Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Identification of Factors Affecting the Success of Next-Generation Sequencing Testing in Solid Tumors.

Author(s): Goswami RS, Luthra R, Singh RR, Patel KP, Routbort MJ, Aldape KD, Yao H, Dang HD, Barkoh BA, Manekia J, Medeiros LJ, Roy-Chowdhuri S, Stewart J, Broaddus RR, Chen H

Publication: Am J Clin Pathol, 2016, Vol. 145, Page 222-37

PubMed ID: 27124905 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated how acquisition method, tumor and tissue type, size of the microdissected area, FFPE block storage duration, and tumor cellularity affects the percentage of specimens for which NGS was successful, as well the impact of reducing DNA input requirements. Areas of solid tumors containing >20% tumor cells were microdissected using an H&E stained slide as a guide, from 5 µm unstained sections of FFPE biopsy (237), surgical (342), and fine-needle aspiration (FNA) cell block (34) specimens. Bone specimens were decalcified in formic acid or remained non-decalcified. DNA was extracted from up to 20 sections of tiny tumors (1-10 mm²), 6-10 sections of small tumors (10-20 mm²), 3-5 sections of medium tumors (20-60 mm²), and 1-2 sections of large tumors (>60 mm²) using the PicoPure DNA extraction kit and purified using the Agencourt AMPure XP kit.  DNA was quantified using Qubit. If initial extraction resulted in insufficient DNA for NGS, extraction was repeated using additional sections before a specimen was considered an NGS failure.  NGS libraries were constructed using the Ion AmpliSeq Cancer Hotspot Panel v2.

   Summary of Findings:

   Of the 614 specimens from which DNA was extracted, 67 (11%) failed NGS because less than 10 ng DNA was available, 3 (0.5%) failed library construction due to lack of amplification and 4 (0.7%) failed due to variably-sized amplicons.  NGS success rates were significantly higher for melanomas than hepatocellular carcinomas (95% versus 55%, P=0.001), gynecologic organs (100%) than for bone (63%, P<0.001) or liver (77%, P=0.002), and for resection specimens (333 of 342, 97%) than biopsy specimens (189 of 237, 80%, P<0.001) or FNA specimens (17 of 34, 50%, P<0.001), while NGS success rates were not significantly different among other tumor types nor were they different between metastasis and primary tumors. Success rates were also lower for specimens when the microdissected area was tiny (<10mm2, 69%) than when small (10-20mm2, 97%; P < 0.001), medium (20- 60mm2, 98%, P<0.01), or large (60mm2, 99.6%, P<0.01); and among small compared to  large specimens (97% versus 99.6%, P<0.01); but was not affected by block storage duration (<1 year, 1-2 years, 3-6 years, or 7-12 years). Decalcification of specimens resulted in decreased NGS success compared to non-decalcified specimens (62% versus 89%, P=0.013), but the effect may be dependent on solution used as the success rate was higher for formic acid decalcified specimens than OSH decalcified specimens (73% versus 0%). Further, NGS success rates were comparable for non-decalcified (3 of 5, 60%) decalcified (8 of 13, 62%) bone specimens.

   Importantly, the size of the microdissected area may be a confounding factor to NGS success, as DNA yield was significantly correlated with the size of the microdissected area (P<0.01). The percentage of specimens that had a microdissected area classified as tiny differed based on procurement method: 6% of resection specimens, 68% (160 of 237) of biopsy specimens, and 59% of FNA specimens (10 of 17). Invasive tumor cellularity was also associated with the size of the microdissected area, as less than 30% cellularity was observed in 60% of specimens with a tiny microdissected area and 33% of specimens with a medium or large microdissected area.

   Of the 22 specimens that were originally excluded from NGS analysis due to insufficient DNA (<10 ng), NGS was successful for 16 while 6 failed library preparation. NGS library construction was possible for 15 of the 17 specimens with 1.2 to 10 ng DNA but was successfully generated for only 1 of the 5 (20%) specimens with <1.2 ng DNA. Sanger sequencing was used to confirm variants in 10 cases with eight allowing for complete verification and two only partial verification. Using another cohort of 408 specimens, NGS was successful in 88% of specimens including 28 of 33 specimens (85%) with 1.2-10 ng DNA. Further, exclusion of specimens with <10 ng increased the NGS success rate to 95%. The effect of excluding specimens with <10 ng had the biggest effect on cytology blocks where success increased from 58% to 87% (P<0.01) and biopsy specimens where success increased from 84% to 95% (P<0.01). Even with the increased success observed after exclusion of specimens with <10 ng, a small difference remained between FNA and resection specimens (87% versus 96%, P=0.03). Block age did not affect NGS success rates.

   Biospecimens

   • Tissue - Bone
   • Tissue - Skin
   • Tissue - Breast
   • Tissue - Colorectal
   • Tissue - Ovary
   • Tissue - Vagina
   • Tissue - Uterus
   • Tissue - Kidney
   • Tissue - Head and Neck
   • Tissue - Liver
   • Tissue - Lung
   • Tissue - Lymph Node
   • Tissue - Esophagus
   • Tissue - Stomach
   • Tissue - Brain

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Sarcoma
   • Neoplastic - Benign
   • Neoplastic - Other
   • Neoplastic - Melanoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Melanoma Astrocytic tumor Unspecified brain tumor Sarcoma Adenocarcinoma Squamous cell carcinoma Serous carcinoma Hepatocellular carcinoma Neuroendocrine carcinoma Unspecified Carcinoma
   Biospecimen Acquisition	Biospecimen location	Bone Brain Breast Lung Liver Head/Neck Other deep-seated organs Colorectal Genitourinary organs Gynecologic organs Lymph Skin Soft tissue Upper gastrointestinal organs Primary tumor Metastasis
   Analyte Extraction and Purification	Decalcification solution/ duration	Decalcified Not decalcified
   Next generation sequencing Specific	Technology platform	Sanger Sequencing
   Storage	Storage duration	<1 year 1-2 years 3-6 years 7-12 years
   Biospecimen Aliquots and Components	Aliquot size/volume	<10mm2 10-20 mm2 20-60 mm2 >60 mm2
   Next generation sequencing Specific	Template/input amount	>10 ng DNA 1.2-10 ng DNA

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