Patients previously diagnosed with advanced NENs underwent molecular profiling (ctDNA) using the FoundationLiquid® testing platform (72 cancer-related genes) between April and November 2019, in the framework of a collaboration between The Christie NHS Foundation Trust (Manchester, UK) and Foundation Medicine (Roche®, Basel, Switzerland). This platform allows for the identification of pathogenic and likely pathogenic somatic and germline variants, herein defined as "pathological alterations," including base substitutions, insertions, deletions, copy number alterations and chromosomal rearrangements. It also reports on high microsatellite instability (MSI-h). Patients provided written informed consent for molecular profiling to be performed; in addition, the retrospective analysis of these data was approved by the institutional Audit Committee (approval number 19/2634).

Patients with a histologically confirmed WdNET diagnosis, as per the 2019 World Health Organisation Classification parameters (WHO editorial Board, 2019), were included in this analysis; patients diagnosed with non-WdNETs, such as paraganglioma, goblet cell adenocarcinoma or PdNECs, were used for comparative purposes only. Clinical baseline characteristics, demographic and treatment data were collected. Molecular profiling information was extracted, including the success of sample analysis, the presence or absence of pathological alterations and the mutant allele frequency (MAF) for pathological alterations.

The aim of the study was to assess the feasibility and the clinical impact of ctDNA molecular profiling in WdNETs. The primary end-point was to assess the percentage of WdNET ctDNA samples that failed testing (defined as those scenarios where insufficient DNA was isolated for analysis). Secondary end-points included defining the proportion of the sample in which pathological findings were identified, and the percentage of patients for whom management changed based on molecular profiling results.

Descriptive statistical analysis using STATA v.12 was performed. The Chi-Square test, Fisher's exact test and *t*-test were used, when appropriate. A two-sided *p*-value < 0.05 was considered statistically significant.

#### **3. Results**

Samples from 45 patients were included: 15 WdNETs and 30 non-WdNETs.

#### *3.1. Patient Characteristics*

Within the total of the 15 individual patients with WdNETs (accounting for 18 ctDNA samples) (Table 1), 8 were female (53.33%), with a median age of 63.2 years (range 23.5–86.8). Most were small-bowel-primary patients (8 patients; 53.33%) (pancreas (5; 33.33%), gastric (1; 6.67%) and unknown primary (1; 6.67%)) and grade 2 patients (9; 60.00%) (grade 1 (5; 33.33%), grade 3 (1; 6.67%)), with a median Ki-67 of 5% (range 1–30). All patients with WdNET had a metastatic disease and seven were on treatment (three somatostatin analogues; four chemotherapy) at the time of the ctDNA sample acquisition.

**Table 1.** Baseline characteristics for patients who underwent ctDNA-based molecular profiling (WdNETs).

*3.2. Feasibility and Main Findings of ctDNA-Based Molecular Profiling*

A total of 5 WdNETs samples (27.78%) failed analysis (vs. 17.65% in non-WdNETs; *p*-value 0.395) (Figure 1).

**Figure 1.** Failure and success rate of molecular profiling analysis by population being analysed.

Of the 13 WdNET samples with a successful ctDNA analysis, pathological alterations were identified in 6 (46.15%) (vs. 82.14% in non-WdNETs; *p*-value 0.018) (Figure 2). In addition, there was a lower maximum MAF in WdNETs (mean 0.33) vs. non-WdNETs (mean 26.99), even though differences did not reach statistical significance (*p*-value 0.0584) (Figure 3). The rate of findings of unclear significance was similar between WdNETs (69.23%) and non-WdNETs (78.57%) (*p*-value 0.517).

**Figure 2.** Presence and absence of pathological alterations in ctDNA by population being analysed.

**Figure 3.** Maximum mutant allele frequency (mMAF) by population analysed.

Within the WdNET cohort, there was a higher presence of pathological mutations in G2 tumours (grade 1: 0%, grade 2: 66.67%; *p*-value 0.07) and in patients who were not receiving ongoing, concomitant anti-cancer systemic therapy at the time of the ctDNA sampling (no treatment: 66.67%, on treatment: 28.57%; *p*-value 0.286; Figure 4).

#### *3.3. Identified Pathological Alterations and Impact on Management*

A total of six pathological alterations were identified within the WdNET samples, including the *CDKN2A* mutation (one sample), *CHEK2* mutation (one sample), *TP53* mutation (two samples), *FGFR2* amplification (one sample), *IDH2* mutation (one sample), *CTNNB1* mutation (one sample), *NF1* mutation (one sample) and *PALB2* mutation (one sample). Concomitant alterations were identified in two samples (one had two alterations (the *CHEK2* and *TP53* mutations) and another had three (the *CTNNB1, NF1* and *PALB2* mutations)). The other four samples had one unique pathological alteration each.

None (0% of samples) of the identified pathological findings were considered potentially targetable. Thus, ctDNA-based molecular profiling did not change therapeutic management for any of the patients with a WdNET (0% of patients).

**Figure 4.** Presence and absence of pathological alterations in ctDNA by concomitant systemic treatment (WdNETs cohort).

#### **4. Discussion**

Although feasible, the role of molecular profiling using ctDNA seems to be limited in clinical decision making for patients with advanced WdNETs. The rate of identification of pathological alterations and the reported mMAF were significantly lower than in non-WdNETs. This is despite WdNETs having a similar sample failure rate to that described for non-Wd NETs, suggesting that the results are not associated with an increased rate of analysis failure in WdNETs (or a small amount of tumour-derived DNA in the bloodstream), but rather with a lower prevalence of significant alterations in this group of patients. Therefore, molecular profiling may be more relevant in non-WdNETs than in WdNETs. This is in line with the findings of genomic profiling studies of NEN tumour tissue showing that somatic mutations are less frequent in WdNETs than in PdNECs [6,12].

Although molecular profiling of WdNETs has been widely utilised to better understand the biology of these malignancies, true precision medicine therapeutic approaches in this patient group are currently non-existent [13]. Other series exploring targetable alterations in WdNETs have reported a higher rate of targetable alterations [6,10], which may be due to the differing definitions of "targetable" used, which ideally should follow evidence-based definitions [14]. It would also be of interest to understand how many of those patients were actually matched to a specific treatment based on the molecular alteration identified. While there are widely available data on this for other malignancies, data in WdNETs are scarce. The MOSCATO-01 clinical trial prospectively evaluated the clinical benefit of utilising high-throughput genomic analyses to identify actionable molecular alterations and match patients with a specific targeted therapy [15]. Of the total of 1035 patients included, 199 patients were matched with a specific treatment; within the group that received matched treatment, 11% of patients achieved an objective response, and a progression-free survival (PFS)2/PFS1 ratio of >1.3 was identified in 33% of patients. Ten patients with "thyroid and other endocrine glands" were included in this study. Of these, only two received a "matched" treatment. This corroborates the challenges of identifying targetable alterations in NENs.

Despite in-depth research on the identification of relevant molecular pathways in NETs [16], the development of precision medicine approaches represents one of the most relevant challenges in the current management of patients with NENs [17]. Beyond developments in the arena of nuclear medicine, which is rapidly developing new theragnostic approaches [18], predictive biomarkers for systemic therapy selection in WdNETs are lacking [19–21].

Interestingly, the findings of this study corroborate previous evidence suggesting that the dysregulation of cell-cycle/DNA damage repair (e.g., *TP53, CDKN2A, CHEK2, PALB2*) is a recurrent, critical biological vulnerability of WdNETs [5], highlighting the rationale for its therapeutic exploitation. Ongoing trials are evaluating the potential role of targeting

such alterations. In addition, trials evaluating peptide-receptor radionuclide therapies in combination with DNA damage repair inhibitors, for patients with WdNETs expressing somatostatin receptors (e.g., ClinicalTrials.gov NCT04086485, NCT05053854), do also exist.

In addition to the potential therapeutic impact of molecular alterations, the identification of specific, presumed somatic mutations in ctDNA should trigger germ-line testing in selected cases, where there is the potential for a known underlying hereditary syndrome in patients with WdNETs, such as multiple endocrine neoplasia (MEN) syndromes, Von Hippel–Lindau disease (VHL), Neurofibromatosis 1 (NF1) syndrome and Tuberous sclerosis (TS) [22].

The limitations of this study include the small sample size, the heterogeneity of the tumour type and treatment administered and the potential selection bias at the time of selecting patients for molecular profiling, as non-consecutive patients were considered for this. In addition, the series included a mix of advanced-stage and prior-line therapy patients, and there was no access to concomitant tissue profiling, which would have been of interest. However, a strength of this study was that all patients were tested with identical technologies and within the same time frame. In addition, the presence of a cohort of patients with non-WdNETs allowed us to put the findings into context, providing our results with more robustness and allowing for clinical interpretation.

Finally, the NGS platform used here included 70 'pan-cancer'-related genes, yet excluded a number of genes commonly altered in WdNETs, such as *MUTYH, ATRX, DAXX* and *MEN1*; a WdNET-specific gene panel, developed on the basis of more recent NGS data from large NEN datasets, may allow for the increased sensitivity of ctDNA detection in these patients.

#### **5. Conclusions**

The use of molecular profiling utilising ctDNA in WdNETs is feasible, but the results are currently unlikely to identify targetable alterations that may impact patient management. While patients with WdNETs should still be offered molecular profiling (if available and reimbursed), it is important to manage patient expectations in relation to the likelihood of the results impacting their management. It is possible that, due to the nature of these malignancies, which have generally low numbers of somatic mutations, the evolution of the field from exclusive ctDNA profiling to a combination of mutational analyses and epigenetic changes (including methylation analyses) will have an impact on the expansion of molecular profiling's use as a tool for neuroendocrine tumours, expanding from prognosis to the uncovering of new targets.

**Author Contributions:** Study design: A.L.; Data collection: A.L., J.B. and M.F.; Data analysis: A.L.; Preparation of first manuscript draft: A.L.; Recruitment of patients: A.L., J.B., M.F., Z.K., W.M., M.G.M., R.A.H. and J.W.V.; Review and approval of final manuscript: A.L., J.B., M.F., Z.K., W.M., M.G.M., R.A.H. and J.W.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding; access to FoundationOne Liquid was provided by Roche®.

**Institutional Review Board Statement:** The retrospective analysis of these data was approved by the institutional Audit Committee (approval number 19/2634) on 4 December 2019.

**Informed Consent Statement:** Patients provided written informed consent for molecular profiling to be performed.

**Data Availability Statement:** Data can be made available upon request.

**Acknowledgments:** Angela Lamarca has been partially-funded by The Christie Charity. Access to FoundationOne Liquid was provided by Roche®. The salary of Zain Kapacee was funded by The Christie Charity. The salary of Melissa Frizziero was funded by a European Neuroendocrine Tumour Society Centre of Excellence Young Investigator grant.

**Conflicts of Interest:** The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Access to FoundationOne Liquid® was provided by Roche®. Angela Lamarca: travel and educational support from Ipsen, Pfizer, Bayer, AAA, Sirtex, Novartis, Mylan and Delcath; speaker honoraria from Merck, Pfizer, Ipsen, Incyte, AAA, QED, Servier, Astra Zeneca and EISAI; advisory and consultancy honoraria from EISAI, Nutricia Ipsen, QED, Roche, Servier, Boston Scientific and Albireo Pharma; member of the Knowledge Network and NETConnect Initiatives funded by Ipsen. Melissa Frizziero has no conflicts of interest to declare. Jorge Barriuso: J.B. reports grants, personal fees and nonfinancial support from Ipsen; personal fees and non-financial support from Pfizer, Novartis; nonfinancial support from AAA, Nanostring, Roche; grants and personal fees from Servier; and personal fees from Nutricia outside the submitted work. Zainul Kapacee has received educational support from EISAI. Mairéad G McNamara: received research grant support from Servier, Ipsen and NuCana. She has received travel and accommodation support from Bayer and Ipsen and speaker honoraria from Advanced Accelerator Applications (UK & Ireland) Ltd., Pfizer, Ipsen, NuCana and Mylan. She has served on advisory boards for Celgene, Ipsen, Sirtex and Baxalta. Richard Hubner has served on the advisory board for Roche, BMS, Eisai, Celgene, Beigene, Ipsen and BTG. He has received speaker fees from Eisai, Ipsen, Mylan and PrimeOncology, and has received travel and educational support from Bayer, BMS, IPSEN and Roche, all outside of the scope of this work. Juan W Valle: consulting or advisory role for Agios, AstraZeneca, Delcath Systems, Keocyt, Genoscience Pharma, Incyte, Ipsen, Merck, Mundipharma EDO, Novartis, PCI Biotech, Pfizer, Pieris Pharmaceuticals, QED and Wren Laboratories; member of the Speakers' Bureau for Imaging Equipment Limited Ipsen Novartis Nucana; and received travel grants from Celgene and Nucana.

#### **References**

## *Article* **Predictive and Prognostic Role of Pre-Therapy and Interim 68Ga-DOTATOC PET/CT Parameters in Metastatic Advanced Neuroendocrine Tumor Patients Treated with PRRT**

**Rexhep Durmo 1,2,\*, Angelina Filice 1, Federica Fioroni 3, Veronica Cervati 4, Domenico Finocchiaro 3, Chiara Coruzzi 1, Giulia Besutti 5, Silvia Fanello 6, Andrea Frasoldati 7 and Annibale Versari 1**

**Simple Summary:** Although a significant improvement has been achieved in the management of metastatic neuroendocrine tumor (NET), disease progression is observed in 20–30% of patients treated with peptide receptor radionuclide therapy (PRRT). Therefore, the early identification of patients who are at high risk of treatment failure is important to avoid futile therapy toxicities. The aim of this study was to identify biomarkers derived from baseline and interim 68Ga-DOTATOC PET/CT in patients undergoing PRRT. In 46 metastatic NET patients with available baseline and interim PET, only baseline total tumor volume (bTV) was able to discriminate responders to PRRT (partial response or stable disease) vs. non-responders. Patients with high bTV had also the worst overall survival. bTV, an imaging biomarker, integrated in the initial workup of NET patients could improve risk stratification and contribute to a tailored therapy approach.

