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Pre-analytical variables for the genomic assessment of the cellular and acellular fractions of the liquid biopsy in a cohort of breast cancer patients.

Author(s): Shishido SN, Welter L, Rodriguez-Lee M, Kolatkar A, Xu L, Ruiz C, Gerdtsson AS, Restrepo-Vassalli S, Carlsson A, Larsen J, Greenspan EJ, Hwang ES, Waitman KR, Nieva J, Bethel K, Hicks J, Kuhn P

Publication: J Mol Diagn, 2020, Vol. , Page

PubMed ID: 31978562 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of collection tube type, time to analysis, and analytical method on analysis of CTCs from patients with metastatic and non-metastatic breast cancer. The effects of time to analysis on single-cell sequencing of CTCs from Streck BCT was also investigated. Blood was collected from 98 patients with non-metastatic treatment naïve breast cancer or 65 patients with metastatic breast cancer. Blood was collected from patients with non-metastatic breast cancer before any treatment and 3-8 weeks post-surgical excision of the tumor and from patients with metastatic cancer 14 times with an interval of 8-12 weeks between visits. The effects of collection tube were investigated by collecting blood from 33 patients into CellSave and Streck BCT tubes and processing after 24 h. The effect of time to analysis was investigated by collecting blood from 35 patients into Streck tubes and processing after 24, 48, 72, and 96 h and then by analysis of 167 matched specimens from 84 patients processed after 24 and 48 h. Blood was centrifuged at 2000 x g for 10 min and the blood cells were reconstituted in PBS. Cells were analyzed using the CellSearch system with a positive result defined as >5 cells/7.5 mL. Replicate slides were prepared for rare cell enumeration and stored at -80°C until immunofluorescence analysis using antibodies to pan-cytokeratin (CK), anti-CK19, CD45, and estrogen receptor as well as DAPI staining. Total number of cells was determined by DAPI staining. HD-CTCs were identified as CK+, CD45- cells with intact nucleus and generally larger and morphologically distinct from surrounding WBCs. Cells that only partially met the HD-CTC criteria were classified as CTC-small if they were CK+, CD45- but had nuclei that were smaller or comparable to those in WBC; as CTC-LowCK if the CK levels were lower than that in HD-CTCs; and as CTC-cfDNA producing if they were CK+, CD45- with a DAPI pattern consistent with nuclear condensation and fragmentation. For single-cell sequencing, CTCs identified were removed using a micropipette and placed into a tube containing TE buffer and frozen at -80°C. Whole genome amplification (WGA) was performed on CTCs using the WGA4 Genomeplex Single Cell Whole Genome Amplification Kit. Sequencing libraries were prepared from WGA DNA using the NEBNext Ultra II DNA Library Prep Kit, size-selected using AMPure Beads, PCR enriched, purified with AMPure XP beads, and sequenced using an Illumina NextSeq 500 or the HiSeq2500 PE100. SNV analysis was conducted using the NEBNext Direct Cancer HotSpot Panel and the Illumina HiSeq2500 PE100 run.

   Summary of Findings:

