Why Formalin-fixed, Paraffin-embedded Biospecimens Must Be Used in Genomic Medicine: An Evidence-based Review and ConclusionMathieson, William; Thomas, Geraldine A.
doi: 10.1369/0022155420945050pmid: 32697619
Fresh-frozen tissue is the “gold standard” biospecimen type for next-generation sequencing (NGS). However, collecting frozen tissue is usually not feasible because clinical workflows deliver formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Some clinicians and researchers are reticent to embrace the use of FFPE tissue for NGS because FFPE tissue can yield low quantities of degraded DNA, containing formalin-induced mutations. We describe the process by which formalin-induced deamination can lead to artifactual cytosine (C) to thymine (T) and guanine (G) to adenine (A) (C:G > T:A) mutation calls and perform a literature review of 17 publications that compare NGS data from patient-matched fresh-frozen and FFPE tissue blocks. We conclude that although it is indeed true that sequencing data from FFPE tissue can be poorer than those from frozen tissue, any differences occur at an inconsequential magnitude, and FFPE biospecimens can be used in genomic medicine with confidence:
The Expression Pattern of Epidermal Differentiation Marker Keratin 10 in the Normal Human Breast and Breast Cancer CellsKim, Jiyoung; Villadsen, René
doi: 10.1369/0022155420940220pmid: 32618487
Cells of the human breast gland express an array of keratins, of which some are used for characterizing both normal and neoplastic breast tissue. However, the expression pattern of certain keratins has yet to be detailed. Here, the expression of a differentiation marker of epidermal epithelium, keratin 10 (K10), was investigated in the human breast gland. While in normal breast tissue generally less than 1% of luminal epithelial cells expressed K10, in women >30 years of age glandular structures with K10-positive (K10pos) cells were found at higher frequency than in younger women. K10pos cells belong to a mature luminal compartment as they were negative for cKIT, positive for Ks20.8, and mostly non-cycling. In breast cancer, around 16% of primary breast carcinomas tested were positive for K10 by immunohistochemistry. Interestingly, K10pos tumor cells generally exhibit features of differentiation similar to their normal counterparts. Although this suggests that K10 is a marker of tumor differentiation, data based on gene expression analysis imply that high levels of K10 dictate a worse outcome for breast cancer patients. These findings can form the basis of future studies that should unravel which role K10 may play as a marker of breast cancer:
Post-diaminobenzidine Treatments for Double Stainings: Extension of Sulfide-Silver-Gold Intensification for Light and Fluorescent MicroscopyTörök, Ibolya; Seprényi, György; Pór, Erzsébet; Borbély, Emőke; Szögi, Titanilla; Dobó, Endre
doi: 10.1369/0022155420942213pmid: 32660313
Double staining protocols using the most popular immunoperoxidase techniques may raise difficulties. The two ordinary detection systems may cross-talk, when the primary antibodies are derived from phylogenetically closely related animals. A color shift of the 3,3′-diaminobenzidine (DAB) polymer may occur during the second development, resulting in poor distinction between the two kinds of deposits. A post-DAB technique, sulfide-silver-gold intensification, was fine tuned to eliminate these difficulties, which may be especially suitable for colocalization of cell nuclei and perikarya of the same cells. The revised method was probed in combination with a subsequent other immunoperoxidase step or fluorochrome-tagged reagents. The nuclear antigens (BrdU, c-Fos, and Prox-1) were first visualized with DAB polymer, which were then treated with SSGI, turning the deposit black. Thereafter, cytoplasmic antigens (doublecortin, neuronal nuclei, and calbindin) were detected with either another immunoperoxidase using DAB again or immunofluorescence labeling. In both approaches, the immunopositive nuclei and cytoplasmic sites could be easily distinguished even at low magnifications. Different shielding or eluting posttreatments were compared for consecutive acetylcholinesterase histochemistry terminated with DAB development and immunohistochemistry in the same sections. In conclusion, we recommend post-DAB treatments that abolish interactions between detection systems and allow clear distinction between the two signals under various conditions: