or more (P = 0.01), age 60 years and older (P = 0.01), and the presence of 3 or more medical comorbidities (P = 0.04). Both groups improved significantly from baseline in SRS subscores; however, patients with PSO started off worse but improved more than VCRs in both the pain (+1.0 vs. +0.1; P < 0.001) and function (+0.6 vs. +0.2; P = 0.01) domains, with no differences in final satisfaction (4.1 vs. 4.3; P = 0.54). PSO and VCR patients with no complications had slightly higher satisfaction scores than patients with minor-only complications, major transient complications, and major permanent complications. There were no significant differences among the groups with respect to change in SRS subscores from baseline, and all complication groups improved significantly from baseline (P = 0.04). Conclusion. Major complications occurred in 35% of 3-column osteotomies and at similar rates for both PSO (38%) and VCR (22%) procedures. The presence of a major complication did not affect the ultimate clinical outcomes at 2 years or more.

Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens

Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of >99.99%, with 96.8% agreement in the single-nucleotide variants detected and >98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing.

Percutaneous needle biopsy in diagnosis and identification of causative organisms in cases of suspected vertebral osteomyelitis

BACKGROUND Biopsy with demonstration of the infectious organism is the gold standard for diagnosing spondylodiscitis. The purpose of this study is to evaluate the positive culture rate of image-guided percutaneous biopsy in cases of radiologically suspected and unsuspected spinal osteomyelitis and to assess the role of pathology in diagnosis. METHODS With IRB approval and in compliance with HIPAA regulations, the charts of patients undergoing 323 consecutive image-guided percutaneous spinal biopsies performed by one musculoskeletal radiology department between January 2001 and March 2007 were reviewed. Image guidance was via fluoroscopy or computed tomography. Radiological and clinical suspicion, cultures, and pathology were assessed and compared to previously published reports. RESULTS In 92 cases radiographically and clinically consistent with infection (high probability of infection), 28 specimens yielded positive cultures (30.4%). Positive cultures resulted from 5 of 31 cases (16.1%) radiographically indeterminate for infection (intermediate probability of infection versus tumor). When radiographically not suggestive of infection (low probability of infection, i.e. suspicious for tumor), 10 of 200 cultures were positive (5.0%). From 113 cases sent to pathology with an intermediate or high suspicion for infection, 63 were histopathologically diagnosed as such (55.8%). Cultures were positive in 19 of those 63 cases (30.2%). Culture and/or pathology was positive in 73 (64.6%) of the 113 cases. There were no significant differences in rates of positive culture or pathology by vertebral region (p=0.51, p=0.81). The most frequently identified organisms were Staphylococcus aureus (13) and coagulase negative staphylococci (13). CONCLUSIONS Our results suggest that the positive culture rate of percutaneous spinal biopsy specimens is 30.4% with radiographically high probability for infection, which is lower than previously published. Infection may also be present in cases with imaging characteristics atypical for infection (5.0%). Careful consideration must be given to the interpretation of negative culture results. LEVEL OF EVIDENCE This retrospective review of 323 consecutive percutaneous spine biopsies is level III evidence.

Detection of viral pathogens in high grade gliomas from unmapped next-generation sequencing data.

Viral pathogens have been implicated in the development of certain cancers including human papillomavirus (HPV) in squamous cell carcinoma and Epstein-Barr virus (EBV) in Burkitts lymphoma. The significance of viral pathogens in brain tumors is controversial, and human cytomegalovirus (HCMV) has been associated with glioblastoma (GBM) in some but not all studies, making the role of HCMV unclear. In this study we sought to determine if viral pathogen sequences could be identified in an unbiased manner from previously discarded, unmapped, non-human, next-generation sequencing (NGS) reads obtained from targeted oncology, panel-based sequencing of high grade gliomas (HGGs), including GBMs. Twenty one sequential HGG cases were analyzed by a targeted NGS clinical oncology panel containing 151 genes using DNA obtained from formalin-fixed, paraffin-embedded (FFPE) tissue. Sequencing reads that did not map to the human genome (average of 38,000 non-human reads/case (1.9%)) were filtered and low quality reads removed. Extracted high quality reads were then sequentially aligned to the National Center for Biotechnology Information (NCBI) non-redundant nucleotide (nt and nr) databases. Aligned reads were classified based on NCBI taxonomy database and all eukaryotic viral sequences were further classified into viral families. Two viral sequences (both herpesviruses), EBV and Roseolovirus were detected in 5/21 (24%) cases and in 1/21 (5%) cases, respectively. None of the cases had detectable HCMV. Of the five HGG cases with detectable EBV DNA, four had additional material for EBV in situ hybridization (ISH), all of which were negative for expressed viral sequence. Overall, a similar discovery approach using unmapped non-human NGS reads could be used to discover viral sequences in other cancer types.

Diagnostic utility of targeted next-generation sequencing in problematic cases.

Targeted next-generation sequencing (NGS) provides predictive and prognostic information in the routine care of patients with cancer. However, with increasing knowledge of the biological basis of cancer, NGS of the same gene sets can also provide diagnostic information in challenging cases, on the basis of identification of both known and novel variants, including single-nucleotide variants, insertions and deletions, copy number alterations, and translocations. Here, we present 3 clinical cases in which targeted NGS of hybrid-capture–enriched DNA from formalin-fixed, paraffin-embedded tumor samples provided unique and clinically important diagnostic and/or staging information in 3 different challenging clinical scenarios. In the first patient, NGS played a key role in both diagnosis and staging in a patient with multiple tumors of the same histologic type. The second case demonstrates the ability of NGS to clarify the tumor tissue type in a single mass involving multiple organs, and thereby guide appropriate chemotherapy. The third case illustrates that information regarding susceptibility to targeted therapeutics can also clarify the original histologic diagnosis.