   There was a median of 0 (0-2832) high definition CTCs/mL with 60.1% of patients (98) having detectable CTCs in at least one blood draw. When broken down by diagnosis, the median HD-CTC was 0 in both groups, but 63.3% of patients with non-metastatic breast cancer and 55.4% of patients with metastatic breast cancer had at least one specimen positive for HD-CTCs. There were comparable numbers of HD-CTCs, CTC-LowCK, CTC-Small, and CTC-cfDNA producing cells in metastatic and non-metastatic patients. HD-SCA detected significantly more CTCs than CellSearch (P=0.0028) and more patient specimens were found to be CTC positive (>5 CTCs/7.5mL) when HD-SCA was used than Cell Search (15 versus 5), but the results were strongly correlated between methods (r=0.9992, P<2.2e^-16). Importantly, a count of >5 using the HD-SCA method had a sensitivity of 80% and specificity of 82.86-97.14% or positivity using the CellSearch method (>5 cells/7.5 mL). CellSave tubes had 10% fewer cells retained (DAPI staining) than those in Streck BCT (P=0.0005), but the number of HD-CTCs was comparable among the tube types. Further examination showed fewer WBCs in the CellSave tubes than the Streck BCT specimens and that the nuclei of the WBCs in CellSave tubes were more elliptical than the circular nuclei observed for specimens in the Streck tubes. The HD-CTCs in the CellSave tube had a smaller nuclear area, less round nuclei, lower nuclear area ratio, and less nuclear solidity than those in Streck BCTs (P<0.0001, all). Increased time to analysis resulted in decreasing numbers of HD-CTCs with 8%, 29%, and 42% losses observed after 48, 72, and 96 h; respectively, compared to 24 h. Further investigation using more specimens comparing the 24 versus 48 h timepoints found no difference in the number of candidate cells or the distribution of HD-CTC and marginal cell populations (CTC-small, CTC-LowCK, or CTC cfDNA-producing), but there was a 6.5% decrease in cell retention at 48 h (P=0.0088). Not surprisingly, the specimens that were CTC positive when analyzed after 24 h but negative when analyzed after 48 h had lower number of candidate cells, HD-CTCs, and lower numbers of each of the marginal cell populations. Single-cell sequencing success rates were comparable in specimens analyzed after 24 and 48 h but DNA concentration, percent aligned reads, percent coverage, and variant counts were higher (P<0.0001, P=0.0055, P<0.0001, and P<0.0001; respectively) and total read and quality score were lower (P=0.0062 and P<0.0001; respectively) in specimens analyzed after 48 h than those analyzed after 24h. The authors attribute the lower DNA concentration to the longer interval between immunofluorescence and cell isolation in the 24 specimens than the 48 h specimens. While more unique variants were identified in one patient with a time to analysis of 48 h than 24 h or both (P=0.0289), neither the number of unique variant calls nor the gene region (exonic versus intronic) were significantly different.

   Biospecimens

   • Cell - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light microscopy
   DNA	Next generation sequencing
   Cell count/volume	Immunoassay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Metastatic breast cancer Non-metastatic breast cancer
   Immunoassay Specific	Technology platform	CellSearch HD-SCA
   Storage	Storage duration	24 h 48 h 72 h 96 h
   Biospecimen Acquisition	Type of collection container/solution	Streck BCT CellSave BCT

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2. Study Purpose

   The purpose of this study was to investigate the effects of frozen plasma storage on mutation detection by next generation sequencing (NGS) of cfDNA. Blood was collected from 98 patients with non-metastatic, treatment naïve breast cancer or 65 patients with metastatic breast cancer. Blood was collected from patients with non-metastatic breast cancer before any treatment and 3-8 weeks post-surgical excision of the tumor and from patients with metastatic cancer 14 times with an interval of 8-12 weeks. The effects of collection tube was investigated by collecting blood from 23 patients into Streck BCT tubes. Blood was centrifuged at 2000 x g for 10 min and the plasma was transferred to a new tube and centrifuged twice at 14000 x g for 10 min. cfDNA was extracted from fresh plasma and matched plasma stored at -80°C for 9-24 days (median of 14 days) using the QIAamp Circulating Nucleic Acid Kit. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit, size-selected using AMPure Beads, PCR enriched, purified with AMPure XP beads, and sequenced using Illumina NextSeq 500 or the HiSeq2500 PE100. SNV analysis was conducted using the QIASeq Target DNA Breast Cancer Panel and the Illumina HiSeq2500 PE100 run.

   Summary of Findings:

   All sequencing metrics including cfDNA concentration, number of aligned reads, percentage aligned reads, and CNV quality score were comparable in fresh and frozen plasma. There was a strong correlation in mutation frequencies in fresh and frozen plasma (R²=0.8714, P<0.0001). There were no significant differences in mutation location (exonic versus intronic), type of mutation (missense, synonymous, or frameshift) or predicted function (tolerated, damaging, or activating) between fresh or frozen plasma.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 days 9-24 days
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)

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