ERG Immunohistochemistry as an Endothelial Marker for Assessing Lymphovascular Invasion

Abstract

Background: ERG, a member of the ETS family of transcription factors, is a highly specific endothelial marker. We investigated whether the use of ERG immunostaining can help pathologists detect lymphovascular invasion (LVI) and decrease interobserver variability in LVI diagnosis.

Methods: Fifteen cases of surgically resected colorectal cancers with hepatic metastasis were selected and the most representative sections for LVI detection were immunostained with ERG, CD31, and D2-40. Eight pathologists independently evaluated LVI status on hematoxylin and eosin (H&E) and the corresponding immunostained sections and then convened for a consensus meeting. The results were analyzed by kappa (κ) statistics.

Results: The average rate of LVI positivity was observed in 43% with H&E only, 10% with CD31, 29% with D2-40, and 16% with ERG. Agreement among pathologists was fair for H&E only (κ=0.27), D2-40 (κ=0.21), ERG (κ=0.23), and was moderate for CD31 (κ=0.55). Consensus revealed that ERG nuclear immunoreactivity showed better visual contrast of LVI detection than the other staining, with improved agreement and LVI detection rate (κ=0.65, LVI positivity rate 80%).

Conclusions: The present study demonstrated a superiority with ERG immunostaining and indicated that ERG is a promising panendothelial marker that might help pathologists increase LVI detection and decrease interobserver variability in LVI diagnosis.

Keywords: CD31; ERG; Endothelial marker; Immunohistochemistry; Lymphovascular invasion.

Fig. 1

Comparison of kappa values among pathologists for lymphovascular invasion (LVI) detection in colorectal cancers. While the average of LVI detection rate for each pathologist was 43% with hematoxylin and eosin (H&E) only, 10% with CD31, 29% with D2-40, and 16% with ERG, the consensus reached 80% of LVI detection after a joint discussion about ERG patterns with LVI. ^aInterpreted by ERG

Fig. 2

The cases that have differential diagnoses between presence of lymphovascular invasion (LVI) and retraction artifact tend to be diagnosed as positive LVI by hematoxylin and eosin examination only (A), but prove clear negative LVI by ERG immunostaining (B).

Fig. 3

Comparison of ERG, CD31, and D2-40 endothelial markers. (A) ERG, panendothelial marker showing nuclear immunoreactivity in artery, vein, and lymphatics. (B) ERG immunostaining specific for endothelial cells without cross-reactivity. (C) D31 immunostaining showing cross-reactivity in inflammatory cells. (D) D2-40 immunostaining showing cross-reactivity in fibroblasts.

Fig. 4

Examples showing high concordance rate of lymphovascular invasion (LVI) positivity in colorectal cancers. (A, B) Tumor cells surrounded by a vascular space with protruding endothelial nuclei well demonstrated by ERG immunostaining (100% concordance rate, arrow). (C) Single-cell metastasis in a single vascular space (100% concordance rate, arrow). (D) Floating single-cell metastasis even in an inflammatory background (75.0% concordance rate, arrow).

Fig. 5

Examples of interpretation issues in colorectal cancers. (A) Lymphovascular invasion (LVI) negativity with presence of thin fibrocollagenous layer between tumor cells and ERG-positive endothelial cells distinguishing from retraction artifact (100% concordance rate). (B) "Kissing pattern" showing tumor cells surrounded by endothelial nuclei interpreted as LVI positivity (87.5% concordance rate). (C, D) "Hugging pattern" highlighted by ERG immunostaining of endothelial nuclei embraced by tumor cells with unknown significance.

Fig. 6

ERG immunostaining in other cancers. (A) Positive lymphovascular invasion (LVI) distinguishing from retraction artifact by highlighted endothelial nuclei of vascular wall in breast cancer. (B) LVI showing tumor cells protruding into a lymphovascular space in gastric carcinoma. (C) Tumor cells attached to the vascular wall distinguishing from retraction artifact in squamous cell carcinoma of thyroid gland.

Fig. 7

Unequivocal or debatable examples of lymphovascular invasion (LVI). (A) Some inflammatory cells in a lymphovascular space in a noninflammatory background as a mimicker of true LVI is considered negative LVI with a 75% concordance rate. (B) Complicated lymphovascular spaces demonstrated by ERG staining with attachment of tumor cells showing a 50% concordance rate for positive LVI. (C) Debatable LVI with 50% concordance rate.