**Abstract:** Peptide receptor radionuclide therapy (PRRT) is an effective therapeutic option in patients with metastatic neuroendocrine tumor (NET). However, PRRT fails in about 15–30% of cases. Identification of biomarkers predicting the response to PRRT is essential for treatment tailoring. We aimed to evaluate the predictive and prognostic role of semiquantitative and volumetric parameters obtained from the 68Ga-DOTATOC PET/CT before therapy (bPET) and after two cycles of PRRT (iPET). A total of 46 patients were included in this retrospective analysis. The primary tumor was 78% gastroenteropancreatic (GEP), 13% broncho-pulmonary and 9% of unknown origin. 35 patients (76.1%) with stable disease or partial response after PRRT were classified as responders and 11 (23.9%) as non-responders. Logistic regression analysis identified that baseline total volume (bTV) was associated with therapy outcome (OR 1.17; 95%CI 1.02–1.32; *p* = 0.02). No significant association with PRRT response was observed for other variables. High bTV was confirmed as the only variable independently associated with OS (HR 12.76, 95%CI 1.53–107, *p* = 0.01). In conclusion, high bTV is a negative predictor for PRRT response and is associated with worse OS rates. Early iPET during PRRT apparently does not provide information useful to change the management of NET patients.

**Keywords:** neuroendocrine tumor; GEP-NET; PET/CT; PRRT; DOTATOC

**Citation:** Durmo, R.; Filice, A.; Fioroni, F.; Cervati, V.; Finocchiaro, D.; Coruzzi, C.; Besutti, G.; Fanello, S.; Frasoldati, A.; Versari, A. Predictive and Prognostic Role of Pre-Therapy and Interim 68Ga-DOTATOC PET/CT Parameters in Metastatic Advanced Neuroendocrine Tumor Patients Treated with PRRT. *Cancers* **2022**, *14*, 592. https://doi.org/10.3390/ cancers14030592

Academic Editor: Elif Hindie

Received: 19 December 2021 Accepted: 22 January 2022 Published: 25 January 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/).

#### **1. Introduction**

Neuroendocrine tumors (NETs) are relatively rare neoplasms that originate from endocrine cells, mostly of the gastroenteropancreatic tract (GEP) and the pulmonary system. Due to their indolent natural course, NETs are identified as locally advanced or with distant metastasis in 40–50% of patients [1].

Peptide receptor radionuclide therapy (PRRT) is an effective systemic therapeutic option for patients with advanced, metastatic, or unresectable NETs with high somatostatin receptor (SSTR) expression. PRRT consists in the intravenous systemic administration of somatostatin analogs (SSAs) labeled with a βminus (β-) emitting radioisotope (90Y and 177Lu), that binds SSTRs overexpressed in tumors with high affinity and specificity. The compound is internalized by endocytosis and retained in lysosomes of cells allowing the delivery of cytotoxic radiation directly on target cells, therefore producing the breakdown of intracellular DNA chains and cell death [2]. Although a significant improvement has been reported in progression free survival (PFS) and overall survival (OS), disease progression is still observed in 20–30% of patients treated with PRRT [3]. Moreover, PRRT can be associated with hematologic, renal, and hepatic toxicities [4]. Therefore, the early identification of patients who are at high risk of treatment failure represents an unmet need.

Positron emission tomography/computed tomography with 68Ga-DOTA-labelled somatostatin analogues (68Ga-DOTA-peptide PET/CT) is the elective imaging technique for diagnosis and management of NETs [5]. In clinical practice PET/CT plays a pivotal role also for properly selecting patients as candidates for PRRT. 68Ga-DOTA-peptide PET imaging is used for both evaluation of somatostatin receptor expression and correct staging of disease [6].

In addition, PET derived parameters, such as the semiquantitative standardized uptake values (SUVs), have been widely studied as prognostic and predictive factors in NET patients treated with PRRT [7]. In several other malignancies, FDG-PET-derived metabolic tumor volume (MTV) and total lesion glycolysis (TLG), which is the product of SUVmean × MTV, have shown a major role in prognosis and treatment monitoring compared to SUVs parameters [8]. MTV and TLG provide a direct estimation of the wholebody tumor burden and have been validated as quantitative imaging biomarkers in several tumors [9]. Furthermore, early changes in tumor burdens are a promising index of response to treatment and could be the basis of a more individualized treatment approach [10,11].

Hence, the aim of this study is to evaluate the predictive and prognostic value of semiquantitative and volumetric parameters calculated from 68Ga-DOTATOC PET/CT performed before PRRT. Moreover, we assessed the potential role of the change (Δ) between baseline and interim PET parameters after two cycles of PRRT for early prediction of response.

#### **2. Materials and Methods**

#### *2.1. Study Design*

We conducted a retrospective subgroup analysis of patients enrolled in a prospective, monocentric, non-randomized phase II clinical trial (EudraCT:2013-002605-65). All patients signed a written informed consent form. For this study we included biopsy-proven, unresectable, metastatic GEP or bronchopulmonary or unknown primary site NET patients, who were treated with PRRT in our institution (Azienda USL-IRCCS of Reggio Emilia, Italy). All patients underwent screening with 68Ga-DOTATOC PET/CT to confirm the adequate expression of SSTR type 2 on tumor sites and an interim PET/CT scan 7 weeks after the second cycle of PRRT. The association between PET parameters and outcomes (PRRT response and OS) was evaluated.

#### *2.2. Ga-DOTATOC PET/CT*

A baseline 68Ga-DOTATOC PET/CT (bPET) 1–3 weeks before PRRT and after two cycles of PRRT (interimPET) was performed with a hybrid scanner (Discovery STE; GE Healthcare). Image acquisition started 60 ± 10 min after administration of 2 MBq/kg of 68Ga-DOTATOC. All DOTATOC-avid lesions were semiautomatically segmented by an experienced board-certified nuclear medicine physician (V.C.) using a commercial software (PET VCAR, GE Healthcare). Subsequently, all regions of physiological or non-disease related uptake were manually removed. From the remaining volume-of interests (VOIs), containing all 68Ga-DOTATOC avid tumor lesions, maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), the somatostatin receptor-derived tumor volume (TV) and total lesion SSR expression (Total Lesion Activity-TLA, defined as SUVmean × TV) were measured. Based on the experience of metabolic tumor volume in FDG PET/CT studies a threshold above 41% of SUVmax was used to calculate TV. The whole-body tumor volume (bTV) was calculated by summing TV measurements of all lesions in each patient. The whole-body TLA (bTLA) was calculated by summing TLA of all lesions. Also, the ratio of the lesion SUVmax to the SUVmax in the spleen (SUVratio T/S) was calculated. Differences (Δ) in SUVmax, SUVmean, bTV, bTLA and SUVratio T/S were evaluated by calculating the percentage in variation of each parameter using the following formula: Delta = (interimPET − bPET)/bPET × 100.

#### *2.3. PRRT*

PRRT was performed according to the joint International Atomic Energy Agency (IAEA; Vienna, Austria), European Association of Nuclear Medicine (EANM; Vienna, Austria), and Society of Nuclear Medicine and Molecular Imaging (SNMMI; Reston, VA, USA) practical guidance ([12]). Briefly, the inclusion criteria for treatment with PRRT were histologically confirmed, unresectable, metastatic GEP or bronchopulmonary or unknown primary site NET patients; high expression of somatostatin receptor on baseline 68Ga-DOTATOC PET/CT (defined as greater than or equal to that of normal liver); a glomerular filtration rate greater than 50 mL/min/1.73 m2; a white blood cell count greater than 2.5 × 109/L; a platelet count greater than 90 × 109/L; bilirubin levels less than 2.5 mg/dL and ECOG performance status ≤ 2. A fractionated treatment protocol, with an activity of 1850–2590 MBq 90Y-DOTATOC or 3700–5550 MBq 177Lu-DOTATOC per treatment cycle every eight weeks, aimed at four to six courses with an interval of eight weeks, was followed. The activity prescription was determined on the basis of the Biological Effective Dose (BED) delivered to kidneys and on the basis of the absorbed dose to bone marrow, considered as the main organs at risk. Dosimetry was scheduled during the first course of therapy after a therapeutic administration of 177Lu-DOTATOC. The cumulative dose limit to kidneys was set to 46 Gy of BED for patients with no risk factors, and at 28 Gy for patients with risk factors, while the absorbed dose limit to bone marrow was set to 2 Gy for both. Documented disease progression at any time led to the cessation of PRRT and the classification of the patient as having progressive disease.

#### *2.4. Assessment of Treatment Response*

Response assessment was performed three months after completion of PRRT courses. Combination of anatomical imaging (CT/MRI), functional (PET) and clinical data in a multidisciplinary tumor board setting was used to define response. Response Evaluation criteria in Solid Tumors (RECIST version 1.1) criteria was used in determining anatomical response to PRRT. PET response was assessed using the European Organization for Research and Treatment of Cancer (EORTC) criteria. For the study analysis, responders were defined as patients with a complete/partial response or stable disease, and non-responders had progressive disease.

#### *2.5. Statistical Analysis*

Quantitative variables were expressed as median with range or mean with SD. Categorical variables are presented with absolute and relative frequencies. Mann-Whitney U test was used for comparing continuous variables between responders and non-responders. Chi-square test was used to analyze differences in discrete variables between responders and non-responders. Binary logistic regression was performed to identify factors predictive

of treatment. Receiver operating characteristics (ROC) analysis was performed to identify optimal cut-off values for PET/CT quantitative parameters. The area under the curves (AUC), sensitivity, specificity and accuracy were reported. Survival curves were estimated according to the Kaplan-Meier method. A log-rank test was used to compare survival curves. Cox proportional-hazards model was used for univariate and multivariate survival analysis and results were reported as hazard ratio (HR), 95% confidence interval (95%CI) and *p*-Value based on statistical Wald-test. OS was defined as the time (months) from the first PRRT cycle to death from any cause. Surviving patients were censored at the last date of follow-up. All statistical tests were two-sided and *p* < 0.05 was considered a statistically significant result. Statistical analysis was performed using IBM SPS Statistics 25 (IBM, New York, NY, USA) and MedCalc Statistical Software 14.8.1 (Ostend, Belgium) for Windows.

#### **3. Results**

#### *3.1. Patients*

A total of 46 patients with available bPET and iPET were included in this retrospective analysis. The median age was 60 (range 25–85) and 21 patients (46%) were female. Primary tumor was for 78% GEP, 13% broncho-pulmonary and 9% of unknown origin. All patients had stage IV disease. Liver and lymph nodes were the most frequent sites of metastasis. Patients' main characteristics are reported in Table 1.

**Table 1.** Main characteristics of the study population.

Abbreviations: GEP: gastroenteropancreatic; PRRT: peptide receptor radionuclide therapy; NET: neuroendocrine tumor.

#### *3.2. PRRT and PET/CT*

Forty-three patients (94%) received four to sixcycles of PRRT. Five patients (11%) were treated with 177Lu-DOTATOC radiopeptide, while the 41 remaining subjects underwent a combination of 90Y-DOTATOC and 177Lu-DOTATOC therapy.

The observed response rates were 21% (*n* = 10) partial response, 55.1% (*n* = 25) stable disease, and 23.9% (*n* = 11) progressive disease for the entire cohort. Considering GEP NET cohort response rates were 27% (*n* = 10) partial response, 51% (*n* = 18) stable disease, and 22% (*n* = 8) progressive disease. Complete response was not observed. For the analysis of this study, 35 patients (76.1%) with either stable disease or partial response after PRRT were classified as responders and 11 (23.9%) patients with progressive disease as non-responders.

The median values of SUVmax, SUVmean, SUVt/s, bTV and bTLA were 34 (IQR, 23.1–55.8), 9.9 (IQR, 7.6–16.4), 1.1 (IQR, 0.7–2.3), 143.8 mL (IQR, 32.9–354), and 1834 (IQR, 342–6309), respectively. The median percentage in variation between baseline and interimPET after two cycles of PRTT reported as ΔSUVmax, ΔSUVmean, ΔSUVratioT/S, ΔbTV, ΔbTLA were 1.1% (IQR, −21.2–22.1), 4.2% (IQR, −17.8–39.4), −1.1% (IQR, −41.5–24.5), 32.4% (IQR, −10.2–70.6), 25% (−12.5–80.9), respectively. Figure 1 shows a baseline and interim PET.

**Figure 1.** Baseline (**A**) and interim (**B**) PET scan of a 54-year-old patient with G1 pancreatic NET. Red arrows indicate the primary pancreatic tumor and the most representative liver metastasis. Interim 68Ga-DOTATOC PET after the second PRRT cycle shows disease progression. The patient was treated with a total of six PRRT courses (four cycles of 177Lu-DOTATOC + 2 cycles of 90Y-DOTATOC) for a total of 28,534 MBq activity and classified as partial response at the assessment following treatment.

The bTV and bTLA values of patients that did not respond to PRRT were significantly higher than those of patients with response to PRRT therapy (496.2 IQR 218.3–2029.4 vs. 77.6 IQR 31–186.6, *p* < 0.001 and 6078.3 IQR 2813–18,959 vs. 1341 IQR 272.3–3865, *p* = 0.001, respectively: Figure 2).

**Figure 2.** Box Plot comparing bTV (**a**) and bTLA (**b**) between PRRT responder and non-responder patients. "-" and "-" symbols represent outlier cases.

No association between semiquantitative PET parameters and response were observed. Percentage variations (Δ) of PET semiquantitative and volumetric parameters were not significantly different between PRRT responders and non-responders. PET parameters and comparison between responders vs. non-responders are summarized in Table 2.

**Table 2.** Characteristic of calculated PET parameters. Data of all patients (*n* = 46) and a comparison between responders (*n* = 35) vs. non-responders (*n* = 11) groups are reported. Values are expressed in mean (SD) and median (IQR). Mann-Whitney U test was used to compare variables. The *p*-Values shown in boldface correspond to *p* < 0.05.

**Table 2.** *Cont.*

Abbreviations: SUV: standardized uptake value; T/S: tumor/spleen; TV: total volume; TLA: total lesion activity.

Logistic regression analysis of bPET derived parameters and ΔPET values identified that only bTV was associated with therapy outcome (OR 1.17; 95%CI 1.02–1.32; *p* = 0.02). No significant association with PRRT response was observed for other variables (Table 3).

**Table 3.** Logistic Regression Analysis.

Abbreviations: SUV: standardized uptake value; T/S: tumor/spleen; TV: total volume; TLA: total lesion activity. The *p*-Values shown in boldface correspond to *p* < 0.05.

#### *3.3. Overall Survival Analysis*

During the follow-up period (mean 31 months; ranged from eight to 86 months), seven (15.2%) patients died. Mean survival time was 69.4 (SD, 5.3) months, the median was non reached for the entire cohort.

