Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample ...requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.
Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments–certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing ...screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions ( n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.
•Stress Fractures in elderly patients are rarely discussed.•Vitamin D deficiency, osteoporosis, and weight are important comorbidities to consider for these stress fractures.•Have a high index of ...suspicion when multiple risk factors are observed in a patient.•Patients can be treated non-operatively or with minimally invasive procedures if the issue is identified early.
Early intervention in femoral neck stress fractures (FNSFs) can be self- limiting, but they have an insidious presentation. High index of suspicion for an occult fracture is necessary to avoid bilateral progression and/or operative interventions.
A 83-year-old female, non-athlete, presents with multiple comorbidities and progressive bilateral FNSFs. Initially the patient presented with groin pain and radiographs demonstrated a non-displaced compression type fracture of the right femoral neck without any inciting events. Subsequently, during a post-operative visit of her right hip, a work-up for new onset contralateral groin pain revealed a non-displaced tension type fracture on the left femoral neck. Both FNSFs were treated with percutaneous cannulated screw fixation.
Vitamin D deficiency, poor nutrition, and osteoporosis have been associated with developing stress fractures. This presents an interesting question of whether these frequently referenced risk factors play an interrelated role. Treatment algorithms are controversial, but have been successful in preventing the progression of occult stress fractures. Yet, identification of FNSFs represents a major challenge in diagnosis for clinicians.
This case report documents an uncommon fracture pattern in the elderly population. With an aging population, it is pertinent to avoid missed opportunities for prompt diagnosis and implementation of noninvasive methods of treatment. Therefore, paying attention to the risk factors with a high index of suspicion would be ideal.
Identification of tumor-specific somatic mutations has had a significant impact on both disease diagnosis and therapy selection. The ability of next-generation sequencing (NGS) to provide a ...quantitative assessment of mutant allele burden, in numerous target genes in a single assay, provides a significant advantage over conventional qualitative genotyping platforms.
We assessed the quantitative capability of NGS and a primer extension-based matrix-assisted laser desorption ionization-time-of-flight (PE-MALDI) assay and directly correlated NGS mutant allele burden determination to morphologic assessment of tumor percentage in H&E-stained slides.
Our results show a 100% concordance between NGS and PE-MALDI in mutant allele detection and a significant correlation between NGS and PE-MALDI for determining mutant allele burden when mutant allele burden is 10% or more.
NGS-based mutation screening provides a quantitative assessment comparable to that of PE-MALDI. In addition, NGS also allows for a high degree of multiplexing and uses nanogram quantities of DNA, thereby preserving precious material for future analysis. Furthermore, this study provides evidence that H&E-based morphologic assessment of tumor burden does not correlate to actual tumor mutant allele burden frequency.
The zoonotic Rift Valley fever virus (RVFV) can cause severe disease in humans and has pandemic potential, yet no approved vaccine or therapy exists. Here we describe a dual-mechanism human ...monoclonal antibody (mAb) combination against RVFV that is effective at minimal doses in a lethal mouse model of infection. We structurally analyze and characterize the binding mode of a prototypical potent Gn domain-A-binding antibody that blocks attachment and of an antibody that inhibits infection by abrogating the fusion process as previously determined. Surprisingly, the Gn domain-A antibody does not directly block RVFV Gn interaction with the host receptor low density lipoprotein receptor-related protein 1 (LRP1) as determined by a competitive assay. This study identifies a rationally designed combination of human mAbs deserving of future investigation for use in humans against RVFV infection. Using a two-pronged mechanistic approach, we demonstrate the potent efficacy of a rationally designed combination mAb therapeutic.
COVID-19 pandemic has multifaceted presentations with rising evidence of immune-mediated mechanisms underplay. We sought to explore the outcomes of severe COVID-19 patients treated with a ...multi-mechanism approach (MMA) in addition to standard-of-care (SC) versus patients who only received SC treatment.
Data were collected retrospectively for patients admitted to the intensive care unit (ICU). This observational cohort study was performed at five institutions, 3 in the United States and 2 in Honduras. Patients were stratified for MMA vs. SC treatment during ICU stay. MMA treatment consists of widely available medications started immediately upon hospitalization. These interventions target immunomodulation, anticoagulation, viral suppression, and oxygenation. Primary outcomes included in-hospital mortality and length of stay (LOS) for the index hospitalization and were measured using logistic regression.
Of 86 patients admitted, 65 (76%) who had severe COVID-19 were included in the study; 30 (46%) patients were in SC group, compared with 35 (54%) patients treated with MMA group. Twelve (40%) patients in the SC group died, compared with 5 (14%) in the MMA group (p-value = 0.01, Chi squared test). After adjustment for gender, age, treatment group, Q-SOFA score, the MMA group had a mean length of stay 8.15 days, when compared with SC group with 13.55 days. ICU length of stay was reduced by a mean of 5.4 days (adjusted for a mean age of 54 years, p-value 0.03) and up to 9 days (unadjusted for mean age), with no significant reduction in overall adjusted mortality rate, where the strongest predictor of mortality was the use of mechanical ventilation.
The finding that MMA decreases the average ICU length of stay by 5.4 days and up to 9 days in older patients suggests that implementation of this treatment protocol could allow a healthcare system to manage 60% more COVID-19 patients with the same number of ICU beds.
Purpose
There is an increasing number of children with in utero exposure to opioids. Knowledge about opioid safety in pregnancy, particularly for outcomes later in childhood is scarce. It has been ...suggested that opioids can modulate immune system and increase the risk of infections. Our goal was to study the impact of in utero opioid exposure on the immune system and the risk of infections in childhood.
Methods
This population‐based cohort study used nationwide registers from Denmark, Norway, and Sweden. Among pregnant women we identified users of opioids for two different indications, opioids used in opioid maintenance therapy (OMT) and opioids used for treatment of pain. We followed the exposed children and studied susceptibility for infections measured as number of antibiotic prescriptions expressed as Incidence rate ratios (IRRs) and diagnoses in specialist health care expressed as hazard ratios (HRs).
Results
After adjustment we did not observe increased risk for filling antibiotic prescriptions in children exposed to OMT opioids compared with OMT discontinuers (IRR, 1.08; 95% CI 0.81‐1.44 in Norway and Sweden, and IRR, 0.74; 95% CI 0.63‐0.88 in Denmark), or for diagnosis of infection in specialist health care (HR 0.83; 95% CI 0.55‐1.26 in Norway and Sweden, and 0.82; 95% CI 0.62‐1.10 in Denmark).
Conclusions
In this population‐based cohort study, we did not observe increased risk of infections among children prenatally exposed to OMT opioids when compared to OMT discontinuers, nor long‐term analgesic opioids exposed when compared to short‐term analgesic opioids exposed.
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