Exploring Theranostic Avenues in Adrenocortical Carcinoma Using Chemokine Receptor and Prostate-Specific Membrane Antigen-Directed PET/CT.

ABSTRACT: We report on an adrenocortical carcinoma (ACC) patient, which has exhausted previous treatment options and was scheduled for prostate-specific membrane antigen (PSMA)- and C-X-C motif chemokine receptor 4 (CXCR4)-targeted PET/CT. We identified PSMA-avid pulmonary metastases exhibiting modest radiotracer accumulation, while chemokine receptor PET/CT provided intense uptake. This dual-tracer molecular imaging approach revealed that chemokine receptor PET appears to be more suitable in patients with advanced ACC, indicating that CXCR4-directed radioligand therapy may be considered in such patients suffering from end-stage disease. Given its dismal prognosis, chemokine receptor-directed theranostics may therefore extend the therapeutic armamentarium as last-line option in advanced ACC.

Multiple TP53 p.R337H haplotypes and implications for tumor susceptibility.

The germline TP53 p.R337H mutation is reported as the most common germline TP53 variant. It exists at a remarkably high frequency in the population of southeast Brazil as founder mutation in two distinct haplotypes with the most frequent co-segregating with the p.E134∗ variant of the XAF1 tumor suppressor and an increased cancer risk. Founder mutations demonstrate linkage disequilibrium with neighboring genetic polymorphic markers that can be used to identify the founder variant in different geographic regions and diverse populations. We report here a shared haplotype among Brazilian, Portuguese, and Spanish families and the existence of three additional distinct TP53 p.R337H alleles. Mitochondrial DNA sequencing and Y-STR profiling of Brazilian carriers of the founder TP53 p.R337H allele reveal an excess of Native American haplogroups in maternal lineages and exclusively European haplogroups in paternal lineages, consistent with communities established through male European settlers with extensive intermarriage with Indigenous women. The identification of founder and independent TP53 p.R337H alleles underlines the importance for considering the haplotype as a functional unit and the additive effects of constitutive polymorphisms and associated variants in modifier genes that can influence the cancer phenotype.

Fluorinated aldosterone synthase (CYP11B2)-inhibitors for differential diagnosis between bilateral and unilateral conditions of primary aldosteronism.

The enzyme aldosterone synthase (CYP11B2) is specifically expressed in aldosterone-producing tissue of the adrenal cortex and is overexpressed in aldosterone-producing adenomas (APA). It therefore represents an ideal target for molecular imaging, particularly for the differential diagnosis between bilateral hyperplasia and unilateral APA in primary aldosteronism. However, the presence of the cortisol-producing enzyme 11ß-hydroxylase (CYP11B1) in the adrenal cortex remains very challenging owing to its high homology to CYP11B2. Within this study, we efficiently synthesized a variety of disubstituted fluorinated pyridines and pyrazines by Suzuki coupling reactions. These compounds were evaluated for their ability to inhibit CYP11B1 and CYP11B2 in transfected Y1 cells and in NCI-h295 cells. Several compounds were found to exhibit excellent affinity (IC50 < 10 nM) to CYP11B2 as well as strong selectivity (up to 125-fold) over CYP11B1. These findings support the further development of an analogous 18F-labelled PET tracer.

Targeting 11-Beta Hydroxylase With [131I]IMAZA: A Novel Approach for the Treatment of Advanced Adrenocortical Carcinoma.

CONTEXT: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with limited treatment options. Theranostic approaches with adrenal specific radiotracers hold promise for improved diagnostics and treatment. OBJECTIVE: Here, we report a new theranostic approach to advanced ACC applying (R)-1-[1-(4-[123I]iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinyl amide ([123I]IMAZA) for diagnostic imaging and [131I]IMAZA for radionuclide therapy. METHODS: Sixty-nine patients with nonresectable, metastatic ACCs were screened using a diagnostic [123I]IMAZA scan. Patients with significant uptake in all tumoral lesions were offered treatment with [131I]IMAZA. Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST version 1.1), and adverse effects were assessed by Common Toxicity Criteria (version 5.0). RESULTS: After screening, 13 patients were treated with a median of 25.7 GBq [131I]IMAZA (range 18.1-30.7 GBq). Five individuals received a second treatment course. Best response was a decrease in the RECIST target lesions of -26% in 2 patients. Five patients with disease stabilization experienced a median progression-free survival of 14.3 months (range 8.3-21.9). Median overall survival in all patients was 14.1 months (4.0-56.5) after therapy. Treatment was well tolerated, in other words no severe toxicities (CTCAE grade ≥3) were observed. CONCLUSION: In patients with advanced ACC refractory to standard therapeutic regimens, [131I]IMAZA treatment was associated with disease stabilization and nonsignificant tumor size reduction in a significant patient fraction and only limited toxicities. High [131I]IMAZA-uptake in tumor lesions was observed in 38.5% of patients with advanced ACC, rendering [131I] IMAZA a potential treatment option in a limited, well-defined patient fraction. Further clinical trials will be necessary to evaluate the full potential of this novel theranostic approach.

