Rapid EGFR Mutation Detection Using the Idylla Platform: Single-Institution Experience of 1200 Cases Analyzed by an In-House Developed Pipeline and Comparison with Concurrent Next-Generation Sequencing Results

Mutations in the epidermal growth factor receptor (EGFR) are the most common targetable alterations in lung adenocarcinoma. To facilitate rapid testing, the Idylla EGFR assay was incorporated as a screening method before next-generation sequencing (NGS). Validation and experience using an in-house developed analysis pipeline, enhanced with a manual review algorithm is described. Results are compared with corresponding NGS results. In all, 1249 samples were studied. Validation demonstrated 98.57% (69/70) concordance with the reference methods. The limit of detection varied from 2% to 5% variant allele frequency for total EGFR quantitation cycle between 20 and 23. Of the 1179 clinical cases, 23.41% were EGFR-positive by Idylla. Concurrent NGS was successfully performed on 94.9% (799/842) requests. Concordance of Idylla with NGS was 98.62% (788/799) and 98.50% (787/799) using our in-house and Idylla analysis pipelines, respectively. Discordances involved missed mutations by both assays associated with low tumor/low input. Incorporating a manual review algorithm to supplement automated calls improved accuracy from 98.62% to 99.37% and sensitivity from 94.68% to 97.58%. Overall reporting time, from receipt of material to official clinical report, ranged from 1 to 3 days. Therefore, Idylla EGFR testing enables rapid and sensitive screening without compromising subsequent comprehensive NGS, when required. Automated calling, enhanced with a manual review algorithm, reduces false-negative calls associated with low tumor/low input samples.

Mutations in the epidermal growth factor receptor (EGFR) are the most common targetable alterations in lung adenocarcinoma (LUAD). In the United States, sensitizing mutations are present in roughly 15% to 20% of LUAD patients,¹ but higher rates of up to 50% can be seen in select populations, such as those of Eastern Asian descent and some Latin American subsets.2, 3, 4 Prompt and robust identification of these alterations is a critical step in guiding initial treatment decisions in patients with advanced disease.In addition to EGFR, a complex array of clinically relevant molecular tumor biomarkers is rapidly emerging, making comprehensive testing of multiple genes a requirement. Current guidelines^5,6 propose that testing algorithms should prioritize assessment for mutations in EGFR and fusions involving ALK and ROS1 over other markers to provide target turnaround times of ≤14 days^5,7 but also endorse the use of next-generation sequencing (NGS) methods over single-gene assays to allow concurrent comprehensive assessment of other targetable biomarkers. Specifically for EGFR, methods should be molecular based and able to detect all sensitizing mutations with a population frequency of at least 1%.⁵Despite the agreement on the importance of biomarker assessment, molecular testing is still not performed in many patients⁸ with LUAD, even for EGFR, which has been part of standard of care analysis for over a decade. Inadequate or insufficient tissue for testing and long turnaround times are the most common barriers affecting compliance with the established guidelines. Obtaining sufficient tumor tissue for sampling from LUAD patients is challenging, placing distinct restrictions on clinical laboratories to perform comprehensive testing. To provide rapid and local testing for select markers, many laboratories still perform testing as a series of single-gene assays, which limits the number of tests that can be run before tissue exhaustion. Alternatively, many laboratories are adopting NGS, but often at the expense of longer turnaround time due to test complexity.Given the importance of both rapid and comprehensive assessment in this setting, a multitest approach, combining rapid screening for key biomarkers followed by broad NGS assessment, constitutes a suitable compromise. The success rate of this approach is contingent on highly optimized protocols and proper choice of technology to ensure that both tests can be performed with a high success rate. In this work, we describe our validation and clinical implementation of the Idylla EGFR assay (Biocartis, Mechelen, Belgium) as a rapid screening method for EGFR mutations before comprehensive NGS testing. The Idylla system has been recently introduced in both Europe and the United States as a simple, fully automated, quantitative real-time PCR (qPCR) platform that takes unextracted formalin-fixed, paraffin-embedded (FFPE) tissue sections as input material. Its cartridge-based design allows the analysis of single samples on demand, without the need for batching or for trained operators and, as such, can be used in most laboratories with minimal infrastructure. Analysis is performed through proprietary analysis pipelines with automatically generated reports.Because of the high variability imparted by the unguided input of unextracted material and the black box nature of the analysis using this system, the false negativity or false positivity of this assay when using minimal tissue is unclear. There is a growing body of evidence related to the assay and clinical experience with the system^9,10; however, objective determination of performance metrics that new laboratories adopting the system as well as in-depth investigation of testing failure have remained limited. For the assessment of this platform, a custom in-house analysis pipeline was developed to analyze the raw data from the Idylla consoles and a set of assessment criteria were developed to facilitate mutation calling and flagging of samples with borderline quality characteristics and at risk for false-positive and false-negative calls. Our 1-year experience with the assay using a broad range of tissue types, beyond FFPE is described, with side-by-side comparisons with NGS testing and some of the lessons learned in the process are shared.     

