Formalin Fixation, Delay to Fixation, and Time in Fixative Adversely Impact Copy Number Variation Analysis by aCGH.
Author(s): Li J, Greytak SR, Guan P, Engel KB, Goerlitz DS, Islam M, Varghese RS, Moore HM, Ressom HW
Publication: Biopreserv Biobank, 2022, Vol. , Page
PubMed ID: 36169416 PubMed Review Paper? No
Purpose of Paper
This paper compared copy number variation (CNV) results from array comparative genomic hybridization (aCGH) between patient-matched blood, and frozen and formalin-fixed paraffin-embedded (FFPE) renal cell carcinoma (RCC) specimens and among FFPE tumor specimens that experienced different delays to fixation (DTF; 1, 2, 3, 12 h at room temperature) and times in fixative (TIF; 6, 12, 23, 72 h).
Conclusion of Paper
When aCGH data was examined by PCA and all specimens were considered together, specimens did not cluster consistently by patient, tumor, or whether DNA purity or DLRSD thresholds were met. FFPE tumor specimens had nearly 2-fold more regions containing CNVs than were detected in snap-frozen tumor specimens. The number of CNVs that were detected in at least 20% or 50% of patients was also consistently higher in FFPE specimens from the TIF time course than respective snap-frozen specimens.
DTF-dependent effects on aCGH profile were progressive, with the number of CNV-containing regions declining by 27% after a 12h DTF compared the 1 h DTF control. The number of regions with a detectable CNV in at least 20% or 50% of patients also declined over the DTF time course, which resulted in a reduction in the average number of CNVs identified per patient from 8.2 after a 1 h DTF to 4.9 patients after a 12 h DTF. When specimens from the DTF time course were plotted separately, a subgroup of 12 h DTF specimens was observable. TIF-mediated effects were less clear, with fewer CNV-containing regions and fewer CNVs identified per patient in 6, 12, and 72 h TIF specimens compared to those fixed for 23 h. However, PCA plots of specimens from the TIF time course clustered 72 h TIF specimens as a subgroup apart from other TIF time points and specimen types (blood and snap-frozen).
A greater number of differences in CNV status (unchanged, amplification, deletion) were observed when FFPE tumor specimens were compared to case-matched blood specimens then when they were compared to the same tumor that was snap-frozen for both DTF (1-3 h; p<0.001) and TIF (6012 h; p<0.001) FFPE specimens. The magnitude and statistical significance of DTF and TIF associated effects depended on whether blood or snap-frozen tumor was used for comparison and the stringency of the statistical test. Overall, the number of regions with a CNV with a different progressively declined with longer TIFs when blood (p<0.01) or snap-frozen tumor (P<0.001) was used for comparison and increased with longer DTFs when blood or snap-frozen tumor was used for comparison (p<0.001 for both). Notably, there was little overlap in the affected regions among TIF or DTF timepoints, regardless of whether comparisons were to blood or snap-frozen tumor. The average GC content of segments containing a CNV with an altered status was lower than in segments that remained stable in 12 or 72 h TIF FFPE specimens and all DTF FFPE specimens (p<0.005 for all), but differences between affected and stable segments were not significant in 6 or 23 h TIF FFPE specimens. The percentage of short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs) were lower and higher, respectively, in segments containing a CNV with an altered status than in stable segments, although the difference in LINEs was not observed among all FFPE TIF timepoints.
Studies
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Study Purpose
This study compared array comparative genomic hybridization (aCGH) profiles from FFPE RCC specimens with those from patient-matched blood (for germline comparisons) and snap-frozen tumor specimens. Blood and surgically resected RCC tumor specimens were collected from 38 patients as part of the National Cancer Institute’s Biospecimen Pre-analytical Variables (BPV) Program. Blood was collected in PAXgene Blood Collection Tubes prior to the initiation of anesthesia and stored at -20°C for 24-72 h before transfer to -80°C. RCC tumor specimens were divided into six equally-sized aliquots that were snap-frozen in the vapor phase of liquid nitrogen within 1 h (snap-frozen control), fixed in 10% neutral buffered formalin (NBF) for 12 h within 1 h of collection, and subjected to four different delays to fixation (DTF; 1, 2, 3, 12 h) or times in fixative (TIF; 6, 12, 23, 72 h). For the DTF time course, tumor aliquots were held in a humidified chamber at room temperature for the specified duration prior to fixation in 10% NBF for 10-12 h. For the TIF time course, within 1 h of resection tumor aliquots were fixed in 10% NBF for the specified duration. DNA was extracted using the QIAsymphony DNA Mini Kit from blood, FFPE sections (10 µm -thick, 4-6 sections) and snap-frozen tumor specimens (25 mg). FFPE sections were deparaffinized, demodification (1 h at 90°C), and RNase-treated prior to extraction. DNA purity was assessed by Nanodrop spectrophotometer and DNA concentration was determined by Qubit fluorometer. DNA samples were shipped on dry ice and stored at -80°C until whole genome CNV analysis with an Agilent SurePrint G3 Human CNV Microarray 400K using reference DNA. Derivative log ratio standard deviation (DLRSD) scores and the following specimen-specific quality thresholds were to qualify samples for aCGH analysis: <0.4 (FFPE tumor), <0.3 (snap-frozen tissue), <0.2 (blood). Diploid copy number changes (relative to the reference DNA) were classified as unchanged, an amplification, or a deletion. Segmentation analysis was used to quantify CNV-containing regions, which were then evaluated by principal component analysis (PCA). The statistical significance of differences in the frequency of copy number classifications (unchanged, amplification, deletion) between specimen types and DTF/TIF timepoints was determined by McNemar’s test. Guanine-cytosine (GC) content was also determined for CNV-containing regions.
