Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumors suitable for in vivo somatostatin receptor targeting

Abstract

High overexpression of somatostatin receptors in neuroendocrine tumors allows imaging and radiotherapy with radiolabeled somatostatin analogues. To ascertain whether a tumor is suitable for in vivo somatostatin receptor targeting, its somatostatin receptor expression has to be determined. There are specific indications for use of immunohistochemistry for the somatostatin receptor subtype 2A, but this has up to now been limited by the lack of an adequate reliable antibody. The aim of this study was to correlate immunohistochemistry using the new monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 with the gold standard in vitro method quantifying somatostatin receptor levels in tumor tissues. A UMB-1 immunohistochemistry protocol was developed, and tumoral UMB-1 staining levels were compared with somatostatin receptor binding site levels quantified with in vitro I-[Tyr]-octreotide autoradiography in 89 tumors. This allowed defining an immunohistochemical staining threshold permitting to distinguish tumors with somatostatin receptor levels high enough for clinical applications from those with low receptor expression. The presence of >10% positive tumor cells correctly predicted high receptor levels in 95% of cases. In contrast, absence of UMB-1 staining truly reflected low or undetectable somatostatin receptor expression in 96% of tumors. If 1% to 10% of tumor cells were stained, a weak staining intensity was suggestive of low somatostatin receptor levels. This study allows for the first time a reliable recommendation for eligibility of an individual patient for in vivo somatostatin receptor targeting based on somatostatin receptor immunohistochemistry. Under optimal methodological conditions, UMB-1 immunohistochemistry may be equivalent to in vitro receptor autoradiography.

Conflict of interest statement

Disclosures: A.S. is currently receiving a grant from NIH. The other authors have no conflicts of interest or funding to disclose.

Figures

Figure 1

Scoring of immunohistochemical staining intensity in typical examples (A: ileal neuroendocrine carcinoma, B: neuroendocrine carcinoma of the gall bladder, C: pulmonary carcinoid tumor). A: 3+ staining intensity represents strong staining at low magnification and fully circumferential staining of tumor cells membranes at high magnification (inset). B: 2+ staining intensity equals strong staining at low magnification, but no staining of the entire tumor cell circumference at high magnification (inset). C: 1+ staining intensity is defined by weak staining of the tumor cell membranes at low and high (inset) magnification.

Figure 2

UMB-1 Western blot analysis providing evidence of the specificity of UMB-1 staining for sst2A in an insulinoma. There is a single broad band (arrow) migrating at approximately 53 kb, the expected size of sst2A. This band is completely abolished when adding the immunogen peptide.

Figure 3

Relation between immunohistochemical UMB-1 staining (concentration 1:20, pre-treatment PC-C) and 125I-[Tyr3]-octreotide binding levels in 89 tumors. A cut-off value of 3’500 dpm/mg is applied to distinguish between no or low levels and high levels of 125I-[Tyr3]-octreotide binding. A: If a tumor exhibits UMB-1 staining in more than 10% of tumor cells, the likelihood that it expresses 125I-[Tyr3]-octreotide binding levels above 3’500 dpm/mg and, therefore, is eligible for somatostatin receptor targeting is very high (high positive predictive value). B: Almost all tumors with 125I-[Tyr3]-octreotide binding above 3’500 dpm/mg exhibit strong (>1+) staining of tumor cells (high sensitivity).

Figure 4

Comparison of UMB-1 immunohistochemistry (concentration 1:20, pre-treatment PC-C; first two columns) with 125I-[Tyr3]-octreotide receptor autoradiography (last column; bars indicate 1mm) in typical examples of tumors with high somatostatin receptor levels (rows 1 and 2), low somatostatin receptor levels (row 3) and no somatostatin receptor expression (row 4). A–E, malignant insulinoma: Strong membranous UMB-1 staining of most tumor cells (A) and often of entire tumor cell circumference (3+; B). Receptor autoradiography on serial tissue sections of the same case showing the tumor tissue stained with HE in C, very strong total 125I-[Tyr3]-octreotide binding to the entire tumor sample in D and complete displacement of 125I-[Tyr3]-octreotide by cold octreotide, providing evidence of specificity of somatostatin receptor binding in E (ns = non-specific 125I-[Tyr3]-octreotide binding in presence of excess cold octreotide). There is an excellent correlation between strong UMB-1 staining and high 125I-[Tyr3]-octreotide binding levels. F–K, ileal neuroendocrine carcinoma: Strong membranous UMB-1 staining of a large proportion of tumor cells (F), in some tumors cells affecting the entire tumor cell circumference (3+; G). This corresponds well to the strong specific 125I-[Tyr3]-octreotide binding to the entire tumor (asterisks) in the autoradiography experiment (H–K). L–P, ileal neuroendocrine carcinoma: Weak membranous UMB-1 staining at low (L) and high (M) magnification, correlating well with weak 125I-[Tyr3]-octreotide binding to the tumor tissue (asterisks; N–P). Q–U, benign insulinoma: No UMB-1 staining (Q, R), which matches well with negative receptor autoradiography (S–U).

Figure 5

Cases requiring cautious interpretation of immunohistochemical UMB-1 staining (concentration 1:20; pre-treatment PC-C; columns 1–3) in view of 125I-[Tyr3]-octreotide autoradiography results (last column; bars indicate 1mm). A–F, meningioma: Wide-spread and strong UMB-1 positivity at low magnification (A) that is completely abolished by the immunogen peptide (B). At high magnification (C), there is often a blurred staining in the area of the cell membrane, but only rarely a clear membranous staining. This staining pattern is probably due to prominent intercellular interdigitations. Autoradiographically, there is strong specific 125I-[Tyr3]-octreotide binding to the entire tumor tissue (D–F). Although <10% of tumor cells show membranous staining, this tumor is an excellent candidate for in vivo somatostatin receptor targeting. G–M, ganglioneuroblastoma: UMB-1 staining is present in single ganglionic tumor cells (arrow; G) in a membranous distribution (I, high magnification), but not in larger areas of neuroblastic tumor cells (asterisk; G). UMB-1 staining of ganglionic tumor cells is completely abolished by the immunogen peptide (H), providing proof of specific UMB-1 staining. No specific 125I-[Tyr3]-octreotide binding with receptor autoradiography (K–M). Despite strong tumor cell staining, this tumor is apparently not suitable for in vivo somatostatin receptor targeting, as the total receptor number per tumor mass is too low.