**1. Introduction**

Intra-tumoural heterogeneity (IH) is regarded as a major determinant of resistance to therapy and patients' prognosis but remains poorly translated into clinical practice [1–3]. The evaluation of IH has been mostly determined from the laboratory analyses of resected specimens, while, more recently, much of the research has been concentrated on imaging. The possibility of achieving a non-invasive mapping of IH is extremely appealing, because it would allow a better characterisation of the tumour (IH-based biopsies), a more precise prediction of prognosis, and a more effective treatment (IH-based therapies). Progress in

**Citation:** Viganò, L.; Lopci, E.; Di Tommaso, L.; Destro, A.; Aghemo, A.; Rimassa, L.; Solbiati, L.; Chiti, A.; Torzilli, G.; Fiz, F. Functional Investigation of the Tumoural Heterogeneity of Intrahepatic Cholangiocarcinoma by In Vivo PET-CT Navigation: A Proof-of-Concept Study. *J. Clin. Med.* **2022**, *11*, 5451. https://doi.org/ 10.3390/jcm11185451

Academic Editor: Hiroki Nishikawa

Received: 31 August 2022 Accepted: 13 September 2022 Published: 16 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

medical imaging modalities open new opportunities for the investigation of IH. Positron emission tomography–computed tomography (PET-CT) offers unique functional imaging of liver tumours [4–6]. Navigation technology systems [7,8] may merge different imaging modalities (morphologic and functional ones) to optimise the identification of the different tumour areas.

Among liver tumours, intrahepatic cholangiocellular carcinoma (ICC) is probably the most adequate to study IH. It is often diagnosed at an advanced stage and presents as large heterogeneous masses with a non-homogeneous uptake at PET-CT [9]. This presentation at imaging corresponds to a major genetic IH [10,11], even if the two have never been associated.

The present study depicts an innovative in vivo technique to functionally study the IH of ICC. Preoperative 18F-fluorodeoxyglucose (FDG) PET-CT images were merged with ultrasonography ones to navigate the tumour and to precisely explore the association between IH at imaging and IH at pathology. In this proof-of-concept study, we tested the procedure during surgery to unequivocally evaluate its reliability and accuracy.