Summary of Findings:
In total, the vast majority of specimens passed the DNA purity threshold (88.5%, OD 260.280 ≥1.6) and generated aCGH data (235/236 specimens). DLRSD score, which quantifies signal to noise ratio, was below the maximum allowable specimen/preservation-specific threshold for 89.8% of the specimens analyzed. The 24 specimens that exceeded the DLRSD threshold also displayed low Cy5 signal intensity, suggesting insufficient labeling, and included both blood, FFPE and snap-frozen tumor specimens. When aCGH data was examined by PCA and all specimens were considered together, specimens did not cluster consistently by patient, tumor, or whether DNA purity or DLRSD thresholds were met. FFPE tumor specimens had nearly 2-fold more regions containing CNVs than were detected in snap-frozen tumor specimens. The number of CNVs that were detected in at least 20% or 50% of patients was also consistently higher in FFPE specimens from the TIF time course than respective snap-frozen specimens (≥20% of specimens: 26,685- 30,446 versus 9,127; ≥50% of specimens: 11,936-20,639 versus 1,580).
DTF-dependent effects on aCGH profile were progressive, with the number of CNV-containing regions declining by 27% after a 12h DTF compared the 1 h DTF control. The number of regions with a detectable CNV in at least 20% or 50% of patients declined by 50% and 81%, respectively, over the DTF time course, which also resulted in reduction in the average number of CNVs identified per patient from 8.2 after a 1 h DTF to 4.9 patients after a 12 h DTF. When specimens from the DTF time course were plotted separately, 12 h DTF specimens formed a subgroup. TIF-mediated effects were less clear, with fewer CNV-containing regions and fewer CNVs identified per patient in 6, 12, and 72 h TIF specimens compared to those fixed for 23 h. PCA plots of specimens from the TIF time course did reveal that 72 h TIF specimens formed a subgroup apart from other TIF time points and specimen types (blood and snap-frozen).
As expected, more differences in CNV status (unchanged, amplification, deletion) were observed when FFPE tumor specimens were compared to case-matched blood specimens than snap-frozen tumor for both the DTF (1-3 h; p<0.001) and TIF (6012 h; p<0.001) timecourse. The number of regions with a CNV with a different status progressively declined with longer TIFs when blood (p<0.01) or snap-frozen tumor (P<0.001) was used for comparison, and increased with longer DTFs when blood or snap-frozen tumor was used for comparison (p<0.001 for both). Notably, there was little overlap in the affected regions among TIF or DTF timepoints regardless of whether comparisons were to blood or snap-frozen tumor. To illustrate, less than 1% of segments that differed significantly between blood and snap-frozen tumor also differed between blood and FFPE tumor, and very few segments differed consistently among all respective DTF or TIF timepoints and snap-frozen tumor (DTF: 152 segments; TIF: 19 segments). The average GC content of segments containing a CNV with an altered status was lower than in segments that remained stable in 12 or 72 h TIF FFPE specimens and all DTF FFPE specimens (p<0.005 for all), but differences between affected and stable segments were not significant in 6 or 23 h TIF FFPE specimens. The percentage of short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs) were lower and higher, respectively, in segments containing a CNV with an altered status than in stable segments, although the difference in LINEs was not observed among all FFPE TIF timepoints.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA CGH DNA Spectrophotometry DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Snap frozen
Formalin (buffered)
Biospecimen Acquisition Cold ischemia time 1 h
2 h
3 h
12 h
Biospecimen Preservation Time in fixative 6 h
12 h
23 h
72 h
Biospecimen Acquisition Biospecimen location RCC tumor
Blood