The Expression of Anti-Müllerian Hormone Type II Receptor (AMHRII) in Non-Gynecological Solid Tumors Offers Potential for Broad Therapeutic Intervention in Cancer

Abstract

The anti-Müllerian hormone (AMH) belongs to the TGF-β family and plays a key role during fetal sexual development. Various reports have described the expression of AMH type II receptor (AMHRII) in human gynecological cancers including ovarian tumors. According to qRT-PCR results confirmed by specific In-Situ Hybridization (ISH) experiments, AMHRII mRNA is expressed in an extremely restricted number of normal tissues. By performing ISH on tissue microarray of solid tumor samples AMHRII mRNA was unexpectedly detected in several non-gynecological primary cancers including lung, breast, head and neck, and colorectal cancers. AMHRII protein expression, evaluated by immunohistochemistry (IHC) was detected in approximately 70% of epithelial ovarian cancers. Using the same IHC protocol on more than 900 frozen samples covering 18 different cancer types we detected AMHRII expression in more than 50% of hepato-carcinomas, colorectal, lung, and renal cancer samples. AMHRII expression was not observed in neuroendocrine lung tumor samples nor in non-Hodgkin lymphoma samples. Complementary analyses by immunofluorescence and flow cytometry confirmed the detection of AMHRII on a panel of ovarian and colorectal cancers displaying comparable expression levels with mean values of 39,000 and 50,000 AMHRII receptors per cell, respectively. Overall, our results suggest that this embryonic receptor could be a suitable target for treating AMHRII-expressing tumors with an anti-AMHRII selective agent such as murlentamab, also named 3C23K or GM102. This potential therapeutic intervention was confirmed in vivo by showing antitumor activity of murlentamab against AMHRII-expressing colorectal cancer and hepatocarcinoma Patient-Derived tumor Xenografts (PDX) models.

Keywords: AMHRII; colorectal cancer; murlentamab; oncofetal antigen; protein expression.

Figure 1

AMHRII expression in normal human tissues determined by RT-qPCR. Relative Quantification (RQ) values were calculated by using the delta Ct method.

Figure 2

RNA in situ hybridization for semi-quantitative determination of mRNA expression levels of AMHRII in normal human tissues (A,C,E) and tumoral human tissues (B,D,F). Normal and tumoral tissues were stained with specific RNA AMHRII probes Hs-AMHRII (30ZZ). (A) Normal ovary tissue, RNAscope score 1, (B) Ovarian cancer, RNAscope score 1, (C) Normal colon, RNAscope score 0, (D) Colon cancer, RNAscope score 1, (E) Normal lung, RNAscope score 0, (F) Lung cancer, RNAscope score 1. Scoring was determined according to the manufacturer criteria: Score 0, no staining or less than 1 dot/10 cells; score 1, 1–3 dot/cell; score 2, 4–9 dots/cell; score 3, 10–15dots/cell; score 4 more than 15 dots/cell.

Figure 3

Immunofluorescence staining with murlentamab antibody of colorectal cancer ((A): A3764 Tp2, (B): A3742 Tc15, patient biopsies), ovarian cancer ((C): 1A717, (D): K583, patient biopsies). All fluorescent images were acquired with a widefield microscope DM5000 (Leica) with ×20 magnification. In Merge pictures, nuclear staining with DAPI was showed in blue and AMHRII staining with GM102 antibody was indicated in yellow.

Figure 4

AMHRII expression by flow cytometry in ovarian and colorectal cancers. (A) The number of RPC was assessed as mentioned in the Material and Methods section. (B) representative images of AMHRII expression in EpCam+ cells in a MSS CRC and in its paired normal colonic mucosa.

Figure 5

In vivo efficacy of murlentamab. (A) Tumor growth inhibition of murlentamab in the hepatocarcinoma PDX model (LI1097). Nude mice (n = 8/group) with established tumors (~145 mm³) were treated twice-weekly during 4 weeks by intravenous injection of murlentamab at 20 mg/kg. Sorafenib was administered p.o. at 50 mg/kg every day for 4 weeks. Open circle: vehicle, black square: murlentamab, Grey triangle: Sorafenib. (B) In vivo efficacy of murlentamab in the colorectal cancer PDX model (CTG-0401). Nude mice (n = 12/group) with established tumor (~175 mm³) were treated twice-weekly for four weeks by intravenous injection of murlentamab at 20 mg/kg. Irinotecan was injected i.p. at 100 mg/kg at day 1, 7 and 14. Open circle: vehicle, black square: murlentamab, Grey triangle: Irinotecan.