ROC analysis showed a best cut-off point for bTV of >244.5 mL (sensitivity 85.7%; specificity 79.5%) and AUC of 0.77 (95% CI 0.62–0.88, *p* = 0.036) for identification of patients with worse OS. For bTLA best cut-off point was 2659 (sensitivity 85.7%; specificity 64.1%) and AUC of 0.71 (95% CI 0.56–0.84, *p* = 0.092). The complete ROC curves analysis of PET parameters is shown in Table S1.

In univariate analysis, higher bTV and bTLA were associated with lower survival probability (HR 13 95%CI 2.6–64.1, *p* = 0.001; HR 9.08 95%CI 1.09–75.76, *p* = 0.04) (Figure 3).

Moreover, a difference in SUVmax above 5.5% between iPET and bPET (ΔSUVmax) was associated with a favourable outcome (HR 0.15 95% CI0.03–0.67; *p* = 0.04). In Cox multivariate analysis, only a high bTV was confirmed as an independent prognostic factor (HR 12.76 95%CI 1.53–107, *p* = 0.01) (Table 4).

**Figure 3.** (**a**) Kaplan-Meier curves for overall survival (OS) among patients with high- and low-bTV defined on the basis of the ROC curve (244 mL). (**b**) Kaplan-Meier curves for OS among patients with high- and low-bTLA defined on the basis of the ROC curve (2658 mL\*SUV).

**Table 4.** Univariate and multivariate Cox regression analysis for OS.

Abbreviations: SUV: standardized uptake value; T/S: tumor/spleen; TV: total volume; TLA: total lesion activity. The *p*-Values shown in boldface correspond to *p* < 0.05.

#### **4. Discussion**

After about 20 years of retrospective and phase I/II trials, PRRT was finally approved by regulatory authorities. Since its approval for the treatment of inoperable or metastatic GEP-NETs with progressive disease by the European Medicine Agency in 2017, PRRT has been widely used [13,14]. The phase III NETTER-1 trial has established that PRRT prolongs PFS compared to high dose octreotide LAR and improved quality of life. [3,15] In our study, 76.1% of NET patients had stable disease or partial response and 23.9% patients showed progressive disease after PRRT. These findings are in line with the response rates reported in a recent large meta-analysis where the disease control rate (complete or partial response and stable disease) was 83% in metastatic NET patients treated with PRRT [16]. Despite the increasing role of PRRT in metastatic NETs, what is clear both from our results and literature is that we need parameters to improve the selection of patient candidates to PRRT.

In this study, we evaluated the predictive and prognostic significance of semiquantitative and volumetric baseline PET imaging parameters in patients with advanced, metastatic NET undergoing PRRT. We also investigated the role of interim PET after two cycles of PRRT as an early predictor.

Several retrospective studies have assessed the role of semiquantitative parameters, mostly SUVmax, as prognostic markers for NETs. However, conflicting results have been published regarding the role of SUV on baseline 68Ga-DOTA-SSA PET in PRRT response prediction. Several authors demonstrated that a high SUVmax in baseline PET is associated with a favorable outcome and a wide range of different cut-off values are reported to separate responders to PRRT vs. non responders [17–20].

In our cohort we did not find any association between SUVs obtained in the baseline PET and PRRT response. This finding is in line with published results by Gabriel et al. and Soydal et al., who similarly found that SUVs showed no additional value for PRRT response prediction [21,22]. This controversial result may be because SUVs are measured in a single region of interest and thus they are not representative of the total tumor burden. Indeed, the lesion heterogeneity in NET patients, and the well-known limits of semiquantitative parameters, might further limit SUVs utility [23].

We found that patients who did not respond to PRRT had significantly higher bTV compared to responders. Moreover, bTV was the only PET parameter that confirmed a predictive role for PRRT response on logistic regression analysis (OR 1.17). Furthermore, bTV was an independent prognostic factor associated with worse OS rates in Cox regression analysis.

To our knowledge, only two other studies have previously evaluated the role of baseline volumetric PET parameters in patients treated with PRRT. Ohlendorf et al. found that PFS was shorter in patients with high bTV and high bTLA in 32 NET patients treated with PRRT [22]. Pauwels et al. reported that a bTV higher than 578 mL was associated with worse OS [23]. In our study population, the bTV cut-off that better identified patients with shorter OS was 244 mL. This difference in cut-offs could be explained by different segmentation methods. Indeed, Pauwels used a SUV threshold customized per patient through visual inspection to segment the tumor. In contrast, we used a semiautomatic method applying a threshold above 41% of the SUVmax for calculation of TV and TLA, based on the experience with FDG PET studies [24]. Further studies are warranted to define and harmonize 68Ga-Dota-peptide PET/CT tumor burden segmentation.

Our findings are in line with several previous studies that assessed the prognostic significance of PET volumetric parameters in settings other than PRRT [25–30]. Thus, bTV seems to be a powerful prognostic parameter in NET patients. However, bTV should be validated in further prospective studies including more homogeneous populations in terms of primary site, disease course and treatment setting.

In contrast, we did not find any relationship between changes in PET semiquantitative and volumetric parameters between baseline and interim PET after two cycles of PRRT. However, changes in SUVmax (ΔSUVmax) showed a significance in OS univariate analysis (*p* = 0.04) that was not confirmed in multivariate analysis. These results, in accordance with previous studies, show no utility of iPET in guiding the management of metastatic NET patients treated with PRRT [11,27].

There are several limitations of the present study that should be acknowledged. First are the retrospective nature and the small sample size. Second, we included patients with different NET sites. Furthermore, we did not evaluate FDG PET findings in our analysis. It is known that some patients with grade 2–3 NET can show higher affinity for FDG rather than 68Ga-DOTATOC PET [28]. This can result in an underestimation of the actual tumor burden by 68Ga-DOTATOC PET. A combination of these two tracers can be considered to address this issue. In addition, it should be noted that the generalizability of our findings with 68Ga-DOTATOC to the other often used 68Ga-DOTATATE PET ligand need to be confirmed. Indeed, while 68Ga-DOTATATE shows only affinity to SSTR2, 68Ga-DOTATOC also exhibits some affinity to SSTR5. However, despite differences in receptor affinities, a head-to-head comparison showed no clinically significant difference between the two tracers [29]. Finally, it is worth mentioning that other PET tracers like 64Cu-DOTATATE, providing better resolution and potentially a better lesion detection rate than 68Ga-DOTATOC, may improve tumor burden quantification [30]. However, the 64Cu-labeled tracers are not in use in the clinical routine and require further work.

#### **5. Conclusions**

In metastatic NET patients addressed to PRRT, higher total body tumor volume measured from baseline 68Ga-DOTATOC PET/CT scans is a negative predictor for therapy response and is associated with worse OS rates. Interim 68Ga-DOTATOC PET/CT after two cycles of PRRT did not allow the identification of patients with poorer prognosis that would justify a change in treatment strategy.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/cancers14030592/s1, Table S1: ROC curve analysis for OS.

**Author Contributions:** Conceptualization and supervision A.V. and A.F. (Angelina Filice); data acquisitions, V.C. and C.C.; data analysis, F.F. and D.F.; writing—original draft preparation, R.D.; revision of the article for important intellectual content, A.F. (Andrea Frasoldati), S.F. and G.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of AUSL-IRCCS of Reggio Emilia (protocol code 24871).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy-related issues.

**Conflicts of Interest:** A.V.: Novartis and Advanced Accelerator Applications for advisory role. The other authors declare no conflict of interest.

#### **References**

## *Article* **Active Surveillance in** *RET* **Gene Carriers Belonging to Families with Multiple Endocrine Neoplasia**

**Alessandro Prete 1, Antonio Matrone 1, Carla Gambale 1, Valeria Bottici 1, Virginia Cappagli 1, Cristina Romei 1, Liborio Torregrossa 2, Laura Valerio 1, Elisa Minaldi 1, Maria Cristina Campopiano 1, Loredana Lorusso 1, Laura Agate 1, Eleonora Molinaro 1, David Viola 1, Teresa Ramone 1, Chiara Mulè 1, Raffaele Ciampi 1, Fulvio Basolo 2 and Rossella Elisei 1,\***

**Simple Summary:** MEN2 has a very high penetrance for the development of medullary thyroid cancer. However, intra- and inter-familial variabilities have been described. Accordingly, in this precision medicine era, a personalized approach should be adopted in subjects harboring *RET* mutations. In these subjects, we showed that thyroid surgery could be safely timed according to basal and stimulated calcitonin, especially in children who can reach adulthood, avoiding the risks of thyroid surgery and decreasing the period of a long-life hypothyroidism treatment.

**Abstract:** Multiple Endocrine Neoplasia 2 (MEN2) is a hereditary cancer syndrome for developing medullary thyroid cancer (MTC) due to germline mutations of *RET* gene. Subjects harboring a germline *RET* mutation without any clinical signs of MTC are defined as gene carriers (GCs), for whom guidelines propose a prophylactic thyroid surgery. We evaluate if active surveillance of GCs, pursuing early thyroid surgery, can be safely proposed and if it allows safely delaying thyroid surgery in children until adolescence/adulthood. We prospectively followed 189 GCs with moderate or high risk germline *RET* mutation. Surgery was planned in case of: elevated basal calcitonin (bCT) and/or stimulated CT (sCT); surgery preference of subjects (or parents, if subject less than 18 years old); other reasons for thyroid surgery. Accordingly, at *RET* screening, we sub-grouped GCs in subjects who promptly were submitted to thyroid surgery (Group A, *n* = 67) and who were not (Group B, *n* = 122). Group B was further sub-grouped in subjects who were submitted to surgery during their active surveillance (Group B1, *n* = 22) and who are still in follow-up (Group B2, *n* = 100). Group A subjects presented significantly more advanced age, bCT and sCT compared to Group B. Mutation *RET*V804M was the most common variant in both groups but it was significantly less frequent in Group A than B. Analyzing age, bCT, sCT and genetic landscape, Group B1 subjects differed from Group B2 only for sCT at last evaluation. Group A subjects presented more frequently MTC foci than Group B1. Moreover, Group A MTCs presented more aggressive features (size, T and N) than Group B1. Accordingly, at the end of follow-up, all Group B1 subjects presented clinical remission, while 6 and 12 Group A MTC patients had structural and biochemical persistent disease, respectively. Thank to active surveillance, only 13/63 subjects younger than 18 years at *RET* screening have been operated on during childhood and/or adolescence. In Group B1, three patients, while actively surveilled, had the possibility to reach the age of 18 (or older) and two patients the age of 15, before being submitted to thyroid surgery. In Group B2, 12 patients become older than 18 years and 17 older than 15 years. In conclusion, we demonstrated that an active surveillance pursuing an early thyroid surgery could be safely recommended in GCs. This patient-centered approach permits postponing thyroid surgery

**Citation:** Prete, A.; Matrone, A.; Gambale, C.; Bottici, V.; Cappagli, V.; Romei, C.; Torregrossa, L.; Valerio, L.; Minaldi, E.; Campopiano, M.C.; et al. Active Surveillance in *RET* Gene Carriers Belonging to Families with Multiple Endocrine Neoplasia. *Cancers* **2021**, *13*, 5554. https:// doi.org/10.3390/cancers13215554

Academic Editors: Alfredo Berruti, Vito Amoroso and Nicola Fazio

Received: 30 September 2021 Accepted: 4 November 2021 Published: 5 November 2021

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

**Copyright:** © 2021 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/).

in children until their adolescence/adulthood. At the same time, we confirmed that genetic screening allows finding hidden MTC cases that otherwise would be diagnosed much later.

**Keywords:** medullary thyroid cancer; calcitonin; MEN2; gene carriers

#### **1. Introduction**

Multiple Endocrine Neoplasia 2 (MEN2) is an hereditary cancer syndrome characterized by the development of medullary thyroid cancer (MTC), variably associated with other endocrine neoplasia, such as pheochromocytoma and primary hyperparathyroidism [1–3]. MEN2 is an autosomal dominant disease with a very high penetrance due to missense gainof-function mutation of the *RET* gene (Rearranged during Transfection) [4,5]. Germline *RET* mutation is present in about 99% of familial and about 6.0% of apparently sporadic cases of MTC [6]. Accordingly, germline *RET* screening must be offered to all patients with MTC and, if positive, all first-degree relatives should be screened [7,8]. Subjects harboring a germline *RET* mutation without any clinical signs of MTC are defined as Gene Carriers (GCs) [8].

In the case of a GC, guidelines propose a prophylactic thyroid surgery as "the removal of the thyroid before MTC develops or while it is clinically unapparent and confined to the gland" [8]. Its timing is essentially based on subject *RET* mutation and age; in cases of *RET* mutation at highest risk (*M918T*) surgical therapy must be performed within the first year, in cases at high risk (*C634F/G/R/S/W/Y* and *A883F*) the timing of thyroidectomy can be based on serum calcitonin (CT). However, in any case before 5 years and in cases at moderate risk (other mutations), basal and stimulated CT (bCT and sCT) should guide thyroid surgery timing [8]. This latter suggestion is not always followed in the real clinical world and several centers still follow the indication to operate immediately after the *RET* screening, warning against the use of serum CT in this clinical scenario [9].

By many years, in the case of GCs harboring high and moderate risk mutations, in our center we are performing an active surveillance by timing the thyroid surgery on bCT and sCT levels, regardless of *RET* mutation and age, pursuing an early, instead of a prophylactic, thyroid surgery [10]. The main reasons are related to both the higher risk of surgical complications in children, particularly permanent hypoparathyroidism that implies long-life therapy [11], and to the need of early medication with levothyroxine during childhood and adolescence in subjects who actually have normal thyroid function.

In this study, we evaluated if an active surveillance with an early thyroid surgery can be safely proposed in *RET* GCs and for how many years the surgery could be safely delayed in children. Moreover, we looked also at the relevance of genetic screening in finding hidden MTC cases that, otherwise, would be diagnosed much later.

#### **2. Materials and Methods**

#### *2.1. Subjects*

After 1993, we performed *RET* genetic screening in all patients with diagnosis of MTC, either familial or apparently sporadic and, if positive, to all their first-grade relatives [6].

All adult patients signed informed consent to perform *RET* genetic screening. Parents or guardians signed the informed consent in the case of subjects less than 18 years of age. As per the policy of the University Hospital, all patients provided written informed consent to both the genetic screening and the use of their clinical and biochemical data for scientific purposes.

