Adequacy of cytology and small biopsy samples obtained with rapid onsite evaluation (ROSE) for predictive biomarker testing in non-small cell lung cancer.

Complete biomarker workup of non-small cell lung cancer (NSCLC) specimens is essential for appropriate and timely clinical management decisions. This can be challenging to achieve from small cytology and histology specimens, with increasing numbers of molecular and immunohistochemical biomarkers required. We conducted a 5 year retrospective audit of cases at our institution to assess the diagnostic and biomarker testing adequacy rates, particularly those specimens obtained with rapid onsite evaluation (ROSE), performed by a cytopathologist and a cytology scientist or pathology trainee, including all endobronchial ultrasound guided transbronchial needle aspirations (EBUS-TBNA), CT guided lung fine needle aspirations (FNA) and CT guided lung core biopsies. A total of 5,354 cases were identified, of which 92.2% had sufficient material for diagnosis. Of the 1506 cases identified with a recorded diagnosis of lung adenocarcinoma or NSCLC, not otherwise specified, 1001 (66.5%) had biomarker testing requested. Sufficient material was available in 89.5% of cases for a complete biomarker workup which included EGFR and KRAS mutational testing (all cases), ALK, ROS1 and PD-L1 immunohistochemistry (all cases), and ALK and ROS1 FISH (as required). For EGFR and KRAS mutational testing across both cytology and histology specimens, 99% of cases were sufficient. Of the samples in which a complete biomarker workup was unable to be performed, approximately half were only insufficient due to inadequate numbers of tumour cells for PD-L1 immunohistochemistry. Excluding PD-L1 IHC, 952 (95.1%) of samples obtained with ROSE were sufficient for the remainder of the testing requirements. Next generation sequencing using a 33 gene custom AmpliSeq panel was achieved in up to 72% of cases. In conclusion, small cytology and histology specimens obtained with ROSE are suitable for predictive biomarker testing in NSCLC, although attention needs to be paid to obtaining sufficient cells (>100) for PD-L1 immunohistochemistry.

The Effect of Nonsense Mediated Decay on Transcriptional Activity Within the Novel ß-Thalassemia Mutation HBB: c.129delT.

Premature termination codons (PTCs) are caused by mutations in the coding sequences of functional genes resulting in an incorrect assignment of a stop codon. Abnormal and truncated proteins are prevented from being translated due to the rapid degradation of mRNA carrying these mutations by an RNA surveillance mechanism referred to as nonsense mediated decay (NMD). Recently, a novel mutation in a patient from Thailand with the clinical diagnosis of Hb E (HBB: c.79G > A)/ß(0)-thalassemia (Hb E/ß(0)-thal) and whose molecular analysis demonstrated a novel mutation in the ß-globin gene, HBB: c.129delT, was reported. The result of this deletion is a frameshift (FSC) resulting in a PTC at codon 60. We have analyzed the impact of this mutation on transcription and translation of the affected ß-globin gene using an in vitro model. The quantitative real-time polymerase chain reaction (qReTi-PCR) analysis revealed that this nucleotide mutation resulted in marked mRNA degradation, which we attributed to the NMD mechanism and as such, the expected deleterious truncated HBB was not generated. This result highlights a valuable application of our in vitro gene expression model that can be used to predict possible molecular pathology for any given nucleotide mutations.