Factors affecting KRAS mutation detection in colorectal cancer tissue.
Author(s): Yun HG, Lee HJ, Kim BR, Lee JH, Lee JH, Lee MY, Kim DH, Sohn JH, Chae SW, Do IG, Do SI, Kim K
Publication: Pathol Res Pract, 2019, Vol. 215, Page 1071-1075
PubMed ID: 30846412 PubMed Review Paper? No
Purpose of Paper
This paper investigated potential effects associated with cold ischemia time and the duration of formalin fixation on the quality of DNA extracted from non-case matched formalin-fixed, paraffin-embedded (FFPE) colon specimens and the detection rate of (KRAS) by qPCR. Both biopsy and surgically resected specimens were evaluated, and specimens were not case-matched.
Conclusion of Paper
Although, significantly more specimens yielded optimal DNA when cold ischemia was less than 1 h than greater than 1 h, the difference was not significant when only surgical specimens were considered. The number and percentage of specimens with optimal DNA quality as determined by real-time PCR cycle threshold value (Ct) was comparable between surgical specimens regardless of cold ischemia time (≤1 h, 1-3 h, or > 3 h) or fixation duration (≤24 h, 24-48 h, 48-72 h, 72-96 h, or >96 h). Prolonged formalin fixation (≥30 d) of case-matched surgically resected colorectal cancer specimens resulted in significantly higher Ct values (p≤0.015). The rate of KRAS mutation detection was significantly higher among specimens with optimal compared to acceptable (30≤Ct<34) DNA quality, but did not differ among biopsy and resected specimens.
Studies
-
Study Purpose
This study investigated potential effects associated with cold ischemia time and the duration of formalin fixation on DNA quality and KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutation detection rate in 405 colorectal carcinoma specimens, which included 74 biopsy and 327 surgically resected specimens. The cold ischemia time and fixation duration were recorded for each specimen and ranged from 0-29 h and 8-120 h, respectively; specimens were not case-matched. Biopsy specimens were immediately placed in 10% neutral buffered formalin (NBF), while resection specimens were held in a refrigerator until processing. Pathologist-defined areas of tumor were isolated from 5 µm-thick FFPE sections and DNA was extracted with the Maxwell FFPE Purification Kit for DNA. DNA concentration and purity were determined by Nanodrop spectrophotometer and quantity was determined by PicoGreen. The Panagene PNAClamp KRAS Mutation Detection kit was used to detect KRAS mutations based on ΔCt values, and without the PNA mix to assess DNA quality. DNA quality was categorized as optimal (22 < Ct < 30), acceptable (30 ≤ Ct < 34), or invalid (Ct ≤ 22 or Ct ≥ 34). The potential influence of the following factors on DNA quality were also evaluated, prior influence with chemotherapy, tumor shape and size, and histologic differentiation.
Summary of Findings:
When biopsy and resection specimens were analyzed together, significantly more specimens yielded optimal DNA when cold ischemia was less than 1 h (p=0.038); however differences were not significant when analysis was limited to surgically resected specimens. While not statistically significant, the percentage of specimens that yielded DNA of optimal quality increased with the duration of fixation from 24 (86.6%) to 72 h (95.7%), although the percentage declined with fixation for 72-96 h (90.5%) and >96 h (88.9%). Notably, significant differences among biopsy and resected specimens were present for mean cold ischemia time (0 vs 6.5 h, respectively; p<0.001) and the duration of formalin fixation (29 vs 36.6 h, respectively; p=0.03) but not tumor cellularity (63.92 vs 66.20%). The KRAS mutation detection rate was significantly higher among specimens with optimal DNA quality than those with acceptable quality (38.8 vs 17.5%; p=0.027), although no difference was observed between biopsy and resected specimens.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative ≤24 h
24-48 h
48-72 h
72-96 h
96-120 h
Biospecimen Acquisition Cold ischemia time 0-1 h
>1-3 h
>3-29 h
-
Study Purpose
Effects of prolonged formalin fixation were investigated in five colorectal carcinoma specimens. Surgically resected specimens were dissected into three equally sized pieces and fixed in 10% neutral buffered formalin for 30, 60, and 90 d. Pathologist-defined areas of tumor were isolated from 5 µm-thick FFPE sections and DNA was extracted with the Maxwell FFPE Purification Kit for DNA. DNA concentration and purity were determined by Nanodrop spectrophotometer and quantity was determined by PicoGreen. The Panagene PNAClamp KRAS Mutation Detection kit was used to detect KRAS mutations based on ΔCt values, and without the PNA mix to assess DNA quality. DNA quality was categorized as optimal (22 < Ct < 30), acceptable (30 ≤ Ct < 34), or invalid (Ct ≤ 22 or Ct ≥ 34).
Summary of Findings:
While DNA was of sufficient quality for KRAS mutation detection in all specimens, mean Ct values increased with fixation duration. Mean Ct values were significantly higher after formalin fixation for 30 d (p=0.015), 60 d (p=0.004), and 90 d (p<0.001) compared to baseline specimens (fixation duration of baseline specimens was not specified by the authors).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative Baseline (unspecified)
30 d
60 d
90 d