Expression of the Chemokine Receptor CCR7 in the Normal Adrenal Gland and Adrenal Tumors and Its Correlation with Clinical Outcome in Adrenocortical Carcinoma.

BACKGROUND: The chemokine receptor CCR7 is crucial for an intact immune function, but its expression is also associated with clinical outcome in several malignancies. No data exist on the expression of CCR7 in adrenocortical tumors. METHODS: CCR7 expression was investigated by qRT-PCR and immunohistochemistry in 4 normal adrenal glands, 59 adrenocortical adenomas, and 181 adrenocortical carcinoma (ACC) samples. RESULTS: CCR7 is highly expressed in the outer adrenocortical zones and medulla. Aldosterone-producing adenomas showed lower CCR7 protein levels (H-score 1.3 ± 1.0) compared to non-functioning (2.4 ± 0.5) and cortisol-producing adenomas (2.3 ± 0.6), whereas protein expression was variable in ACC (1.8 ± 0.8). In ACC, CCR7 protein expression was significantly higher in lymph node metastases (2.5 ± 0.5) compared to primary tumors (1.8±0.8) or distant metastases (2.0 ± 0.4; p < 0.01). mRNA levels of CCR7 were not significantly different between ACCs, normal adrenals, and adrenocortical adenomas. In contrast to other tumor entities, neither CCR7 protein nor mRNA expression significantly impacted patients' survival. CONCLUSION: We show that CCR7 is expressed on mRNA and protein level across normal adrenals, benign adrenocortical tumors, as well as ACCs. Given that CCR7 did not influence survival in ACC, it is probably not involved in tumor progression, but it could play a role in adrenocortical homeostasis.

Novel CYP11B-ligand [123/131I]IMAZA as promising theranostic tool for adrenocortical tumors: comprehensive preclinical characterization and first clinical experience.

PURPOSE: Adrenal tumors represent a diagnostic and therapeutic challenge. Promising results have been obtained through targeting the cytochrome P450 enzymes CYP11B1 and CYP11B2 for molecular imaging, and [123/131I]iodometomidate ([123/131I]IMTO) has even been successfully introduced as a theranostic agent. As this radiopharmaceutical shows rapid metabolic inactivation, we aimed at developing new improved tracers. METHODS: Several IMTO derivatives were newly designed by replacing the unstable methyl ester by different carboxylic esters or amides. The inhibition of aldosterone and cortisol synthesis was tested in different adrenocortical cell lines. The corresponding radiolabeled compounds were assessed regarding their stability, in vitro cell uptake, in vivo biodistribution in mice, and their binding specificity to cryosections of human adrenocortical and non-adrenocortical tissue. Furthermore, a first investigation was performed in patients with known metastatic adrenal cancer using both [123I]IMTO and the most promising compound (R)-1-[1-(4-[123I]iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide ([123I]IMAZA) for scintigraphy. Subsequently, a first endoradiotherapy with [131I]IMAZA in one of these patients was performed. RESULTS: We identified three analogues to IMTO with high-affinity binding to the target enzymes and comparable or higher metabolic stability and very high and specific accumulation in adrenocortical cells in vitro and in vivo. Labeled IMAZA exhibited superior pharmacokinetic and imaging properties compared to IMTO in mice and 3 patients, too. An endoradiotherapy with [131I]IMAZA induced a 21-month progression-free interval in a patient with rapidly progressing ACC prior this therapy. CONCLUSION: We developed the new radiopharmaceutical [123/131I]IMAZA with superior properties compared to the reference compound IMTO and promising first experiences in humans.

Impact of the Chemokine Receptors CXCR4 and CXCR7 on Clinical Outcome in Adrenocortical Carcinoma.

