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Updated: Jun 10, 2026
Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment
Published on: August 18, 2023
Novel classification based on immunohistochemistry combined with hierarchical clustering analysis in non-functioning
Shinya Iida1, Yasuhiro Miki, Katsuhiko Ono
1Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Abstract:
Somatostatin analogues ameliorated many symptoms caused by neuroendocrine tumors (NET), but their antitumor activities are limited especially in non-functioning cases. An overactivation of signaling pathways under receptor tyrosine-kinase (RTK) has been recently demonstrated in some NET patients, but its details have remained largely unknown. Therefore, in this study, we immunolocalized therapeutic factors and evaluated the data to study the clinical significance of the molecules in non-functioning Japanese gastrointestinal NET. Fifty-two NET cases were available for examination in this study and expression of somatostatin receptor (sstr) 1, 2A, 2B, 3 and 5, activated form of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4-binding protein 1 (4EBP1), ribosomal protein s6 (S6), extracellular signal-regulated kinase (ERK) and insulin-like growth factor 1 receptor (IGF-1R) was evaluated using immunohistochemistry. We then studied the correlation among the immunohistochemical results of the individual cases using hierarchical clustering analysis. Results of clustering analysis demonstrated that NET cases were basically classified into Cluster I and II. Cluster I was associated with higher expression of sstr1, 2B and 3 and Cluster II was characterized by an activation of the PI3K/Akt pathway and IGF-1R and higher proliferative status. Cluster II was further classified into Cluster IIa and IIb. Cluster IIa was associated with higher expression of sstr1 and 5 and higher proliferative status and Cluster IIb was characterized by ERK activation. Hierarchical clustering analysis of immunoreactivity of the therapeutic factors can classify NET cases into three distinctive groups and the medical treatment may be determined according to this novel classification method for non-functioning NET patients.
Insights
This study classifies non-functioning neuroendocrine tumors (NET) into three groups using molecular markers. This novel classification may guide personalized treatment for gastrointestinal NET patients.
Area of Science:
- Oncology
- Molecular Biology
- Gastroenterology
Background:
- Somatostatin analogues offer limited antitumor activity in non-functioning neuroendocrine tumors (NET).
- Receptor tyrosine-kinase (RTK) pathway overactivation is implicated in some NETs, but details remain unclear.
- Understanding molecular drivers is crucial for improving non-functioning NET treatment.
Purpose of the Study:
- To immunolocalize therapeutic factors and evaluate their clinical significance in non-functioning Japanese gastrointestinal NET.
- To explore the correlation among molecular markers using hierarchical clustering analysis.
- To establish a novel classification method for non-functioning NET based on molecular profiles.
Main Methods:
- Immunohistochemistry was used to evaluate the expression of somatostatin receptors (sstr 1-3, 5), mTOR, 4EBP1, S6, ERK, and IGF-1R in 52 NET cases.
- Hierarchical clustering analysis was applied to correlate the expression patterns of these molecules.
- NET cases were classified into distinct clusters based on molecular marker immunoreactivity.
Main Results:
- NET cases were primarily classified into two clusters: Cluster I (higher sstr1, 2B, 3 expression) and Cluster II (PI3K/Akt pathway activation, IGF-1R, higher proliferation).
- Cluster II was further divided into Cluster IIa (higher sstr1, 5, proliferation) and Cluster IIb (ERK activation).
- Hierarchical clustering identified three distinctive molecular groups within non-functioning NETs.
Conclusions:
- Molecular profiling via hierarchical clustering can effectively classify non-functioning NETs into distinct prognostic groups.
- This novel classification holds potential for guiding personalized medical treatment strategies for non-functioning NET patients.
- Further research into RTK pathway signaling in NETs is warranted to optimize therapeutic interventions.
