*Article* **Functional Investigation of the Tumoural Heterogeneity of Intrahepatic Cholangiocarcinoma by In Vivo PET-CT Navigation: A Proof-of-Concept Study**

**Luca Viganò 1,2,\*,†, Egesta Lopci 3,†, Luca Di Tommaso 2,4, Annarita Destro 4, Alessio Aghemo 2,5, Lorenza Rimassa 2,6, Luigi Solbiati 2,7, Arturo Chiti 2,3, Guido Torzilli 2,8 and Francesco Fiz 3,9**


**Abstract:** Intra-tumoural heterogeneity (IH) is a major determinant of resistance to therapy and outcomes but remains poorly translated into clinical practice. Intrahepatic cholangiocarcinoma (ICC) often presents as large heterogeneous masses at imaging. The present study proposed an innovative in vivo technique to functionally assess the IH of ICC. Preoperative 18F-FDG PET-CT and intraoperative ultrasonography were merged to perform the intraoperative navigation of functional tumour heterogeneity. The tumour areas with the highest and the lowest metabolism (SUV) at PET-CT were selected, identified during surgery, and sampled. Three consecutive patients underwent the procedure. The areas with the highest uptake at PET-CT had higher proliferation index (KI67) values and higher immune infiltration compared to areas with the lowest uptake. One of the patients showed a heterogeneous presence of FGFR2 translocation within the samples. Tumour heterogeneity at PET-CT may drive biopsy to sample the most informative ICC areas. Even more relevant, these preliminary data show the possibility of achieving a non-invasive evaluation of IH in ICC, paving the way for an imaging-based precision-medicine approach.

**Keywords:** intra-tumoural heterogeneity; intrahepatic cholangiocarcinoma; positron emission tomography–computed tomography; immunology; FGFR2 translocation; imaging fusion; navigation technology