Occult Specimen Contamination in Routine Clinical Next-Generation Sequencing Testing.

OBJECTIVES To evaluate the extent of human-to-human specimen contamination in clinical next-generation sequencing (NGS) data. METHODS Using haplotype analysis to detect specimen admixture, with orthogonal validation by short tandem repeat analysis, we determined the rate of clinically significant (>5%) DNA contamination in clinical NGS data from 296 consecutive cases. Haplotype analysis was performed using read haplotypes at common, closely spaced single-nucleotide polymorphisms in low linkage disequilibrium in the population, which were present in regions targeted by the clinical assay. Percent admixture was estimated based on frequencies of the read haplotypes at loci that showed evidence for contamination. RESULTS We identified nine (3%) cases with at least 5% DNA admixture. Three cases were bone marrow transplant patients known to be chimeric. Six admixed cases were incidents of contamination, and the rate of contamination was strongly correlated with DNA yield from the tissue specimen. CONCLUSIONS Human-human specimen contamination occurs in clinical NGS testing. Tools for detecting contamination in NGS sequence data should be integrated into clinical bioinformatics pipelines, especially as laboratories trend toward using smaller amounts of input DNA and reporting lower frequency variants. This study provides one estimate of the rate of clinically significant human-human specimen contamination in clinical NGS testing.

Copy number variants in clinical next-generation sequencing data can define the relationship between simultaneous tumors in an individual patient.

Targeted next-generation sequencing (NGS) cancer panels have become a popular method for the identification of clinically predictive mutations in cancer. Such methods typically detect single nucleotide variants (SNVs) and small insertions/deletions (indels) in known cancer genes and can provide further information regarding diagnosis in challenging surgical pathology cases, as well as identify therapeutic targets and prognostically significant mutations. However, in addition to SNVs and indels, other mutation classes, including copy number variants (CNVs) and translocations, can be simultaneously detected from targeted NGS data. Here, as proof of methods, we present clinical data which demonstrate that targeted NGS panels can separate synchronous liver tumors based on CNV status, in the absence of distinct SNVs and indels. Such CNV-based analysis can be performed without additional cost using existing targeted cancer panel data and publically available software.

Ovarian complete hydatidiform mole: case study with molecular analysis and review of the literature

Ectopic complete molar pregnancy in the ovary is an exceptionally rare event. Here we present a case of ovarian complete hydatidiform mole in a 20-year-old gravida 2 para 1 woman. At presentation, the patient underwent excision of a hemorrhagic left ovarian cyst, with routine sections demonstrating a hemorrhagic corpus luteum with a single microscopic focus of detached atypical trophoblast, without chorionic villi. Subsequent left salpingo-oophorectomy for persistently elevated human chorionic gonadotropin led to a final diagnosis of complete hydatidiform mole arising in the ovary. The fallopian tube was unremarkable. Zygosity was determined using short tandem repeat analysis, confirming the diagnosis of monospermic complete mole. In the clinical setting of a markedly elevated human chorionic gonadotropin level and an ovarian mass, histopathologic examination is critical in distinguishing ectopic pregnancy from choriocarcinoma. Short tandem repeat analysis can be a useful adjunct to histologic diagnosis in challenging cases.

Somatic Diseases (Cancer): Whole Exome and Whole Genome Sequencing

Exome and genome sequencing are often applied to the study of cancer as a discovery tool in the investigative setting. Because of limitations related to result interpretation and technical factors like depth of coverage and sensitivity, these broad approaches have not been widely adopted in clinical cancer testing. In this chapter, issues related to variant interpretation in clinical cancer testing will be addressed. Specifically, the limited utility of sequencing genes without established clinical significance will be discussed. The advantages and limitations of exome and genome sequencing in cancer from a technical perspective, including depth of coverage and variant detection, also will be considered.Exome and genome sequencing are often applied to the study of cancer as a discovery tool in the investigative setting. Because of limitations related to result interpretation and technical factors like depth of coverage and sensitivity, these broad approaches have not been widely adopted in clinical cancer testing. In this chapter, issues related to variant interpretation in clinical cancer testing will be addressed. Specifically, the limited utility of sequencing genes without established clinical significance will be discussed. The advantages and limitations of exome and genome sequencing in cancer from a technical perspective, including depth of coverage and variant detection, also will be considered.

Cervical Spinal Cord Dimensions and Clinical Outcomes in Adults with Klippel-Feil Syndrome: A Comparison with Matched Controls

Study Design Retrospective case–control study. Objectives To confirm the fact that spinal cord dimensions are smaller in adults with Klippel-Feil syndrome (KFS) than in pediatric patients with KFS and to compare the clinical characteristics and outcomes of neurologic complications in patients with KFS with matched controls. Methods We performed an independent 1:2 case–control retrospective radiographic and chart review of a consecutive series of adults with KFS who underwent surgical intervention. The control group consisted of consecutive non-KFS surgical patients. Patients were matched in 1:2 case–control manner. Their charts were reviewed and the clinical characteristics were compared. Axial T2-weighted magnetic resonance imaging (MRI) was used to measure the anteroposterior and mediolateral axial spinal cord and spinal canal at the operative levels and measurements were compared. Results A total of 22 patients with KFS and 44 controls were identified. The KFS group had a tendency of more myeloradiculopathy, and the control group had a tendency toward more radiculopathy. Both tendencies, however, were not significantly different. MRIs of 10 patients from the KFS group and 22 controls were available. There was no difference in the area of both spinal cord and canal at the operative levels. Conclusion Contrary to the finding in previous reports on pediatric patients, there were no differences between KFS and well-matched control groups in terms of age of onset, presentation, revision rate, complication rate, surgical outcome, and cross-sectional spinal cord and canal dimensions at the operative level.