The inflammatory radicular cysts have higher concentration of tnf-alpha in comparison to odontogenic keratocysts (odontogenic tumour)

TNF-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. We investigated levels of TNF-alpha in 43 radicular cysts and 15 odontogenic keratocysts, obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. TNF-alpha is elevated in both cysts' fluid, but higher values were found in radicular cysts in comparison to keratocysts. The significantly higher concentration of TNF-alpha was associated with smaller radicular cysts, higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, and the degree of vascularisation and cysts wall thickness (Mann-Whitney U-test, p < 0.05). No correlation was found based on these parameters in odontogenic keratocyst, but all cysts have detectable concentrations of TNF-alpha. We here for the first time present that a difference in the concentration of TNF-alpha exists between these two cystic types.     

Difference in expression of collagen type I and matrix metalloproteinase-1 in uterosacral ligaments of women with and without pelvic organ prolapse

Objectives

The objective of this study was to determine whether or not the angiotensin-converting enzyme insertion/deletion (ACE I/D), angiotensin II type 1 receptor (AT1R), and angiotensinogen (AGT) gene polymorphisms are associated with idiopathic recurrent spontaneous abortions (RSAs) in Korean women.

Study design

A total of 251 patients with unexplained consecutive pregnancy losses, and 126 healthy controls with at least one live birth and no history of pregnancy loss.

Result

The odds ratios (ORs) of the ACE ID (OR = 2.423; 95% confidence interval (CI) = 1.417–4.142; p = 0.001) and the ACE II (OR = 2.050; 95% CI = 1.143–3.675; p = 0.018) for the ACE DD genotype were significantly different between patients with idiopathic RSA and controls; however, there were no significant differences between patients and controls with respect to the AT1R 1166A>C and AGT M235 T polymorphisms. In a haplotype-based analysis of I-A (p = 0.010), D-A (p = 0.004), I-A-T (p = 0.033), D-A-T (p = 0.0005), and D-C-T (p = 0.013) polymorphism pairs with synergistic effects derived by the MDR method in patients and in controls showed significant results.

Conclusion

This study suggests that ACE, AT1R and AGT polymorphisms and haplotypes are a genetic determinant for the risk of idiopathic RSA in Korean women.     

Is it possible to overcome antiapoptotic API2/MALT1 events in tumor B-cells by influencing Tregs in MALT lymphoma?

Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) comprises approximately 50% of primary gastric lymphoma. Proliferation of tumor cells infected with Helicobacter pylori is facilitated by the presence of T cells activated by H. pylori antigens. Unlike the majority of MALT lymphomas, tumors bearing the t(11;18)(q21;q21) resulting in production of a chimeric protein API2/MALT1 are often resistant to H. pylori eradication therapy, and require more aggressive therapeutic approach including chemotherapy. The authors hypothesize that a subset of patients with translocation-positive MALT lymphoma might benefit from a novel therapeutic approach that would address intercellular communication pathways between various cell types in the tumor microenvironment. A subset of T cells called regulatory T cells (Tregs) are one of the major immunomodulators of antitumor response mechanisms. There are several potential tools that could have a substantial impact on this particular T cell population, such as interleukin (IL)-15, indoleamine 2,3-dioxigenase (IDO), anti-CD25 antibodies. Introducing some of these components into treatment protocols for patients with API2/MALT1 translocation-positive MALT lymphomas might also prove to be benefitial for other lymphomas with increased number of intratumoral Tregs.     

