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RNA-Seq Analysis in Non-Small Cell Lung Cancer: What Is the Best Sample from Clinical Practice?

Author(s): Nibid L, Sabarese G, Andreotti L, Canalis B, Righi D, Longo F, Grazi M, Crucitti P, Perrone G

Publication: J Pers Med, 2024, Vol. 14, Page

PubMed ID: 39202042 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to evaluate the influence of tissue collection method (fine needle aspiration, FNA; multiple biopsy and surgical resection techniques), site of collection (primary tumor, lymph node, metastatic site), duration of formalin fixation, and the age of the FFPE block on the concentration, fragmentation, and RNAseq success rate of RNA isolated from FFPE specimens from patients diagnosed with non-small cell lung cancer (NSCLC). The tumor specimens used in the study were collected from 140 patients (1 specimen per patient) diagnosed with lung adenocarcinoma (124 patients), squamous cell carcinoma of the lung (2 patients), or one of the following non-adeno/non-squamous histotypes (14 patients): adenosquamous lung cancer, large cell lung cancer/NSCLC, Not-Otherwise Specified, pleomorphic carcinoma, and non-small cell neuroendocrine carcinoma; patientsdid not received neoadjuvant therapy prior to specimen collection. Tumor specimens were collected from the primary tumor (102 specimens), lymph node metastases (14 specimens), or distant metastases (24 specimens) and were procured by biopsy (69 specimens), surgical resection (63 specimens: lobectomy, 34 specimens; wedge resection, 18 specimens; metastasectomy, 11 specimens), or FNA (8 specimens, cell block). All 140 tumor specimens were retrospectively collected, and while the duration of formalin fixation was available for 116 specimens (54 specimens underwent optimal fixation, 62 specimens underwent suboptimal fixation) additional details on fixation, tissue processing, storage conditions, and sectioning were not provided; optimal fixation was defined as fixation for 1 d for biopsies and cell blocks and 2 d for surgical resections, while suboptimal fixation exceeded those thresholds. “Storage duration”, defined by the authors as the time between surgery and analysis, likely reflects several pre-analytical factors, including fixation, tissue processing, storage of the FFPE block and storage of FFPE sections; additional details of conditions of specimen storage were not provided. An unspecified number of 10 µm-thick FFPE sections per extraction were deparaffinized in xylene, incubated overnight in proteinase K at 55°C, and bone biopsies were decalcified in EDTA before total RNA was isolated using the High Pure FFPE RNA Isolation Kit. The Myriapod NGS Cancer Panel RNA Kit was used in conjunction with qPCR to determine the concentration and fragmentation of the isolated RNA. The EasyPGX ready ALK/ROS1/RET/MET exon skipping and EasyPGX ready NTRK1/NTRK2/NTRK3 Kits were used with EasyPGX Analysis Software for RT-PCR analysis. Next-generation sequencing libraries were prepared using the Myriapod NGS Cancer Panel RNA NG034 Kit and an Illumina MiSeq platform. Hematoxylin and eosin-stained sections of all FFPE specimens were evaluated by two pathologists with expertise in lung.

   Summary of Findings:

   When the RNA concentration (<0.063 ng/µl) and fragmentation index (<0.05) specified by the Myriapod NGS Cancer Panel were used as thresholds, 102 of the 140 (72.9%) RNA samples from FFPE specimens were determined to be suitable for molecular (RNAseq or RT-PCR) analysis. RNA from 102 specimens were analyzed by RNAseq, with a success rate of 52.9% (54/102), while RNA from only 27 specimens were analyzed by RT-PCR analysis, with a success rate of 92.6% (25/27). RT-PCR was also success for 73.7% (28/38) of the samples that failed to meet concentration and fragmentation index thresholds for molecular analysis. RNAseq success rate was positively and significantly correlated to both RNA concentration (p < 0.0001; r = 0.509) and fragmentation index (p < 0.0001; r = 0.556). The authors state that they modestly improved the RNAseq success rate when they adjusted the RNA fragmentation index threshold to ≥ 0.14 (42/55 samples, 76.4%), with only 25.5% (12/47) success rate among samples with a fragmentation index below that threshold.

   RNAseq success rate was influenced by specimen collection method, with significant differences observed among RNA from biopsies (60.3%, 35/58), surgical resections (48.7%, 19/39), and cell blocks (0%, 0/5) (p = 0.028). RNAseq success rates also varied by biopsy and resection technique, with the highest success rates observed among RNA from EBUS/EUS biopsies (62.5%, 10/16), followed by thoracoscopic biopsies (55.6%, 5/9), and CT-guided biopsies (50%, 6/12) whereas for RNA from surgical specimens, RNAseq success rates were higher for specimens from wedge resections than lobectomies (10/15, 66.6% vs. 5/17, 29.4%). Similarly, the method used for specimen collection method resulted in significant differences in the percentage of samples that passed the RNA fragmentation threshold of ≥ 0.14 (p = 0.002), as  57% (40/69) of biopsies passed the RNA fragmentation threshold whereas just 29% (18/63) of surgical resections and 25% (2/8) of cell blocks did. The percentage of samples that met the RNA fragmentation index threshold did not differ among the specimen types examined, as 42% of primary tumors, 42% of lymph node metastases, and 40% of distant metastases passed the RNA fragmentation threshold 40%. While RNA from the different biopsy techniques examined did not differ from one another in RNA fragmentation index, they did differ significantly in mean RNA concentration (p = 0.017), with the highest RNA concentration observed among thoracoscopic biopsies (65.9 µg/mL), followed by US-guided biopsies (17.4 µg/mL), endoscopic (EBUS/EUS) biopsies (17.2 µg/mL), and CT-guided biopsies (4.4 ng/mL).

   Storage for 30 days between surgery and analysis, resulted in a non-significant decline in RNAseq success rate (46.4%, 32/69), and the median storage duration was longer for samples that failed RNAseq than those that were successful (46 vs. 37 days).  RNAseq success rate was negatively, albeit weakly, correlated to RNA fragmentation index (r = −0.206; p = 0.037). Effects of storage duration on RNA fragmentation were progressive, with the percentage of samples classified as “optimal” (≥ 0.14) decreasing from 68% when stored for <30 days, to 55% when stored for 31-60 days, to 38% when stored for >60 days.  Notably, RNA fragmentation index was also negatively correlated with time in formalin (r = −0.333, p < 0.001).

   Biospecimens

   • Tissue - Lung
   • Tissue - Bone
   • Tissue - Lymph Node
   • Tissue - Adrenal Gland
   • Cell - Adrenal Gland
   • Cell - Lung
   • Cell - Lymph Node
   • Cell - Bladder
   • Tissue - Bladder

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Method of tissue acquisition	Fine needle aspiration Ultrasound-guided biopsy CT-guided biopsy Endoscopic biopsy Lobectomy Surgical resection
   Biospecimen Preservation	Time in fixative	1 d 2 d 3 d 4 d 5 d 6 d
   Biospecimen Acquisition	Locale of biospecimen collection	Primary tumor Lymph node metastasis Distant metastasis
   Storage	Storage duration	≤30 d 31-60 d >60 d

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