[1918] 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.

Molecular Findings Using Stained Histology Slides
CaseSpecimenEGFR (H&E)KRAS (H&E)ALK (neg ctrl slides)
1CB, lungExon 19 delwtn
2Bone curettingsExon 19 delwtn
3FNA lymph nodeExon 19 delwtn
4CB, lungExon 21 mutwtn
5CB, lungExon 21 mutwtn
6CB, lungwtG12C mutn
7LobectomywtG12C mutn
8CB, lungwtwtPos
9CB, liverwtwtPos
10LobectomywtwtPos
CB-core biopsy, wt-wild type, n-normal signal


Conclusions: Tumor cells derived from existing H&E-stained slides can be used for PCR-based assays (EGFR and KRAS), and cells on existing IHC negative control slides (rather than H&E) can be used for ALK FISH testing, even in small biopsies. This approach can obviate the need for repeat biopsies in cases in which the paraffin block is depleted by histology and/or IHC.
Category: Pulmonary

Monday, March 4, 2013 1:00 PM

Poster Session II # 284, Monday Afternoon

 

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