Wartime open globe eye injuries

Background  

Open globe injuries are the most serious eye injuries in war as in peace time. The purpose of this study is to analyze wartime open globe eye injuries in 72 patients treated at the Department of Ophthalmology, Clinical Hospital of Split from July 1991 to April 1993, during the intensive war in Croatia and Bosnia and Herzegovina, and to evaluate crucial factors responsible for the functional success of the treatment.     

Short tail with skin lesion phenotype occurs in transgenic mice with keratin-14 promoter-directed expression of mutant CXCR2

CXCR2 plays an important role during cutaneous wound healing. Transgenic mice were generated using the keratin-14 promoter/enhancer to direct expression of wild-type human CXCR2 (K14hCXCR2 WT) or mutant CXCR2, in which the carboxyl-terminal domain (CTD) was truncated at Ser 331 and the dileucine AP-2 binding motif was mutated to alanine (K14hCXCR2 331T/LL/AA/IL/AA). Our results indicate that K14hCXCR2WT transgenic mice exhibited a normal phenotype, while K14hCXCR2 331T/LL/AA/IL/AA transgenic mice were born with tails of normal length, but three to eight days after birth their tails degenerated, leaving only a short tail stub. The tissue degeneration in the tail started between caudal somites with degeneration of bone and connective tissue distal to the constriction, which was replaced with stromal tissue heavily infiltrated with inflammatory cells. The tail lesion site revealed coagulation in enlarged vessels and marked edema that eventually led to loss of the distal tail. Moreover, 66% of the mice exhibited focal skin blemishes and inflammation that exhibited an increase in the number of sebaceous glands and blood vessels, enlargement of the hair follicles due to increased number of keratinocytes, reduction in the connective tissue content, and a thickening of the epidermis. Furthermore, immunohistochemical staining of the epidermis from tail tissue in the transgenic mice indicated a loss of the cell adhesion markers E-cadherin and desmoplakin. These data suggest that keratinocyte expression of a CTD mutant of CXCR2 has effects on homeostasis of the connective tissue in the tail, as well as the maintenance of the epidermis and its appendages.     

Neurophysiological methods in headache diagnosis

Neurophysiological methods used in the diagnosis of headache, especially migraine are: electroencephalography (EEG), evoked cortical potentials (VEP, BAER, ERP), reflex responses, autonomic tests and transcranial magnetic stimulation (TMS). Interpretation of EEG can be important for the differential diagnosis of some disorders with headache as a presenting symptom. Noninvasiveness, accessibility and ability to repeat the test due to exposure to harmful ionization are the main advantages of EEG. The role of thorough medical history and clinical assessment in patients with headache should not be underestimated. Interictal EEG (between headache attacks) is not significant in routine evaluation of these patients, but can be useful in patients with unusual symptoms suggesting epilepsy or migraine. It is indicated in patients with an abrupt onset of headache, in patients with migraine followed by neurological signs, in basilar migraine, migraine with extended duration of aura and in cases where epilepsy is suspected. Headache as a symptom is present in various brain and systemic diseases and metabolic disorders. EEG changes seen in headache patients are not specific for a particular disorder, but can suggest additional evaluation and accelerate accurate diagnosis and earlier treatment. Visual evoked cortical potentials (VEP) and cognitive evoked potentials (ERP) in patients with migraine in interictal periods have shown differences in sensory processing between patients with headache and healthy controls. Neurophysiological methods (VEP, ERP) between migraine attacks show cortical hyperactivity and predisposition for further attacks. Brainstem auditory evoked responses (BAER) are a sensitive method for the detection of central nervous system damage. Activation of the brainstem during the migraine attack results in an amplitude increment seen soon after the end of the attack. According to recent studies, R2 component of the blink reflex was six times longer during migraine attack as compared to interictal values. This is thought to be a response to sensitization of the skin nociceptive afferent arch or other neurons in the trigeminal nucleus. In patients with cluster headache, autonomic tests generate cardiovascular and pupillary response suggesting systemic sympathetic hyperactivation connected to concurrent pupillary sympathetic hypofunction and modified opioid modulation. TMS is shown to be very useful for the detection of pathophysiological changes of numerous disorders including migraine, due to its excitatory and inhibitory effects. Recent studies have shown changes in motor and occipital cortex during TMS interictal excitability. Neurophysiological tests are used in differential diagnosis of headache, follow up of possible complications in patients with symptomatic headache as well as in neurorehabilitation. In addition, electrophysiological diagnostic test can contribute to better understand the headache pathophysiology.