Specific Real-time PCR For Detection Research Articles

Spectrum of BRAF mutations in Indian patients with NSCLC: A molecular kaleidoscope.

e21199 Background: Mutations in the BRAF gene (0.8%-8% cases of NSCLC) comprise a molecularly heterogeneous group, with the canonical V600E mutation as well as myriad variants. Traditionally, diagnostics utilize modalities like mutation specific IHC or allele specific real-time PCR for detection of V600E variant and may underestimate the true prevalence of non-V600E variants. Broader panel next generation sequencing (NGS) testing may be a better modality for detecting them and this may have potential therapeutic implications. This retrospective study of patients with BRAF mutated NSCLC looked at their molecular and clinicopathologic spectrum. Methods: 260 patients underwent panel based NGS testing between 2017-2020. BRAF mutant cases, detected at any point during the course of their disease, were reviewed for clinical, pathologic, molecular features; as well as associated treatment and outcomes. Results: BRAF alterations were seen in 14/260 (5.38%) patients. Median age of the cohort was 62 years (range: 39-86 years) with a female predilection (8/14, 57.1%). BRAF V600E mutation was seen in 6 (42.9%) patients and 8 (57.1%) harbored a non-V600E alteration; the spectrum of which included G466E, G469A, N581I, V600_K601delins, D594G, L597Q, G649V and a TRIM24-BRAF fusion. All patients were never smokers with an adenocarcinoma histology.11 (78.6%) patients had extra thoracic metastases, and 2 (14.3%) cases had metastases to the brain at diagnosis. 11/14 (78.6%) patients had presence of liver metastases (V600E vs non V600E: 43% vs 57%, p = 0.09). 11 patients took treatment at our centre and two patients received anti-BRAF therapy. One patient with V600E mutation received vemurafenib. The other patient with a nonV600E mutation (V600_601delins, detected on NGS) received combination dabrafenib-trametinib with an ongoing partial response at 6 months. 8 patients received chemotherapy and median PFS was 4.8 months, similar for all mutation types. Median OS for the entire cohort was not reached owing to high censorship. Conclusions: This single centre study depicts distinct clinicopathologic features in Indian patients with BRAF mutant NSCLC. The higher prevalence of non-V600E mutation may be attributed to increased use of broader panel based NGS testing as opposed to sequential single gene testing. One of the patients with a non-V600E mutation responded to targeted therapy, hence clinicians should be aware of this spectrum of potentially targetable BRAF mutations.

Enterovirus (EV)-D68 in Pediatric Patients with Respiratory Tract Infection: the Circulation of new B3 Clade in Italy

BackgroundEV-D68 is an emerging infectious agent that has been found associated with both mild and severe respiratory diseases and neurological clinical manifestations. Four clades with a number of subclades that can circulate or co-circulate during different periods have been identified. However, molecular evolution of EV-D68 is not known as it is the possible association between specific genetic variants and the development of severe cases. To solve these problems, genetics of strains identified during an outbreak of EV-D68 infection that occurred in Italy during the period March-October 2016 were studied.MethodsNasopharyngeal samples obtained from children admitted to the Emergency Room for respiratory infection were tested with a previously validated specific real-time PCR for detection and quantification of EV-D68. Phylogenetic analysis (PhA) of the virus was performed sequencing the major capsid protein (VP1). Moreover, tests for detection of selective pressure were carried out.ResultsRespiratory samples of 390 children were tested. Twenty-two patients (5.6%; median age, 47 months) were infected by EV-D68. All but three had a lower respiratory tract infection but none of these was severe, although in a few cases a transient reduction of SaO2 level was evidenced at admission. All the strains belonged to the EV-D68 subclade B3. No evidence of increased clinical severity associated with specific molecular signatures of VP1 sequence or viral load was shown. B3 strains had 92.3% and 94.7% nucleotide identity with B3 clades identified in other countries where a number of very severe cases was diagnosed. No sign of selective pressure was found.ConclusionEV-D68 subclade B3 was the only cause of the EV-D68 diseases diagnosed in Italy during 2016.The same subclade was found in Northern Europe, China and the USA in the same period. This suggests that all the outbreaks had a common origin and EV-D68 B3 has become the preeminent EV-D68 strain causing disease worldwide. No specific characteristic of EV-D68 VP1 has been found associated with disease characteristics, in contrast with what has been evidenced with other studies. Further studies on full-genome sequencing in larger cohorts of patients are needed.Disclosures All authors: No reported disclosures.