EGFR, KRAS and ALK Molecular Tests Can Be Performed Using Existing Stained Histologic Slides, Including Small Biopsies: A Proof-of-Concept Study
Sanjay Mukhopadhyay, Jamie Tull, Shengle Zhang. State University of New York Upstate Medical University, Syracuse, NY
Background: Testing pulmonary adenocarcinomas in small biopsies for EGFR and KRAS mutations and ALK gene rearrangements is now standard practice. However, tissue in paraffin blocks is occasionally depleted by histology and/or immunohistochemistry (IHC), leaving no cells for molecular studies. Our aim was to determine whether tumor cells from existing stained histology slides can be used for EGFR, KRAS and ALK testing, especially in small biopsies.
Design: Ten pulmonary adenocarcinomas with known EGFR mutations (5), KRAS mutations (2) or ALK gene rearrangments (3) were re-tested for EGFR, KRAS and ALK using stained histologic slides. For PCR analysis, tumor cells were marked on existing hematoxylin-eosin (H&E) stained slides under a microscope and macro-dissected from the marked areas with a 19 gauge needle. DNA was isolated and tested for EGFR (exon 19 deletion and L858R mutation) and KRAS mutations. Based upon our preliminary data that FISH for ALK on H&E stained slides is uninterpretable due to eosin autofluorescence, FISH for ALK was instead performed on existing negative control slides prepared during initial IHC. After ISH with an ALK break-apart probe, signals were visualized with a fluorescent microscope. ALK FISH was considered positive if ≥15% of tumor cells showed break-apart and/or 5'deletion fluorescent signals.
Results: DNA sufficient for EGFR and KRAS testing was obtained from H&E-stained slides in all 10 cases, yielding 5 EGFR mutations/deletions and 2 KRAS mutations identical to the original results using the paraffin block. Satisfactory FISH signals for ALK testing were obtained from all 10 IHC negative control slides, 3 showing ALK split signals identical to the original results using the paraffin block.
|Case||Specimen||EGFR (H&E)||KRAS (H&E)||ALK (neg ctrl slides)|
|1||CB, lung||Exon 19 del||wt||n|
|2||Bone curettings||Exon 19 del||wt||n|
|3||FNA lymph node||Exon 19 del||wt||n|
|4||CB, lung||Exon 21 mut||wt||n|
|5||CB, lung||Exon 21 mut||wt||n|
|6||CB, lung||wt||G12C mut||n|