#### *2.2. Clinical Evaluation*

We evaluated GCs by using clinical, biochemical (i.e., bCT and sCT (pentagastrin (Pg) stimulation test up to 2013, and then calcium (Ca) stimulation test, as elsewhere described [12]), urinary metanephrine and normetanephrine, serum PTH, calcium and 25-hydroxyvitamin D (25[OH]D) and imaging examinations (i.e., neck and abdominal ultrasound and whenever necessary abdominal MRI).

#### *2.3. Surgery Criteria*

According to the most recent advances carried out by Elisei et al. [10], in our center the surgical treatment for GCs, independently from the type of germline mutation, is planned according to the following criteria:

Otherwise, patients without any of the above-mentioned criteria were followed every 6–12 months with clinical, biochemical, and morphological assays (namely neck ultrasound) as previously described.

#### *2.4. Post-Surgery Follow-Up*

Four/six months after surgery, all patients were submitted to biochemical analysis (bCT and, if necessary, Pg or Ca stimulation test for CT) and neck ultrasound. Whenever indicated, other imaging (e.g., CT scan, MRI etc.) were performed.

#### *2.5. RET Genetic Analysis*

*RET* genetic screening has been performed on DNA extracted from the blood of MTC patients and of their relatives according to a procedure previously reported [6]. MTC patients have been screened for the presence of *RET* mutations in exons 5, 8, 10, 11, 13, 14, 15, and 16 while relatives of *RET* positive index cases have been analyzed only for the presence of the mutation identified in their family. Actually, genomic DNA is amplified using KAPA2G Fast HotStart PCR Kit (Sigma-Aldrich, Saint Louis, MI, USA) in a final volume of 20 μL with 0.5 pmoli/μL of each primer and using a SimplyAmp thermal cycler (Thermofisher, Waltham, MA, USA). Amplification cycle is performed with an initial step of 95 ◦C for 2 min, followed by 35 cycles of 95 ◦C for 15 s, 60 ◦C for 15 s and 72 ◦C for 15 s. A final extension at 72 ◦C for 7 min was performed at the end of the amplification protocol. Sequence analysis was performed, and has been reported on previously. Primers'sequence can be provided upon request. Sequence reactions are performed according to the Sanger method using an ABI Prism 3130XL genetic analyzer (Thermofisher, Waltham, MA, USA).

#### *2.6. Laboratory Evaluation*

In the last 25 years CT measurement has been performed using two immunometric assays (ELSA-hCT, Cis-BioInternational, Gif sur Yvette, France, functional sensitivity 10.0 pg/mL, from 1993 to 2013 and chemoluminescent immunometric Immulite, Siemens Healthcare Diagnostic Products Ltd., Lianberis, Gwynedd LL55 4EL, UK, with analytic sensitivity 2.0 pg/mL reference values of up to 18.2 pg/mL for women and 11.5 pg/mL for men, from 2014 to the present).

#### *2.7. Histopathology*

All the specimens were submitted to routine pathological procedure and were reviewed by two pathologists (LT, FB). Briefly, the surgical specimens were fixed in 10% buffered formaldehyde and embedded in paraffin, and then 4-mm-thick sections were cut and stained with hematoxylin & eosin (H&E). For immunohistochemistry, paraffin sections (3–5 mm) were dewaxed in xylene, dehydrated through graded alcohols, and processed using the diaminobenzidine detection system. All of the immunohistochemical analyses for calcitonin were performed automatically using the Ventana Benchmark® immunostaining system (Ventana Medical Systems, Tucson, AZ, USA) and a rabbit monoclonal

primary antibody direct against calcitonin polypeptide (Ventana Medical Systems, clone SP17; dilution 0.56 μg/mL).

Usually, on routine H&E stained-slides the "neoplastic" or "primary" CCH is easily identified by the presence of clusters of intrafollicular C-cells, composed of cells with mild or moderate cellular atypia, resembling those identified in an MTC [13]. According to the last edition of WHO Classification of Tumours of Endocrine Organs [14] the diagnosis of "primary" CCH is encountered when >6–8 C cells per cluster in several foci with >50 C cells per low power field are identified. Immunostaining for CT was performed in all cases to confirm the recognition of C-cells. Histologically, the main difference between the "primary" CCH and the microfocus of MTC is represented by extension of C cells through the basement membrane into the surrounding thyroid interstitium or when a desmoplastic stromal reaction surrounding the infiltrating neoplastic cells is evident [15].

#### *2.8. Statistical Analysis*

Statistical analysis was performed using Kruskal–Wallis, Mann–Whitney, *t* tests, ROC curves, univariate and multivariate regression analysis, according to the variables to be analyzed, using IBM SPSS Statistics (Armonk, NY, USA) for Macintosh, Version 25.0. A *p* value less than 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Study Groups: Epidemiological, Biochemical and Genetics Data*

*RET* genetic screening allowed us to discover 189 GCs in 84 families. At first clinical evaluation, after the screening, we distinguished two groups of GCs: those who already met surgery criteria (*n* = 67, Group A) and those who did not (*n* = 122, Group B). Epidemiological, biochemical and US data of Group A and B, are reported in Table 1. Group A subjects were significantly older than Group B (median 44 vs. 18 years) (*p* < 0.0001). As expected, at *RET* genetics screening, Group A subjects presented significantly higher bCT (median 24 ng/L vs. below functional sensitivity) as well as sCT (median 276.5 vs. 10.6 ng/L) compared to Group B (*p* < 0.0001). US scan identified thyroid nodule in 71.2% (37/52) of Group A subjects and in 22.1% (23/104) of Group B (*p* < 0.0001) (Table 1).

**Table 1.** Epidemiological, biochemical and US data of Group A and B. bCT: basal calcitonin, sCT: calcitonin upon pentagastrin or calcium stimulation test, US: ultrasound, BFS: below functional sensitivity.

We analyzed the genetic landscape of Group A and B. In agreement with our previous report [6], we confirmed that mutations occurring at 804 codon were the most common mutations in both groups, although they were significantly less frequent in Group A than B (26% vs. 42%, *p* = 0.034) (Figure 1A). Otherwise, we observed that mutations occurring at 634 codon were substantially, although not significantly, more frequent in Group A than B (12% vs. 5%, *p* = 0.083) (Figure 1A). Accordingly, at first evaluation, 57% of patients with *RETC634X* mutation presented the criteria for surgery while only 37% with other

mutations presented these criteria, although this difference was not statistically significant, probably due to relatively low number of subjects with *RETC634X* mutation (*n* = 14) (*p* = 0.22) (Figure 1B).

**Figure 1.** (**A**) Genetic landscape of Group A (upper graph) and B (lower graph). (**B**) Rate of patients submitted to surgery or follow-up, according to RET mutations (634 codon vs. other codons).

#### *3.2. Follow-Up in Group B*

After the *RET* genetics screening assessment, Group B subjects were followed every 6–12 months. During their follow-up, 22/122 (19%) subjects were submitted to surgery (Group B1) after a median time of 1.6 years (IQR 1.1–3.6, range 1.1–10.3 years) and 100/122 (81%) patients are still in follow-up (Group B2) after a median time of 2.9 years (IQR 0.9–6.3, range 0.1–21.8 years). We analyzed epidemiological, biochemical, and US-features of GCs of Groups B1 and B2 both at *RET* screening and last evaluation (either before surgery in Group B1 or at the end of follow-up in Group B2). At *RET* screening evaluation, Groups B1 and B2 subjects did not differ for age, bCT, and sCT (Figure 2). Otherwise, at the last evaluation, Group B1 subjects presented significantly higher levels of sCT compared to Group B2 (median 38 vs. 20 ng/L, respectively, *p* = 0.035), whereas bCT and age were not different (Figure 2). At US scan, thyroid nodules were substantially more frequent in Group B1 than Group B2 at *RET* screening evaluation (42%, 8/19 vs. 17.6%, 15/85; *p* = 0.059) and significantly at last evaluation (50%, 10/20 vs. 25%, 23/92; *p* = 0.022) (Figure 2D). Figure 2E summarized genetics landscapes of both groups. Mutations occurring at 804 codon were substantially, although not significantly, more frequent in Group B1 (61%, 14/23) than B2 (38%, 37/98) (*p* = 0.067). Mutations at 634 codon did not differ between the two groups.

**Figure 2.** (**A**,**B**) basal CT (bCT) and stimulated CT (sCT) in Group B1 and B2 at *RET* screening and at last evaluation. (**C**) Age of subjects of Group B1 and Group B2 at *RET* screening and at last evaluation. (**D**) Rate of nodule at neck ultrasound in subjects of Group B1 and Group B2 at RET screening and at last evaluation. (**E**) Genetic landscape of subjects of Group B1 and Group B2.

#### *3.3. MTC in Group A and B1: Anatomopathological Features, Prognosis and Surgical Complications*

At histology, all cases showed MTC foci and/or CCH. We compared anatomopathological features between Groups A and B1 and we found that MTC foci ± CCH was significantly more present in Group A (58/67, 86.7%) than B1 (9/22, 40.9%), in which the CCH alone was prevalent (Figure 3) (*p* < 0.0001).

**Figure 3.** Rate of MTC + CCH and only CCH in Group A and B1 patients.

Among those patients who had MTC foci, Group A patients had MTC foci significantly larger than Group B1 (median 0.65 vs. 0.40 cm, *p* value = 0.036). At variance, MTC multifocality and bilaterality were not different in Groups A and B1 (Table 2). We, therefore, analyzed TNM classification system in patients of Group A and B1 with MTC (67 patients). Although most of the MTC patients belonging to Group A had T score of 1 (51/58, 88%), a significant portion (7/58, 12%) had T score > 1, whereas all MTC patients of Group B1 had a T score of 1. Lymph node metastasis occurred in 21 patients of Group A, while they did not occur in Group B1 patients (*p* = 0.045). In the case of lymph-node metastasis, they occurred in 15/21 patients (71.4%) in central and in 6/21 in latero-cervical (28.6%) compartments (Table 2). At the time of surgery, only one case of Group A presented metastasis spread to lungs, liver, and bones. All MTC patients of Group B1 experienced clinical remission during the follow-up after surgery (median 4 years, IQR 2–7 years, intervals 3–153 months), while 6/67 (9%) and 12/67 (18%) MTC patients of Group A had structural and biochemical persistent disease, respectively, during their follow-up (median 6.5 years, IQR 2.25–13 years, 3–311 months) (Table 2). All patients of both groups with CCH were cured at the data lock of this study (median follow up 4.6 years, IQR 2.5–11, 1–178) as assessed by undetectable levels of both bCT and sCT.

**Table 2.** Histopathological features of MTCs of Group A and B1 patients.

About surgical complications, they were observed in 15 (22.4%) patients of group A and in only one (6.3%) of group B1 (*p* = 0.059). Among group A patients, 14 of them presented only hypoparathyroidism and one patient both recurrent laryngeal nerve injury and hypoparathyroidism. Patient of group B1 developed only hypoparathyroidism.

#### *3.4. Follow-Up of GCs under the Age of 18*

Looking at GCs younger than 18 years at the time of *RET* genetic screening, we had a total of 63 subjects. Applying the aforementioned surgery criteria, 5/63 patients were submitted to surgery after first evaluation (belonging to Group A), 8/63 during their followup (belonging to Group B1), while 50/63 individuals are still on follow-up (belonging to Group B2). At *RET* genetics screening, there was not any difference between age of subjects belonging to Group A (median age 10 years old, IQR 6–14, intervals 5–15 years), to Group B1 (median age 7 years old, IQR 3–12.5, intervals 2–15 years) or to Group B2 (median age 8 years old, IQR 5–13, intervals 1–17 years). Otherwise, as expected, mutations occurring at 634 or cysteine codon were significantly more common in group A, although present also in Group B1 and B2 (*p* = 0.001 for 634 codon and *p* = 0.021 for cysteines); likewise, *RETC634X* mutations were substantially more common in Group B1 than Group B2 (*p* = 0.075), whereas mutations occurring at cysteine or 804 codons did not differ in Group B1 and Group B2 (*p* = 0.935 and *p* = 0.847, respectively) (Figure 4).

Surgery was performed after a median time of 5 months (IQR 4–7, intervals 4–7 months) in subjects of Group A and of about 3 years (IQR 1.6–9.3, intervals 1.6–10.3 years) in subjects of Group B1. So far, only 11/63 (17.5%) patients have been operated during childhood and/or adolescence. At the study data lock, a total of 15/58 (25.9%) GCs who did not immediately meet the criteria for surgery reached the age of 18 and two of them have been operated at 18 and 22 versus 15 and 11 years at screening. Among Group B1 patients, at

time of surgery, two of them (one patient with *RETC634Y* and one with *RETV804M*) became older than 18 years, one reached 18 years (with *RETV804M*) and two older than 15 (two patients with *RETE768D*) (Figure 5). Only one patient (age at surgery of 17 years) developed a surgical complication (hypoparathyroidism). Among patients who are still in followup (*n* = 50), (median time of 5 years, IQR 3–9, intervals 1–15 years) at study data lock, 12 patients became older than 18 years and 17 older than 15 years (Figure 5).

**Figure 4.** Genetic landscape of subjects younger than 18 years belonging to Group A, B1 or B2.

**Figure 5.** Genetic landscape, and duration of follow-up, of GCs younger than 18 at the time of *RET* genetic screening of Group A, B1 and B2.

#### **4. Discussion**

Oncology was radically revolutionized by screening of hereditary cancer diseases, diminishing the rate of patients with advanced disease at diagnosis and their mortality [16]. According to genetic and clinical characteristics of each hereditary disease, several approaches may be proposed: prophylactic surgery of involved organ, regular biochemical and/or morphological screening to promptly identify an arising neoplasia, and chemoprevention to hinder cancer development [16]. In MEN2, the suggested approach swings between the prophylactic surgery (in case of highest and very high risk *RET* mutations) and the regular follow-up (in case of moderate risk *RET* mutations), while chemoprevention has not gained space so far [8,17]. If the surgical approach must be proposed before 1 year of age in patients harboring *RETM918T*, in case of other *RET* mutations a personalized approach should be persuaded [8,18].