Chemokine receptors have a negative impact on tumor progression in several human cancers and have therefore been of interest for molecular imaging and targeted therapy. However, their clinical and prognostic significance in adrenocortical carcinoma (ACC) is unknown. The aim of this study was to evaluate the chemokine receptor profile in ACC and to analyse its association with clinicopathological characteristics and clinical outcome. A chemokine receptor profile was initially evaluated by quantitative PCR in 4 normal adrenals, 18 ACC samples and human ACC cell line NCI-H295. High expression of CXCR4 and CXCR7 in both healthy and malignant adrenal tissue and ACC cells was confirmed. In the next step, we analyzed the expression and cellular localization of CXCR4 and CXCR7 in ACC by immunohistochemistry in 187 and 84 samples, respectively. These results were correlated with clinicopathological parameters and survival outcome. We detected strong membrane expression of CXCR4 and CXCR7 in 50% of ACC samples. Strong cytoplasmic CXCR4 staining was more frequent among samples derived from metastases compared to primaries (p=0.01) and local recurrences (p=0.04). CXCR4 membrane staining positively correlated with proliferation index Ki67 (r=0.17, p=0.028). CXCR7 membrane staining negatively correlated with Ki67 (r=-0.254, p=0.03) but positively with tumor size (r=0.3, p=0.02). No differences in progression-free or overall survival were observed between patients with strong and weak staining intensities for CXCR4 or CXCR7. Taken together, high expression of CXCR4 and CXCR7 in both local tumors and metastases suggests that some ACC patients might benefit from CXCR4/CXCR7-targeted therapy.

Targeting CXCR4 (CXC Chemokine Receptor Type 4) for Molecular Imaging of Aldosterone-Producing Adenoma.

Primary aldosteronism is the most frequent cause of secondary hypertension and is associated with increased morbidity and mortality compared with hypertensive controls. The central diagnostic challenge is the differentiation between bilateral and unilateral disease, which determines treatment options. Bilateral adrenal venous sampling, currently recommended for differential diagnosis, is an invasive procedure with several drawbacks, making it desirable to develop novel noninvasive diagnostic tools. When investigating the expression pattern of chemokine receptors by quantitative real-time polymerase chain reaction and immunohistochemistry, we observed high expression of CXCR4 (CXC chemokine receptor type 4) in aldosterone-producing tissue in normal adrenals, adjacent adrenal cortex from adrenocortical adenomas, and in aldosterone-producing adenomas (APA), correlating strongly with the expression of CYP11B2 (aldosterone synthase). In contrast, CXCR4 was not detected in the majority of nonfunctioning adenomas that are frequently found coincidently. The specific CXCR4 ligand 68Ga-pentixafor has recently been established as radiotracer for molecular imaging of CXCR4 expression and showed strong and specific binding to cryosections of APAs in our study. We further investigated 9 patients with primary aldosteronism because of APA by 68Ga-pentixafor-positron emission tomography. The tracer uptake was significantly higher on the side of increased adrenocortical aldosterone secretion in patients with APAs compared with patients investigated by 68Ga-pentixafor-positron emission tomography for other causes. Molecular imaging of aldosterone-producing tissue by a CXCR4-specific ligand may, therefore, be a highly promising tool for noninvasive characterization of patients with APAs.

Investigating the Chemokine Receptor 4 as Potential Theranostic Target in Adrenocortical Cancer Patients.

PURPOSE: Adrenocortical carcinoma (ACC) is a rare but aggressive endocrine tumor with limited treatment options. Preclinical studies confirmed overexpression of the chemokine receptor 4 (CXCR4) in this cancer type. This study aimed to analyze the role of CXCR4 imaging using Ga-pentixafor for ACC staging and selection of patients for CXCR4-directed endoradiotherapy. METHODS: Thirty patients with histologically proven advanced, metastasized ACC underwent F-FDG PET/CT and Ga-pentixafor PET/CT within a time interval of 3 ± 4 days to evaluate suitability for CXCR4-directed endoradiotherapy. Scans were analyzed retrospectively for visual extent of ACC and SUVmax/mean of the tumor lesions. Ga-pentixafor PET was compared with F-FDG PET, the reference imaging standard. All patients were rated for suitability of CXCR4-directed endoradiotherapy considering patient's history, previous treatment, and CXCR4 expression of FDG-positive lesions compared with background activity within the same organ. RESULTS: All patients had lesions that were positive for both F-FDG and Ga-pentixafor PET and were rated as positive for disease. In 2 patients (7%), Ga-pentixafor PET identified more lesions compared with F-FDG PET. In 5 patients (17%) and 10 patients (33%), complementary and comparable information, respectively, was provided by dual-tracer imaging. In 13 patients (43%), more tumor lesions were identified by F-FDG PET compared with Ga-pentixafor PET. The F-FDG uptake of the malignant lesions was significantly higher (P < 0.01) than the SUVmax/mean for Ga-pentixafor. Overall, 70% of the patients were rated as suitable or potentially suitable for CXCR4-directed treatment. CONCLUSIONS: Ga-pentixafor allows in vivo imaging of CXCR4 expression in patients with advanced ACC and may serve as companion diagnostic tool in selecting patients for potential CXCR4-directed endoradiotherapy. Seventy percent of the patients with advanced, metastasized ACC may be suitable for a CXCR4-directed treatment after failure of standard treatment options.

Less common genotype variants of TP53 polymorphisms are associated with poor outcome in adult patients with adrenocortical carcinoma.

CONTEXT: The Li-Fraumeni tumor syndrome is strongly associated with adrenocortical carcinoma (ACC) and is caused by germline mutations in TP53 in 70% of cases. Also, TP53 polymorphisms have been shown to influence both cancer risk and clinical outcome in several tumor entities. We, therefore, investigated TP53 polymorphisms in a cohort of adult patients with ACC. OBJECTIVE: Evaluation of the role of TP53 polymorphisms in adult patients with ACC. SUBJECTS AND METHODS: Peripheral blood for DNA extraction was collected from 72 ACC patients. Polymorphism analysis was carried out by amplification and sequencing of exons and adjacent intron sections of TP53. Results were correlated with clinical data and the distribution of the polymorphisms was compared with published Caucasian control groups. RESULTS: Compared with control groups, genotype frequencies of analyzed TP53 polymorphisms among ACC patients were significantly different in three out of four polymorphisms: IVS2+38G>C (G/G, P=0.0248), IVS3ins16 (NoIns/NoIns, P<0.0001; NoIns/Ins, P<0.0001), and IVS6+62A>G (G/G, P<0.0001; G/A, P<0.0001). Overall, the survival of ACC patients, which harbored at least one of the less frequent genotype variants of four analyzed polymorphisms (n=23), was significantly inferior (median survival: 81.0 months in patients with the common homozygous genotypes vs 20.0 months in patients with the less frequent genotypes, HR 2.56, 95% CI 1.66-7.07; P=0.001). These results were confirmed by multivariable regression analysis (HR 2.84, 95% CI 1.52-7.17; P=0.037). CONCLUSION: Some TP53 polymorphisms seem to influence overall survival in ACC patients. This effect was observed for a combination of polymorphic changes rather than for single polymorphisms.

TP53 germline mutations in adult patients with adrenocortical carcinoma.

CONTEXT: Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome associated with germline mutations in TP53. According to the Chompret criteria for LFS, any patient with adrenocortical cancer (ACC), irrespective of age and family history, is at high risk for a TP53 germline mutation. However, whereas such mutations have been detected with high frequency in childhood ACC, a large cohort of adult patients with ACC has never been investigated for TP53 germline mutations. OBJECTIVE: The aim of the study was to evaluate the prevalence of TP53 germline mutations in adult patients with ACC. SUBJECTS AND METHODS: In 103 adult Caucasian patients with ACC, TP53 germline mutation analysis was performed. In patients with a TP53 germline mutation, tumor tissue was analyzed for loss of heterozygosity of TP53 and p53 immunohistochemistry. Family history and clinical course were also evaluated. RESULTS: In four patients, a total of five TP53 germline mutations were found. Two mutations occurred in exon 10 (R337H and I332M, respectively), outside the hot spot region. Here, three mutations are described for the first time in ACC, and one, which occurred combined with a second mutation (R202C) on the same allele, has never been reported before in the context of LFS. This combined mutation was associated with a remarkable family history of ACC also affecting the mother and uncle of the index patient. In the 23 patients with ACC below the age of 40 yr, 13% (95% confidence interval, 3.7-32.9%) carried a TP53 germline mutation, whereas such mutations were rare in older patients with ACC. CONCLUSION: Our findings indicate a need to revise the Chompret criteria. However, in younger adults (<40 yr old) with ACC, screening for TP53 germline mutations may be justified.