Targeted deep sequencing of effusion cytology samples is feasible, informs spatiotemporal tumor evolution, and has clinical and diagnostic utility

Abstract

During the course of disease, many cancer patients eventually present with metastatic disease including peritoneal or pleural spread. In this context, cytology specimens derived from ascites or pleural effusion may help to differentiate malignant from benign conditions and sometimes yield diagnosis of a malignancy. However, even when supported by immunohistochemistry, cytological interpretation can be challenging, especially if tumor cellularity is low. Here, we investigated whether targeted deep sequencing of formalin-fixed and paraffin embedded (FFPE) cytology specimens of cancer patients is feasible, and has diagnostic and clinical impact. To this end, a cohort of 20 matched pairs was compiled, each comprising a cytology sample (FFPE cell block) and at least one biopsy/surgical resection specimen serving as benchmark. In addition, 5 non-malignant effusions were sequenced serving as negative-controls. All samples yielded sufficient libraries and were successfully subjected to targeted sequencing employing a semiconductor based next-generation sequencing platform. Using gene panels of different size and composition, including the Oncomine Comprehensive Assay, for targeted sequencing, somatic mutations were detected in the tissue of all 20 cases. Of these, 15 (75%) harbored mutations that were also detected in the corresponding cytology samples. In four of these cases (20%), additional private mutations were detected in either cytology or tissue samples, reflecting spatiotemporal tumor evolution. Of the five remaining cases, three (15%) showed wild type alleles in cytology material whereas tumor tissue had mutations in interrogated genes. Two cases were discordant, showing different private mutations in the cytology and in the tissue sample, respectively. In summary, sequencing of cytology specimens (FFPE cell block) reflecting spatiotemporal tumor evolution is feasible and yields adjunct genetic information that may be exploitable for diagnostics and therapy.

RNA-Based Detection of Gene Fusions in Formalin-Fixed and Paraffin-Embedded Solid Cancer Samples

“…In case of requests for gene fusion analysis only, analysis was performed with the Archer ® DX Fusion Plex ® Solid tumor panel based on anchored multiplex polymerase chain reaction (PCR) [19] (AMP; AMP-cohort, n = 285 patients). The Oncomine™ Comprehensive Assay v3 [20,21,22] (OCA, a combination of DNA- and RNA-targeted sequencing, was used for molecular profiling beyond fusions; OCA-cohort, n = 232 patients) (Table 1). Gene fusions detected by these assays were further analyzed by orthogonal Sanger sequencing, immunohistochemistry or fluorescence in situ hybridization (FISH) (Table 2 and Table S1).…”

Performance analysis of SiRe next-generation sequencing panel in diagnostic setting: focus on NSCLC routine samples

“…This small cohort of patients (n=20) with matched cytological and histological specimens showed discordances in five cases; in particular, in three samples, the mutation was identified only in the histological specimens and two samples showed discordant mutation, respectively, on histological and cytological samples. These discordances should be related to the lower abundance of tumour cells (<10%) in effusion material than the other samples 28. In a larger cytological cohort (n=207) adopting the AmpliSeq Cancer Hotspot V.2 panel (50 genes), there was a 21% failure rate due to either a low DNA yield or a template/library preparation failure 29…”

Comparative genetic profiling aids diagnosis and clinical decision making in challenging cases of CUP syndrome

“…DNA extraction, library preparation and semiconductor sequencing were conducted as described previously . Briefly, tumor tissue was microdissected to enrich for tumor tissue (at least 10%).…”