In this prospective study looking at 189 GCs with high and moderate risk *RET* mutations, we showed that thyroid surgery might be safely planned following bCT and sCT. In particular, GCs who were submitted to surgery after a regular follow-up (Group B1) did not experience neither lymph-node nor distant metastasis, and neither biochemical nor structural persistence was observed, at least at study data lock (median follow-up 4 years). Although the median follow-up is rather short, we should consider that all these patients showed a negative CT stimulation test at 3–6 months after surgery, which implies a negligible risk of possible recurrence [19].

The disease status of GCs who already had the criteria for surgery at the time of *RET* genetic screening (Group A) was indeed more advanced with 21/67 (31.3%) patients having lymph-node and 1/67 (1.5%) distant metastasis. Despite the prompt thyroidectomy and lymphadenectomy, 9.0% and 18% of them had structural and biochemical persistent disease, respectively, after a median follow-up of 6.5 years. However, if we consider that the percentage of MTC patients with lymph-node metastasis and/or distant metastasis in big series of MTC is around 45.1–53% and 10–11.4%, respectively [19–21], our findings demonstrate that, even in already affected GCs, the *RET* genetic screening can anticipate the diagnosis when the MTC is still clinically silent. This evidence confirmed that *RET* genetic screening should be offered and solicited to all first-degree relatives of patients with MEN2, as recommended by MTC guidelines [7,8].

We also found a relevant difference of both disease stage and outcome between group A and B1, demonstrating that timing surgery according to the increase of bCT and sCT allows performing an early, but not prophylactic, thyroid surgery that is still safe, since all patients in group B1 were cured at the time of data lock but also justified since microfoci of MTC were already present in more than 40% of cases [10].

Recently, Machens et al. showed that the risk of lymph-nodes metastasis in patients harboring *RET* germline mutations increased by age and by *RET* risk category (e.g., lowmoderate vs. moderate-high and high risk) [22]. In our series, patients of Group A, were effectively older than those of Group B while no significant differences were found in the type of RET mutations except for the fact that V804M was more frequent in Group B. This finding confirms the role of the advanced age in the development of the disease but reduces the impact of the type of *RET* mutation. New evidence showed that RETV804M mutation harbors a moderate risk of MTC development [23], although in our cohort this risk seems to not be negligible. Although age and *RET* mutation seem to be two milestones of MEN2 phenotypic variability, it is far long to be completely enlightened and inter- and intra-familial variability has been shown by many authors [24–28]. Accordingly, patients in Groups B1 and B2 did differ neither in age nor in *RET* genetics while they differ in the biological behavior of the tumor whose growth was faster in Group B1. These data argued that MEN2 genotypic-phenotypic relation is less stiff than imagined in the past and might be influenced by other factors: genetics (e.g., unbalanced expression of mutant and wild *RET* gene), epigenetics (e.g., DNA methylation, histone modification, or chromatin remodeling) and non-genetics (e.g., environmental factor) [29]. In this nebulous scenario, *RET* mutation and age should certainly guide clinical decisions, but these data argued that

each clinical management must be individualized, and thyroid surgery should be timed according to bCT and sCT, avoiding prophylactic surgery that is necessarily followed by the medicalization of the patients and, sometimes, by surgical complications, especially in children.

Using this approach, 15 out 58 patients, who were younger than 18 years of age at the time of screening, reached adulthood without thyroid surgery, postponing the beginning of a long-life therapy with levothyroxine (LT4). LT4 is the only current recommended therapy for patients undergone to total thyroidectomy, both in adults and children [7,30]. However, although a biochemical euthyroidism is generally reached, LT4 seems to do not guarantee an euthyroidism state in all tissues [31,32]. In addition, biochemical features in athyreotic patients seem to be different from those in euthyroid ones, as demonstrated by Gullo and colleagues, who showed that athyreotic patients treated with LT4 had higher fT4 and lower fT3 levels than euthyroid control and about one third of them had lower than reference fT4/fT3 ratio [33]. It is unknown if this not physiological thyroid state might play a role in children growth.

Transient or permanent disruption of calcium metabolism may occur after thyroid surgery in more than 25% and 5% of patients, respectively [34]. De Jong and colleagues collected clinical and biochemical data of 106 children (younger than 18 years) submitted to thyroid surgery and described a hypocalcemia at discharge in 49.3% and at 6 months 21.7% of them [35]. The higher risk of hypoparathyroidism in children compared to adult was confirmed by other authors [36,37], in particular in those younger than 3 years old [37]. In our cohort of GCs younger than 18 years who were submitted to surgery (13), only one patient (1/13, 7.7%) is experiencing a permanent hypoparathyroidism. Otherwise, this risk seems to be minimized in high-volume facilities [38,39], especially in patients who do not need central neck dissection [40]. Accordingly, in order to minimize this risk, GCs should be referred to surgical centers experienced in pediatric surgery for thyroid cancer. In this scenario, safely postponing thyroid surgery across the childhood could be a winning choice.

A recent review observed that subjects who experienced cancer diagnosis during their childhood seemed to be at higher risk of impaired psychological development [41], as well as manifestations of anxiety, depression, inattention, and antisocial behavior [42]. Adult survivors of childhood cancer were described to be at higher risk of depression and anxiety symptoms, even many years after the end of therapies [43]. At the same time, adults with a history of cancer during childhood presented poorer social outcomes, such as the capacity of living independently or psychosexual milestones in both females and males [43–45]. According to this evidence, the psychological impact of thyroid surgery should be carefully evaluated in children, especially after this demonstration that by taking children in active surveillance once a year and postponing the thyroidectomy to an early phase of the disease development their outcome is still favorable like that obtained with prophylactic thyroidectomy.

Finally, this approach requires an adherence of GCs (and their parents, in case of children) to regular assessments, which could represent a limitation of this active surveillance approach in our mobile society. However, like in other chronic conditions, patient education and participation are vitally important [46,47]. Subjects with RET mutations (and their parents) must be highly informed about the advantages and the disadvantages of this approach. In this highly personalized approach, each GC must not be a passive character but an active and collaborative player and social and psychological needs of each subject should be considered.

#### **5. Conclusions**

Our data showed that an active surveillance pursuing an early thyroid surgery, based upon bCT and sCT, could be safely recommended in high and moderate risk *RET* GCs, both adults and children, thus reducing the lifespan of medicalization and the risk of surgical complications. This is particularly desirable in children and is independent from the type of *RET* mutation even if those with a high risk mutation likely will reach the need to be operated earlier than those with moderate risk mutations. Moreover, we confirmed that genetic screening allows finding hidden MTC cases that otherwise would be diagnosed much later.

**Author Contributions:** Conceptualization, A.P. and R.E.; methodology, R.E.; investigation, A.M., C.G., V.B., V.C., E.M. (Elisa Minaldi), L.A., D.V., C.R., T.R., C.M., and R.C.; resources, L.T. and F.B.; data curation, A.P., L.V., E.M. (Eleonora Molinaro), M.C.C., L.L., and R.E.; writing—original draft preparation, A.P. and R.E.; writing—review and editing, A.P. and R.E.; supervision, R.E.; funding acquisition, R.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Associazione Italiana per la Ricerca sul Cancro (AIRC, Investigator grant 2018, project code 21790).

**Institutional Review Board Statement:** This study has been conducted according to the guidelines of the Declaration of Helsinki. Since it is a retrospective collection and analysis of clinical and biochemical data, it does not require the approval by Ethics Committee.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Parents or guardians signed the informed consent in case of subjects less than 18 years of age.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical issues.

**Acknowledgments:** The authors would thank all the families who had faith in us and in this clinical behavior. Their incessant support was essential for this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Epithelial and Mesenchymal Markers in Adrenocortical Tissues: How Mesenchymal Are Adrenocortical Tissues?**

**Iuliu Sbiera 1, Stefan Kircher 2, Barbara Altieri 1, Martin Fassnacht 1,3,4, Matthias Kroiss 1,4,5,\* and Silviu Sbiera 1,\***

**Simple Summary:** Recent studies have hinted to an involvement of epithelial to mesenchymal transition, a mechanism often associated with metastasis in epithelial cancers, in adrenocortical carcinoma. We assessed, in a large number of normal, benign and malignant adrenocortical tissues, the expression of canonical epithelial and mesenchymal markers and compared it with their expression in typical epithelial and mesenchymal tissues. Surprisingly, both normal and neoplastic adrenocortical tissues lacked expression of epithelial markers but strongly expressed mesenchymal markers, suggesting a higher similarity of adrenocortical tissues to mesenchymal compared to epithelial tissues, reminiscent of the adrenocortical origin from the intermediate mesoderm. Despite their ubiquitous expression in all adrenocortical tissues, mesenchymal markers had a variable expression in ACC, associating either directly or inversely with different clinical markers of tumor aggressiveness. Our data are an important step in better understanding the adrenocortical tissues in general and adrenocortical tumorigenesis in particular, and could be exploited therapeutically in the future.

**Abstract:** A clinically relevant proportion of adrenocortical carcinoma (ACC) cases shows a tendency to metastatic spread. The objective was to determine whether the epithelial to mesenchymal transition (EMT), a mechanism associated with metastasizing in several epithelial cancers, might play a crucial role in ACC. 138 ACC, 29 adrenocortical adenomas (ACA), three normal adrenal glands (NAG), and control tissue samples were assessed for the expression of epithelial (E-cadherin and EpCAM) and mesenchymal (N-cadherin, SLUG and SNAIL) markers by immunohistochemistry. Using real-time RT-PCR we quantified the alternative isoform splicing of FGFR 2 and 3, another known indicator of EMT. We also assessed the impact of these markers on clinical outcome. Results show that both normal and neoplastic adrenocortical tissues lacked expression of epithelial markers but strongly expressed mesenchymal markers N-cadherin and SLUG. FGFR isoform splicing confirmed higher similarity of adrenocortical tissues to mesenchymal compared to epithelial tissues. In ACC, higher SLUG expression was associated with clinical markers indicating aggressiveness, while N-cadherin expression inversely associated with these markers. In conclusion, we could not find any indication of EMT as all adrenocortical tissues lacked expression of epithelial markers and exhibited closer similarity to mesenchymal tissues. However, while N-cadherin might play a positive role in tissue structure upkeep, SLUG seems to be associated with a more aggressive phenotype.

**Keywords:** adrenocortical tissues; EMT; epithelial markers; mesenchymal markers; recurrencefree survival

**Citation:** Sbiera, I.; Kircher, S.; Altieri, B.; Fassnacht, M.; Kroiss, M.; Sbiera, S. Epithelial and Mesenchymal Markers in Adrenocortical Tissues: How Mesenchymal Are Adrenocortical Tissues?. *Cancers* **2021**, *13*, 1736. https://doi.org/10.3390/ cancers13071736

Academic Editor: Christofer Juhlin

Received: 17 March 2021 Accepted: 4 April 2021 Published: 6 April 2021

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

**Copyright:** © 2021 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/).

#### **1. Introduction**

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and its pathogenesis is poorly understood. Complete surgical resection is the treatment of choice in localized ACC and is virtually the only option to achieve a cure. As recurrence is frequent, adjuvant therapy is recommended in most patients [1–4]. Several genomic studies have been performed in adrenocortical tumors with the goal to better understand the mechanisms that lead to tumorigenesis, hormone excess and malignancy [5–8]. Using clustering of genome wide data, these studies consistently identified a subgroup of highly malignant tumors characterized by enhanced genomic variability and altered gene expression [9,10].

In irresectable and metastatic disease, cytotoxic chemotherapy is the standard treatment. The first and largest randomized phase III study in advanced ACC established etoposide, doxorubicin, cisplatin plus mitotane (EDP-M) as the cytotoxic chemotherapy of first choice in metastatic ACC [11]. With a median progression-free survival of only 5.0 months and an overall survival of only 14.8 months in the group receiving EDP-M, the prognosis is still poor. In the meantime, several other therapeutic approaches have been investigated [12–16], but a clinically meaningful breakthrough has not yet been achieved.

Epithelial-mesenchymal transition (EMT) is a process that was first recognized as a feature of embryogenesis and, together with its reverse process MET (mesenchymal-epithelial transition), plays a crucial role in the development of many tissues and organs [17,18]. Most importantly, the mechanism of EMT, which allows the epithelial tumor cells to acquire a motile mesenchymal phenotype [19], is diverted by several types of cancer to promote metastasis and resistance to treatment [20]. This process has been considered to be implicated in metastatic spread of such a large variety of human cancers like breast, prostate, lung etc. [21–23] (Figure 1).

**Figure 1.** Classical EMT in cancer cells. Upper panel: EMT (Epithelial to Mesenchymal Transition) and MET (Mesenchymal to Epithelial Transition) processes in metastatic spread. Lower panel: Canonical markers of epithelial (left) and mesenchymal (right) cells.

Around 90% of all malignancies originate from epithelial tissue. The adrenocortical tissue is also classically categorized as an epithelial tissue. Studies on the adrenal cortex place its origins in the intermediate mesoderm (mesenchymal) [24], but it is considered to have undergone MET to an epithelial tissue [25]. Accordingly, adrenal tumors are also classified as carcinomas (tumors of an epithelial tissue) [26] as opposed to sarcomas (tumors of a mesenchymal tissue) [27]. Two studies have provided a first indication that adrenocortical tissues are expressing some mesenchymal markers [28,29]. However, the number of adrenocortical carcinoma tissues analyzed in these studies was low (24 cases in each study) and a correlation between EMT marker expression and clinicopathological markers indicative of tumor aggressiveness was not possible.

A number of distinct molecular processes participate in EMT like activation of transcription factors, expression of specific cell-surface proteins, reorganization and expression of cytoskeletal proteins etc. In many cases, the factors involved are also used as biomarkers to demonstrate that a specific cell is undergoing EMT [20]. For example, E-cadherin and epithelial cell adhesion molecule (EpCAM) are considered as classical epithelial markers while N-cadherin, SNAIL and SLUG are considered mesenchymal markers [30]. In addition, at mRNA level, the expression of the epithelial (IIIB) and mesenchymal (IIIC) isoforms of FGFR 2 and 3 can be also used to characterize EMT [31] (Figure 1).

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell–cell adhesion in epithelia [32] associating with the actin cytoskeleton via an intermediate molecule [33]. Epithelial tumors are often characterized by strong expression of EpCAM while its expression is downregulated during EMT but then upregulated once the metastasis reaches its future tumor site, where the MET process is supposed to take place [34].

E-cadherin and N-cadherin are classical cadherins and share similar structures. They form cadherin-catenin complex where the cytoplasmic domain consists of EC repeats that bind with catenins to moderate the cytoskeletal filament containing actin. The structural difference between E-cadherin and N-cadherin is that E-cadherin binds with the shorter isoform of p120 catenin while N-cadherin binds with the longer isoform and the switch from E-cadherin expression to N-cadherin, which mediates weaker cell-cell interactions is classically used as a mesenchymal marker to define EMT [35]. N-cadherin is also present in few epithelial tissues such as hepatocytes but only together with a much stronger E-cadherin expression [30].

Snail and Slug (SNAI1 and SNAI 2), are two transcription factors that suppress Ecadherin and lead to a decrease in cell-to-cell adhesion and are also commonly used to detect EMT [36,37]. Knockout models for both SNAIL and SLUG showed significant reduction in cancer invasiveness [38,39].

At mRNA level, the fibroblast growth factors receptors (FGFR) isoform switching is another model that can be used as a marker for EMT. Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases expressed on the cell membrane that play crucial roles in both developmental and adult cells. The fibroblast growth factor receptor family has 4 members, FGFR1, FGFR2, FGFR3, and FGFR4 [40]. The FGFRs consist of three extracellular immunoglobulin-type domains (D1-D3), a single-span trans-membrane domain and an intracellular split tyrosine kinase domain. FGFs interact with the D2 and D3 domains, with the D3 interactions primarily responsible for ligand-binding specificity [40]. The receptors 1 to 3 have the unique feature of having two isoforms due to alternative splicing of the D3 domain which changes the specificity [41]. For the FGFRs 2 and 3 it has been shown that the isoform IIIB is mainly present in epithelial cells while the isoform IIIC is mostly mesenchymal [31,42].

We hypothesized that EMT may be relevant for the subgroup of highly aggressive ACCs and investigated the expression of several EMT markers in a large cohort of adrenocortical carcinoma tissue samples (including also normal adrenals and benign adrenocortical tumors) and correlated them with clinical features and patient outcome.

#### **2. Materials and Methods**

#### *2.1. Patient Material*

Formalin fixed, paraffin embedded (FFPE) tissue samples of ACC (*n* = 138; a total of 122 samples were previously assembled in seven tissue microarrays/TMAs [43]), adrenocortical adenomas (ACA, *n* = 29), as well as normal adrenal glands resulting from kidney cancer surgery (NAG, *n* = 3) were analyzed. All patients gave informed consent and the study was approved by the ethical committee of the University of Würzburg (88/11). This cohort was clinically annotated and the data were collected through the registry of the

European Network for the Study of Adrenal Cancer (ENSAT). A short clinical description of the patients can be found in Table 1. For establishment of the detection of the different epithelial markers we used tissues from two normal thyroid, three thyroid carcinoma and three colon carcinoma. For establishment of the detection of the different mesenchymal markers we used tissues from one osteosarcoma, one liposarcoma, one leiomyosarcoma and one pleomorphic sarcoma.

**Table 1.** Patient clinical characteristics.

For the evaluation of the FGFR 1-3 isoforms at mRNA levels, we used available frozen tissue from 18 ACA and NAG, 21 ACC, 6 sarcoma (2× osteosarcoma, 2× liposarcoma, 1× synovialsarcoma, 1× rhabdomyosarcoma), 5 epithelial tumors (3 colon carcinoma, 1 thyroid carcinoma, 1 ovarian carcinoma), as well as four different ACC cell lines (NCI-H295R [44], MUC-1 [45], CU-ACC1 and CU-ACC2 [46]).

#### *2.2. Immunohistochemistry*

The immunohistochemistry procedure is explained in more detail elsewhere [43]. In short, the FFPE TMA and full tissue slices of ~2 μm thickness were mounted on SuperFrost glass slides (Langenbrinck, Emmendingen, Germany), deparaffinized in xylene, and rehydrated in a series of water in alcohol dilutions. Antigen retrieval was achieved by boiling in 1 mM citrate buffer (pH 6.5) inside a pressure cooker for 13 min. Endogenous peroxidase was blocked with a 3% solution of hydrogen peroxide in methanol for 10 min, followed by the blocking of non-specific binding for another 10 min with the help of a 20% solution of human AB serum in PBS. The primary antibodies used were: E-Cadherin (Sigma-Aldrich, St. Louis, MO, USA, mouse monoclonal, clone CL1172, 1:2250 dilution), EpCAM (abcam, Cambridge, UK, rabbit polyclonal (ab71916), 1:20000 dilution), N-Cadherin (Santa Cruz Biotechnology, Dallas, TX, USA, mouse monoclonal, clone D-4, 1:125 dilution), SLUG (Novus Biologicals, Centennial, CO, USA, mouse monoclonal, clone OTI1A6, 1:300 dilution) and SNAIL (kindly provided as a gift from Dr. A. García de Herreros, University Pompeu Fabra, Barcelona, Spain; clone EC3, subclone EC11, 1:50 dilution [47]). Incubation time for the primary antibodies was 1 h at room temperature in PBS. Signal amplification was done with "HiDef Detection HRP Polymer System" (Cell Marque, Rocklin, CA, USA) and the signal was developed using the chromogen DAB substrate kit (Cell Marque) for 10 min. Counterstaining of nuclei was performed using Meyer's Hematoxylin for 2 min (Carl Roth, Karlsruhe, Germany), followed by washing in running tap water for 5 min. After dehydration, slides were mounted using Entellan (Merck, Darmstadt, Germany) and borosilicate glass coverslips (A. Hartenstein, Würzburg, Germany).

Stained tissue slides were imaged with the Leica Aperio Versa brightfield scanning microscope (Leica, Wetzlar, Germany) and evaluated using a semi-quantitative H-Score [48] that estimated the intensity of the staining [scored as negative (0), low (1), medium (2) and high (3)] and the percentage of positive cells (scored as 0, 0.1, 0.5, or 1 if 0%, 1–9%, 10–49%, or ≥50% of the cells were positive, respectively). All slides were evaluated by two independent investigators blinded to clinical data. Low expression was defined as H-score < 2 and high score as H-score ≥ 2.

#### *2.3. Cell Culture*

NCI-H295R cells were obtained from ATCC and cultured in DMEM/F12 supplemented with 1× Insulin-Transferrin-Selenium and Nu-Serum (2.5%). CU-ACC1 and CU-ACC2 cells were obtained from Katja Kiseljak-Vassiliades and cultured as described [46]. In brief, a 1:3 mixture of F12 Ham and DMEM high glucose (both Thermo Fisher Scientific, Waltham, MA, USA) was supplemented with 10% FCS, 0.4 μg/mL hydrocortisone (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 8.4 ng/mL cholera toxin (Sigma-Aldrich), 24 μg/mL adenine (Sigma-Aldrich) and 10 ng/mL EGF (Thermo Fisher Scientific). MUC-1 were obtained from Constanze Hantel and cultured in DMEM Advance 1% penicilin/streptomycin and 10% FCS as described [45].

#### *2.4. Real-Time PCR*

RNA was first extracted from frozen tissues using the RNeasy Lipid Tissue Kit (Qiagen, Düsseldorf, Germany) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). TaqMan Gene Expression Assay (Thermo Fisher Scientific) was used with the Hs01005396\_m1 (FGFR3 IIIB) and Hs00997397\_m1 (FGFR3 IIIC) probes and the following custom primers/probes for FGFR2 receptor isoforms [49]:

FGFR2 IIIb fw: 5'-GGCTCTGTTCAATGTGACCGA-3 ; rev: 5 -GTTGGCCTGCCCTAT ATAATTGGA-3 ; TaqMan probe: 5 -TTTCCCCAGCATCCGCC-3

FGFR2 IIIc up: 5 -CACGGACAAAGAGATTGAGGTTCT-3 ; low: 5 -CCGCCAAGCA CGTATATTCC-3 ; TaqMan probe: 5 -CCAGCGTCCTCAAAAG-3

The expression of β-actin (Hs9999903\_m1) was used for normalization. Amplification and results evaluation were performed using a Bio-Rad CFX-96 Dx system (Bio-Rad Laboratories, Hercules, CA, USA).

#### *2.5. Statistical Analyses*

The relationship between two categorical variables was determined using the Chi square test. For non-parametrical multiple comparisons between groups the Kruskal-Wallis test with Dunn's post-hoc comparison was used. A *p*-value < 0.05 was considered statistically significant. *p*-values between 0.05 and 0.15 were considered indicative of a statistical trend. All statistical analyses were performed with Graph Pad Prism v 7 for Windows (GrapPad Software Inc., La Jolla, CA, USA). For ACC patients, the Kaplan-Meier method was used to estimate overall survival (OS, in all patients with primary tumors) and recurrence-free survival (RFS, in patients with complete resection of the primary tumor) using IBM SPSS v 26 for Windows (SPSS Inc., Chicago, IL, USA). After ≈10% of subjects remaining at risk the Kaplan-Meier survival was curtailed.

#### **3. Results**

#### *3.1. Typical Epithelial Adhesion Markers Are Not Expressed in Adrenocortical Tissues*

The expression of E-cadherin, was absent in all adrenal tissues analyzed (*n* = 170), both normal and tumoral (Figure 2B–E), while this marker showed a normal, membrane expression in 14 different epithelial tissues analyzed (Figure 2A,E). Similar results were also observed for EpCAM, which was also completely missing in all adrenocortical tissues analyzed (Figure 2G–J) while strong expression of this marker in classical epithelial tissues was observed that were used as positive controls (Figure 2F,J).

**Figure 2.** Expression of canonical immunohistochemical epithelial markers in adrenocortical tissues. Staining of epithelial markers E-cadherin (**A**–**E**) and Epithelial Cell Adhesion Molecule (EpCAM) (**F**–**J**) protein in classical epithelial tissues (**A**,**F**) vs. normal adrenal glands (NAG, *n* = 3; **B**,**G**) vs. adrenocortical adenomas (ACA, *n* = 29; **C**,**H**) vs. adrenocortical carcinomas (ACC, *n* = 138; **D**,**I**). Scale bar = 200 μm. Quantitative evaluation in (**E**,**J**), respectively.

#### *3.2. Adrenocortical Tissues Are Characterized by Relatively High Expression of Mesenchymal Markers*

Surprisingly, membrane N-cadherin expression was expressed in normal adrenocortical tissues at high levels (H-score 2.5 ± 0.5, Figure 3B,E). The expression was distributed rather equally between the three functional regions, with slightly lower expression in the zona fasciculata (Supplementary Figure S1A–C). Most adrenocortical adenomas and carcinomas demonstrated moderate to high expression (ACA mean H-score 1.8 ± 0.7; Figure 3C,E, ACC 1.6 ± 0.9; Figure 3D,E) similar to the mesenchymal sarcomas (1.9 ± 0.8; Figure 3A,E). There were no significant differences between the different adrenocortical tissues, only a trend (NAG vs. ACA: *p* = 0.14, NAG vs. ACC: *p* = 0.09 and ACA vs. ACC: *p* = 0.20), however, interestingly, the variability of expression of N-cadherin increased gradually from NAG to ACA and then to ACC (Figure 3E) as shown by increasing coefficients of variation (NAG 20.00%, ACA 39.25% and ACC 58.53%; mesenchymal 45.54%).

While Snail nuclear expression was found in most mesenchymal tissues tested (Figure 3F,J), detectable expression was not observed in any of the adrenocortical tissues (Figure 3G–I and evaluation in Figure 3J). In contrast, a strong expression of Slug was found in both mesenchymal tissues (mean H-score 2.3 ± 0.5; Figure 3K,O) and normal and benign adrenal tissues without statistically significant differences among groups (NAG mean H-score 2.3 ± 0.5, ACA 2.2 ± 0.7; Figure 3L,M,O) but variable expression in ACC (mean H-score 1.6 ± 1.1; Figure 3N,O).

Only the expression in ACC was significantly different compared to the other two adrenocortical sample sets (NAG vs. ACA: *p* = 0.79, NAG vs. ACC: *p* = 0.02 and ACA vs. ACC: *p* = 0.01\*) but as with N-cadherin, the variability of expression of SLUG increased gradually from NAG to ACA and then to ACC (Figure 3O) as shown by increasing coefficients of variation (NAG 24.74%, ACA 34.29% and ACC 68.77%; mesenchymal 20.16%). Interestingly, in the normal adrenal gland tissue the most nuclei stained positive were localized in the subcapsular region, in the zona glomerulosa (Supplementary Figure S1D–F).

**Figure 3.** Expression of canonical immunohistochemical mesenchymal markers in adrenocortical tissues. Staining of mesenchymal markers N-cadherin (**A**–**E**), Zinc finger protein SNAI1 (SNAIL) (**F**–**J**) and Zinc finger protein SNAI2 (SLUG) (**K**–**O**) in classical mesenchymal cancers (**A**,**F**,**K**) vs. normal adrenal glands (NAG, *n* = 3; **B**,**G**,**L**) vs. adrenocortical adenomas (ACA, *n* = 29; **C**,**H**,**M**) and vs. adrenocortical carcinomas (ACC, *n* = 138; **D**,**I**,**N**). Scale bar = 200 μm. Quantitative evaluation in (**E**,**J**,**O**), respectively.

#### *3.3. FGFR1-3 Isotype Expression Shows a Pattern Similar to Mesenchymal Tissues*

To further elucidate the epithelial vs. mesenchymal phenotype of adrenocortical tumors, we used the ratio between the "mesenchymal" IIIC and the "epithelial" IIIB isotypes of FGFR 2-3 in a subgroup of fresh frozen adrenocortical tissue samples and cell lines. Isoform IIIC of FGFR 2 was expressed on average 4.6 times higher than IIIB in all adrenocortical tissues studied (Figure 4A) (ratio IIIC/IIIB: 5.1 ± 2.6 for the normal adrenal glands and adrenocortical adenomas vs. 4.2 ± 2 for the ACC samples vs. 4.8 ± 1.2 for ACC cell-lines) similar to the mesenchymal sarcomas (2.8 ± 0.8), but in contrast to the epithelial samples where the IIIB isoform was higher expressed than the IIIC isoform, as expected (ratio IIIB/IIIC: 3.9 ± 2.3). For FGFR 3 the IIIC/IIIB ratios were even higher (Figure 4B) (12.2 ± 5.5 for the normal adrenal glands and adrenocortical adenomas vs. 11.9 ± 7.7 for the ACC samples vs. 11.7 ± 3.2 for ACC cell-lines) similar again to the mesenchymal sarcomas (9.1 ± 7.1). The epithelial control tissues showed again, as expected, higher IIIB than IIIC expression (ratio IIIB/IIIC: 25.1 ± 18.4).

**Figure 4.** Differential expression of FGFR splice variants mRNA in adrenocortical tissues. Analysis of the ratios between the "mesenchymal" (IIIC) and "epithelial" (IIIB) splice variants for FGFR-2 (**A**) and 3 (**B**), in normal adrenal glands (NAG), adrenocortical adenomas (ACA) and carcinomas (ACC) as compared to mesenchymal sarcomas and canonical epithelial tissues; for better visualization of the isoform switch, results are represented in log10 base.

#### *3.4. SLUG and N-Cadherine Are Associated in an Opposite Manner with Pathoclinical Tumor Aggressiveness Parameters*

Since expression of NCAD and SLUG showed an increase in variability from normal, to benign, to malignant adrenocortical tissues, this suggested a modulation of these factors during the tumorigenesis and tumor progression. Therefore we looked for possible associations between different expression levels of NCAD and SLUG and indicators of tumoral metastatic potential. The presence of venous infiltration was associated with high (H-score ≥ 2) vs. low (H-score < 2) expression of SLUG (31 vs. 44%, χ2 = 3.6, *p* = 0.05) (Figure 5A), but with lower expression of N-cadherin (28 vs. 46%, χ2 = 6.9, *p* = 0.008) (Figure 5B). Similarly, lymph node infiltration was significantly more often present in tumors with high SLUG expression (23% vs. 12%, χ2 = 4.2, *p* = 0.04) (Figure 5C) and with low N-cadherin expression (26% vs. 9%, χ2 = 10.0, *p* = 0.001) (Figure 5D). Unsurprisingly, also the mixed pathomorphological diagnostic Weiss score, an indicator for tumor malignancy, was significantly higher for samples with low NCAD expression (6.0 ± 1.5 vs. 4.7 ± 1.6, *p* < 0.001) (Figure 5F), and for samples with high SLUG expression (6.0 ± 1.9 vs. 5.1 ± 1.5, *p* = 0.04) (Figure 5E). A Mann-Whitney test of the distribution of N-cadherin and SLUG expression in tumors with low and high expression of the proliferation marker Ki67, the best defined prognostic marker for the ACC [50], confirmed this association. In tumors with high Ki67 expression the SLUG expression was significantly higher (2.2 ± 0.9 vs. 1.5 ± 1.1, *p* = 0.03) (Figure 5G) while the expression of N-cadherin was significantly lower (1.1 ± 0.6 vs. 1.5 ± 0.9, *p* = 0.04) (Figure 5H).

#### *3.5. SLUG and N-Cadherin Expression Have a Divergent Association with ACC Patients' Progression-Free Survival*

We next investigated a potential association of SLUG and N-cadherin expression with patient outcome and found no difference on OS (low vs. high SLUG expression: Average survival time 64.20 ± 10.27 vs. 68.82 ± 9.14 months, HR = 1.15, 95%CI: 0.5–1.5, *p* = 0.79 and low vs. high N-cadherin expression: Mean survival time 65.11 ± 9.49 vs. 71.22 ± 10.66 months, HR = 0.81, 95%CI: 0.48–1.37, *p* = 0.44) (Figure 6A,B) and only a trend that high SLUG expression correlated with a less favorable RFS in ACC patients after complete resection (high vs. low SLUG expression: Mean survival time 25.96 ± 5.40 vs. 49.82 ± 10.12 months, HR = 2.15, 95% CI: 0.96–4.83, *p* = 0.056) (Figure 6C). For N-cadherin the situation was opposite, while again not statistically significant, there was a light trend that high N-cadherin expression correlated with a better progression-free survival (mean survival time 40.12 ± 8.35 vs. 21.32 ± 6.6 months, HR = 0.65, 95% CI: 0.34–1.11, *p* = 0.14) (Figure 6D).

**Figure 5.** Comparison between relevant clinicopathological data and expression levels of mesenchymal markers SLUG and N-Cadherin. (**A**,**B**) venous tumor infiltration, (**C**,**D**) lymph node tumor infiltration, (**E**,**F**) Weiss score distribution and (**G**,**H**) proliferation marker Ki67. "*n*" numbers represent the absolute number of cases in each subgroup. χ2 analyses have been performed between proportions (%) in each staining intensity group. \* *p <* 0.05, \*\* *p <* 0.01, \*\*\* *p <* 0.001.

**Figure 6.** Correlation of patient survival with expression of mesenchymal markers SLUG and N-Cadherin. (**A**,**B**) overall survival (**C**,**D**) recurrence-free survival.

#### **4. Discussion**

In this study we investigated a series of both classical epithelial and mesenchymal markers in a large cohort of normal, benign and malignant adrenocortical tissues, and compared the expression of these markers with that in epithelial and mesenchymal control tissues. Against our hypothesis, our analysis revealed that in adrenocortical tumors EMT indicated by a more frequent occurrence of mesenchymal markers in neoplastic tissue, does not appear to play a role in tumor progression as suggested before in smaller studies [28,29]. Adrenocortical tissues do not express established epithelial markers like E-cadherin and EpCAM but express a series of "classical" mesenchymal markers like Slug and N-cadherin at similar levels as mesenchymal tissues.

By using the more recently discovered marker of alternative mRNA splicing of the FGFR2 and 3 [42,51–53] we confirmed that adrenocortical tissues are more similar to mesenchymal than to epithelial tissues. This may be due to the special case of adrenocortical tissue as it originates during embryogenesis from the intermediate mesoderm, but is considered to undergo MET to result in an epithelial tissue [25]. Obviously, this epithelial transformation is incomplete and the adrenal cortex keeps most of its mesenchymal characteristics at molecular level.

While expressed in all adrenocortical tissues, there may still be a role of mesenchymal differentiation status in tumor aggressiveness. While higher SLUG expression is associated with more aggressive behavior of the tumors as indicated by its association with markers for lymphatic and hematogenic metastasizing and high cell proliferation, N-cadherin appears to play the role of major cell- cell adhesion molecule in the adrenocortical tissue and thus is a counterplayer of SLUG. There are also other tissues where N-cadherin is the prevalent constituent of adherens junctions such as neural tissues [54]. It is hence likely that adherens junctions in adrenocortical tissue are predominantly mediated by N-cadherin instead of the E-cadherin, which is more commonly found in epithelial adherens junctions. However, only future studies at a deeper molecular level on the cell-to-cell interactions in the adrenal will be able to definitely answer this question.

Interestingly, SLUG nuclear expression in the normal adrenal gland was highest in the subcapsular area of the zona glomerulosa. This zone accommodates a subset of cells that have been reported to centripetally migrate towards the center of the gland and are responsible for the permanent renewal of the adrenocortical tissue [55]. The idea of a progenitor cell population that gives rise to all differentiated cell types within the adrenal cortex is old [56,57], but while its subcapsular localization has been clarified using animal models [58,59], there is not yet an universally accepted immunohistochemical marker that can be used to identify this population. It has been shown that Wnt, β-catenin and Shh all play an important role in this process [60–62], however, their expression in the adult adrenal cortex did not coincide with cell-proliferation markers [63] so they cannot be used to identify the progenitor cell population. The best candidate to date is the Notch atypical ligand Delta-like homologue 1 (DLK1) [64].

It would have been especially interesting to correlate in more detail the expression of SLUG and N-cadehrin in metastases with clinicopathological characteristics of the same, especially KI67. However, these data have been inconsistently retrieved in the past and the rarity of ACC renders the collection of such a larger series of clinically well-annotated cases prospectively quite challenging. Another limitation is the perceived limited choice of both epithelial and mesenchymal markers analyzed. While the list of possible specific markers is very long [20], we have concentrated on the best defined and used markers in each category. But we did not limit ourselves to immunohistological staining investigating also markers defined at mRNA level, thus covering quite a broad selection of pathways involved in cell adhesion, migration and response to external stimuli. The results of all these analyses corroborated with each other to give a synchronized picture on the role played by these markers in the adrenal tissues.

#### **5. Conclusions**

We could show that adrenocortical tissues, whether normal, benign or malignant, are characterized by lack of expression of classical epithelial tissues and are closer to mesenchymal tissues through high expression of classical mesenchymal markers like Ncadherin and SLUG. These factors also appear to play a role in cancer progression in ACC: While N-cadherin seems to have a positive role in the tissue structure sustainability and against metastatic spread, SLUG seems to promote this.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/cancers13071736/s1, Figure S1: Expression of canonical immunohistochemical mesenchymal markers in normal adrenal gland.

**Author Contributions:** Conceptualization, M.F., M.K. and S.S.; methodology, I.S.; software, I.S. and S.S.; validation, I.S., S.K. and B.A.; formal analysis, I.S. and S.S.; investigation, I.S.; resources, S.K. and B.A.; data curation, I.S. and B.A.; writing—original draft preparation, I.S., M.F., M.K. and S.S.; writing—review and editing, I.S., S.K., B.A., M.F., M.K. and S.S.; visualization, I.S. and S.S.; supervision, M.F., M.K., S.S.; project administration, S.S.; funding acquisition, M.F., M.K., S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Else Kröner-Fresenius Foundation (Else Kröner-Fresenius-Stiftung; project number: 2016\_A96 to SS and MK and the German Research Foundation (Deutsche Forschungsgemeinschaft; project numbers 314061271—CRC/TRR 205 and 237292849 to MF and MK). **Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the University of Würzburg (approval # 88/11).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** All data presented in this study is contained within the article and supplementary material.

**Acknowledgments:** This publication was supported by the Open Access Publication Fund of the University of Würzburg.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

## *Review* **Anaplastic Thyroid Carcinoma: An Update**

**Arnaud Jannin 1,2, Alexandre Escande 2,3, Abir Al Ghuzlan 4, Pierre Blanchard 5, Dana Hartl 6, Benjamin Chevalier 1,2, Frédéric Deschamps 7, Livia Lamartina 8, Ludovic Lacroix 9, Corinne Dupuy 10, Eric Baudin 8, Christine Do Cao 1 and Julien Hadoux 8,\***

**Simple Summary:** Anaplastic thyroid carcinoma (ATC) has a dismal prognostic. Chemotherapy and radiotherapy are the mainstem options for patients with ATC. In selected cases with actionable genomic alterations or with favorable immune tumor microenvironment, new therapeutic options as targeted therapies and immunotherapy have led to better outcome and raised some hope for treatment of this deadly disease.

**Abstract:** Anaplastic thyroid carcinoma (ATC) is a rare and undifferentiated form of thyroid cancer. Its prognosis is poor: the median overall survival (OS) of patients varies from 4 to 10 months after diagnosis. However, a doubling of the OS time may be possible owing to a more systematic use of molecular tests for targeted therapies and integration of fast-track dedicated care pathways for these patients in tertiary centers. The diagnostic confirmation, if needed, requires an urgent biopsy reread by an expert pathologist with additional immunohistochemical and molecular analyses. Therapeutic management, defined in multidisciplinary meetings, respecting the patient's choice, must start within days following diagnosis. For localized disease diagnosed after primary surgical treatment, adjuvant chemo-radiotherapy is recommended. In the event of locally advanced or metastatic disease, the prognosis is very poor. Treatment should then involve chemotherapy or targeted therapy and decompressive cervical radiotherapy. Here we will review current knowledge on ATC and provide perspectives to improve the management of this deadly disease.

**Keywords:** anaplastic thyroid carcinoma; chemotherapy; immune checkpoint inhibitors; tumors associated macrophages; radiotherapy; molecular targeted therapy

#### **1. Introduction**

Anaplastic thyroid cancer (ATC) is a rare malignancy with a poor prognosis. It is characterized by a rapid onset with local and distant metastases, local progression

**Citation:** Jannin, A.; Escande, A.; Al Ghuzlan, A.; Blanchard, P.; Hartl, D.; Chevalier, B.; Deschamps, F.; Lamartina, L.; Lacroix, L.; Dupuy, C.; et al. Anaplastic Thyroid Carcinoma: An Update. *Cancers* **2022**, *14*, 1061. https://doi.org/10.3390/ cancers14041061

Academic Editors: Alfredo Berruti, Vito Amoroso and Nicola Fazio

Received: 20 January 2022 Accepted: 14 February 2022 Published: 19 February 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/).

and distant evolution [1]. Its treatment is an emergency, based on surgery (if feasible), radiation therapy and chemotherapy [2,3]. However, the prognosis remains very poor with a one-year overall survival (OS) rate between only 20 and 50% [4–7]. Recent results of the dabrafenib/trametinib combination for *BRAF*-mutated patients [8,9] and of immune checkpoint inhibitors (ICI), used alone or in combination with targeted therapies [10–13], have raised some hope toward an improvement of the prognosis of this deadly disease [14].

Here we will review current knowledge on the epidemiology, pathology/biology and standard treatment of ATC and discuss the recent progress and perspectives for their management.

#### **2. Epidemiology and Clinical Presentation: A Rare Disease with a Rapid Onset and Poor Prognosis**

ATC is a rare cancer as defined by the European Union rare cancer surveillance program (RARECARE) with an incidence far below six new cases per 100,000 personyears [15]. Indeed, recent epidemiological studies have confirmed an age-adjusted incidence in the US of 0.12 per 100,000 person-years (95% CI: 0.8–1.6) in 2014 in the Surveillance, Epidemiology, and End Results (SEER) database [16] and 0.1 to 0.3 per 100,000 personyears in Europe according to data from Denmark [4], Wales [17] and the Netherlands [18] registries. The SEER database and the Netherlands registries analyses have suggested an increase in the age-adjusted incidence with an average annual percent change of 3.0% per year (95% CI: 2.2–3.7%) and 1.3% per year (95% CI: 0.4–2.1%), respectively, over a 30–40-year period of time; this increase in incidence was not reported in the Danish and the Welsh databases [4,16–18]. The reasons for the discrepancies are unknown; however, incidence rates are consistent across the different studies in Europe and US. The increase in incidence is unlikely to be related to better screening/diagnosis because all ATC patients end up being diagnosed with cervical compressive symptoms.

ATC usually affects elderly patients, with the majority being over 60 years old with a female predominance (male/female sex ratio = 1.5:2). The advanced stage of the disease is the most common diagnosis presentation (localized (IVa) 10%, locally advanced (IVb) 35% and metastatic (IVc) 55%) displaying extremely aggressive behavior with rapid tumor progression, local invasion and/or distant metastases (lung, bone, liver and/or brain metastases) [1,2,19–21] (Figure 1).

Classically, patients report a rapid transformation of a long-standing goiter (30% of cases) within a few days to a few weeks. The others symptoms include neck pain as well as signs of neck tumoral invasion and compression: dyspnea, dysphonia and dysphagia.

On clinical examination, there is a large cervical mass, hardness on palpation, palpable lymphadenopathy and sometimes skin involvement. Invasion of the aerodigestive tract is frequent. Laryngeal dyspnea, dysphagia or superior vena cava syndrome which may require urgent treatment (placement of a tracheotomy and/or placement of a gastrostomy) as well as local pain can be associated. The repercussions of local tumor spread on the general condition immediately indicate the gravity of the situation.

**Figure 1.** CT scan illustration of anaplastic thyroid carcinoma (ATC) staging.

#### **3. Pathology and Biology: How Do We Understand the Aggressiveness of This Disease?**

#### *3.1. Pathology*

ATC is defined as a highly malignant tumor composed of undifferentiated cells which retain some features of an epithelial origin on morphology and/or immunohistochemical examination [22]. Various and heterogeneous histologic features can be seen in ATC samples including epithelioid and squamous morphology, giant cells, pleomorphic morphology, osteoclast giant cell-rich morphology, and spindle cell morphology which is the most common histotype [23]. The most recent series from the Memorial Sloan Kettering Cancer Center (MKKCC) has examined the clinic–pathologic features of 360 cases from two institutions over a 34-year period. In this study, the most common histological subtypes were spindle cell (26%), pleomorphic (23%) and squamous (21% of the cases) [7]. Tumor necrosis was found in 77% of the cases, atypical mitosis in 77% and a neutrophilic infiltrate was noted in 71% of the cases. Interestingly, the mitotic index was >20 mitoses per 10 highpower fields in only 15% of the cases, and Ki67 was not reported. This study confirmed that thyroglobulin and TTF1 immunohistochemistry is almost always negative (96 and 70% of the cases, respectively), whereas cytokeratins AE1/AE3 are present in 67% of the cases and PAX 8 in up to 70% (with anti-PAX8 antibody 10336-1-AP). A recent immunohistochemical study, with the most commonly used monoclonal anti-PAX8 antibody (MRQ-50), showed lower PAX8 expression in 54.4% of the ATC cases [24]. Therefore, performing PAX8 immunohistochemistry in all samples of thyroid undifferentiated tumors suspicious for ATC, and in particular in squamous subtypes, allows for support of a differential diagnosis with squamous cell carcinoma of the head and neck which is always negative for PAX8 [25].

ATC tumorigenesis may be a multistep process with a biological transformation (synchronous or metachronous) from differentiated thyroid cancer (DTC) to ATC. This assumption is suggested by the common recognition of a concomitant DTC tumor component or a history of DTC observed in 58 to up to 90% of cases [7,26]. Poorly differentiated DTC (PDTC) and the tall cell variant of papillary DTC were the most common subtypes found associated to these transformed ATC subtypes in the MSKCC series [7]. From a molecular point of view the association of additional *TP53* and/or *TERT* promoter mutations is found in up to 80% of ATC cases harboring typical DTC molecular alteration in the *BRAF* and *RAS* genes [27–30]. These data have suggested that additional mutations in *TP53* and *TERT* may drive the tumor progression from DTC to ATC [1] in these transformed ATC subtypes. This transformation process of ATC may occur differently according to the genetic mutation background. Indeed, in *RAS* mutant ATC, a history or concomitant DTC is observed in 38% of cases and it is observed in 75% of cases with *BRAF* mutations (*p* = 0.001) [7]. A whole-exome sequencing analysis of the two tumor components (DTC and ATC) of three mixed ATC tumor samples revealed that most of the somatic mutations identified in the ATC component differed from the ones in papillary DTC. This led to the conclusion of there being very few common mutations and a large genomic divergence between the two components challenging the concept of tumor progression from DTC to ATC [31]. From the clinical point of view, a recent retrospective multicenter and SEER database study on 642 primary (i.e., tumors with no DTC component at diagnosis) and 47 secondary ATC (i.e., tumors with a DTC component at diagnosis), found no statistical differences in terms of demographic, clinical manifestations and patient survival and a more frequent *BRAF* mutation as compared with *RAS* mutation in secondary tumors [32]. However, it must be pointed out that identification of "transformed" ATC requires the knowledge of the detailed clinical history of the patient as well as the detailed pathology assessment of the tumor which may not be optimal in a very large database. Therefore, it is still not known whether primary or "pure" ATC carries a different prognosis as compared with secondary or "transformed" ATC.

#### *3.2. Molecular Biology*

In a recent series of 126 samples of ATC analyzed by Next Generation Sequencing (NGS), the most common molecular alterations were found in *TERT* promoter (75%), *TP 53* (63%), *BRAF* (45%), *RAS* (22%), *PIK3CA* (18%), *EIF1AX* (14%) and *PTEN* (14%) with the first two being more frequent in ATC than the others, which can be seen in either DTC or PDTC [7,28]. Strikingly, *BRAF* mutation frequency in ATC seems to differ between recent series for US and Europe. Indeed, *BRAF* mutations are found in 40–45% of cases in US studies [7,14] whereas they are found in 14–37% of cases in European studies [27,30,33]; furthermore, data from south Korea reported a rate of 41% *BRAF* alterations in a series of 13 ATC cases [34]. Whether these discrepancies are linked to various sequencing techniques and/or to geographical differences in pathophysiology remains an open question. Recent results have revealed that *NTRK* and *RET* fusion can be detected in 2–3% of ATC cases [7,35–37] which is of utmost importance for the few patients who may be offered highly specific targeted therapies. If regulation of cell cycle has a crucial role in oncogenesis and particularly in ATC, protein metabolism control is also involved in tumorigenesis. For example, about 10% of patients with ATC harbor *EIF1AX* mutations, which has recently been involved in deregulating protein synthesis [36]. Interestingly, *EIF1AX* mutations could co-occur with *RAS* mutations in ATC with a positive feedback relationship between RAS and EIF1AX proteins, which reinforces *c-MYC* gene expression [28,38]. Molecular alteration of the Wnt signaling pathway could also be observed, notably with β-catenin gene (*CTNNB1*), *AXIN1* and *APC mutations* [36]. Although increased levels of cytoplasmic β-catenin are observed in most thyroid cancer cells, mutations of β-catenin that lead to nuclear localization of the protein are limited to PDTC and ATC, suggesting a role in tumor

progression [39]. Alterations of epigenetic-related genes such as the chromatin remodeling SWI/SNF complex (*ARID1A*, *SMARCB1*, *PBRM1*, etc.) and histone methyltransferases (*KMT2A*, *KMT2C*, *KMT2D* and *SETD2)* have been found in 36% and 24% of ATC samples, respectively. The DNA Mismatch repair (MMR) pathway may be altered in 10–15% of cases [7,28,29,40–43].

Knowledge of the molecular alterations in ATC patients has been more and more prominently important in the clinics recently, with the advent of targeted therapies. This growing importance has been recognized in the recent ATA and ESMO guidelines which recommends offering molecular testing to all ATC patients with unresectable disease [2,3].

#### *3.3. Immune Infiltrate of ATC*

ATC tumors are characterized by an important infiltration of Tumor-Associated Macrophages (TAMs) which can represent 40 to 70% of the total tumor mass and could play some role as an immunosuppressive tumor stroma, in treatment resistance and in the poor prognosis of the disease [44–47]. ATCs display a very dense network of interconnected "ramified" TAMs which may have metabolic and trophic functions via direct contact with intermingled cancer cells [44]. This macrophage infiltration is composed of M2 pro-tumorigenic tumor-associated macrophages as demonstrated by the identification of the M2-TAMs transcriptomic signature of 78 genes identified in ATC samples, which is able to discriminate them from DTC samples [28]. The co-culture of thyroid cancer cell lines with M2-like TAMs facilitates dedifferentiation, proliferation, migration and invasion in thyroid cancer cells through the Wnt/ß-catenin pathway activation by Wnt1 and Wnt3a secretion [48] but also through insulin-like growth factor (IGF) secretion which promotes thyroid cancer stemness and metastasis by activating the PI3K/AKT/mTOR pathway [48]. A study of 19 ATC samples evaluated by automated digital quantification of CD68 and CD163 immunohistochemistry positivity confirmed the putative importance of macrophage infiltration. The mean macrophage infiltration rate was 17% and 23% for these two markers, respectively, and most of the ATC samples displayed a low to moderate level of the CD47 "don't eat me signal" which physiologically binds to signal regulatory protein α (SIRPα) on macrophages and inhibits phagocytosis of tumor cells. With an anti-CD47 antibody, phagocytosis of ATC cell lines by macrophages could be induced in vitro and in a xenotransplant model [49].

In ATC, this high macrophage infiltration in ATC tumor samples results in an alteredimmunosuppressed immune microenvironment in 50% of cases and a hot immune environment in 34%, with a high expression of several inhibitory immune checkpoint mediators such as anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed deathligand 1–2 (PD-L1/PD-L2), TIGIT, etc., known to inhibit cytotoxic CD8+ T-cell functions [50]. Among these inhibitory immune checkpoint molecules, PD-L1 expression has been identified in 70% of ATC samples in the pre-clinical study by Schürch et al. [49] and in the phase I study of spartalizumab [10]. PD-L1 expression based on the proportion of stained tumor cells according to Tumor Proportion Score (TPS) has been found at ≥5% in 73% of 93 samples in a recent multicenter study from Germany [51]. In the tumor microenvironment, PD-L1 can be upregulated in both tumor cells and immune-microenvironment cells, such as TAMs in ATC samples [52,53]. This dual expression of PD-L1 may have important pathophysiological implications, because although induction of PD-L1 on tumor cells is interferon gamma (IFNg)-dependent and transient, PD-L1 induction on TAMs is of greater magnitude, only partially IFNg dependent and more stable over time, and thus may account for the immunosuppressive microenvironment in ATC [54]. This PD-L1 expression on TAMs may account for the presence of exhausted T-cells in transcriptomic analysis of ATC samples [50]. Moreover, PD-L1 expression on tumor cells, although possibly predictive of a response to immunotherapy in ATC [10], can be induced by the immune microenvironment, especially T-cells and TAMs, by different signaling pathways, and thus may result in differential responses to treatment with immune checkpoint inhibitors (ICI). This PD-L1 induction through different pathways by different immune cells of the

tumor microenvironment may result in either accelerated tumor growth and resistance to doxorubicin and ICI of PD-L1+ tumor cells induced by TAMs; or delayed tumor growth and greater sensitivity to both doxorubicin and ICI when induced by T-cells, as shown in a hepatocellular cell line and a mouse model of hepatoma [55]. Therefore, although PD-L1 expression is high in ATC samples, its clinical impact and the differential expression on either tumor cells and/or TAMs still remains to be refined.

#### **4. Treatment: To Treat Aggressively or to Palliate the Symptoms?**

#### *4.1. Multimodal Therapy or Palliative Care within a Fast-Dedicated Management Track*

In ATC, goals of care may be therapeutic and/or palliative depending on staging and prognosis when considered in the context of available therapies, comorbidities and the patient's wishes. Multimodal therapy refers to the combination of excisional surgery, when possible, external beam radiation therapy (EBRT), chemotherapy and/or targeted therapy (Figures 2–4). This multimodal strategy is associated with a better OS in retrospective studies. In 1990, the mean OS was about two to six months [56,57] and it seems to be nearly the same 20 years later, with a one-year OS of less than 20% [58–61]. However, in a cohort of 479 patients treated in the same institution spanning nearly 20 years, Maniakas et al. found one- and two-year OS of 35/18% in the 2000/13 era (*n* = 227), 47/25% in the 2014/16 era (*n* = 100) and 59/42% in the 2017/19 era (*n* = 152) which suggests an impact of multi-modal treatment strategies on survival [14].

**Figure 2.** Illustration of 18FDG PET/CT anaplastic thyroid carcinoma (ATC) response to chemotherapy and radiotherapy before surgery. The figure represents axial (**A**) and coronal (**B**) slices of the neck region with an ATC volume of 230 mL before initiating treatment comprising chemotherapy (Cisplatin-Doxorubicin, 2 cures) and radiotherapy (IMRT, 50 Gy. (**C**,**D**) represent axial (**C**) and coronal slices (**D**) of the neck region with an ATC volume of 15 mL.

Dedicated fast-track management of these patients may offer a better chance of tumor control, as early management is key according to good practice guidance for fast-growing cancers [2,62]. Although feasible in highly specialized tertiary centers, the applicability of this fast-track management approach in multiple tertiary centers at a whole-country level remains to be demonstrated, as exemplified by the huge differences in reported survival between nationwide ATC database analysis [4,5,16] in comparison with tertiary-center database analysis [14,62–64].

Although effective in providing better OS, multimodal treatment is, in most cases, a palliative aggressive approach with risks of side effects and complications which may hamper the quality of life of the patients. Indeed, in large national series, the proportion of patients unfit for combined treatments varies from 4% (*n* = 4/100) [5] to 15% in the French network on refractory thyroid cancer (ENDOCAN-TUTHYREF) experience (unpublished data). Therefore, a review of the treatment options, risks, benefits and outcomes has to be submitted to a multidisciplinary team (including palliative caregivers and geriatric oncologist) and presented to the patient to create a shared decision-making process about a realistic treatment plan [2]. One critical issue is to clarify with the patient and his family whether tracheostomy should be performed in case of acute respiratory failure versus palliative sedation, because such a procedure would profoundly impact their ability to communicate and their quality of life until death.

Whenever possible, surgery must be performed as it can provide prolonged survival and even a cure in the 10% patients with stage IVa disease, in association with chemoradiotherapy and surgery [63,65]. It should also be performed in patients with advanced disease who may respond to initial medical/radiation treatment [2,14] (Figures 2 and 3).

#### *4.2. Radiation Therapy: Still the Mainstem of ATC Treatment*

The aggressive nature of the disease results in a high rate of local progression and recurrence [66,67] which require achieving local control with surgery, when feasible, and, more often, with EBRT. There has been a high heterogeneity of EBRT reports in the last 25 years in terms of dose administration, fractionation, techniques and combinations [26].