CXCR4-directed PET/CT with [⁶⁸ Ga]Ga-pentixafor in solid tumors—a comprehensive analysis of imaging findings and comparison with histopathology

C-X-C motif chemokine receptor 4 (CXCR4) is a transmembrane G-protein-coupled receptor crucially involved in tumor dissemination of varying malignancies [1, 2]. In this regard, analyses of tumor specimens have already demonstrated a substantial upregulation of this receptor subtype, which then triggered the use of the CXCR4-directed PET agent [⁶⁸ Ga]Ga-pentixafor [3‐5]. Those molecular imaging studies have revealed substantial uptake, including elevated maximum standardized uptake value (SUV_max) or excellent image contrast (provided by increased target-to-background ratios [TBR]), particularly in hematological malignancies [6].

Beyond lymphoma or leukemia, increased chemokine receptor 4 expression on solid tumors was also linked to less favorable outcome, including ovarian, hepatocellular, neuroendocrine, or cholangiocarcinoma [7‐9]. Previous studies on CXCR4-targeted PET/CT, however, included only a limited number of patients or scans. For instance, Vag et al. [10] reported on 21 subjects and revealed a discrepancy between the chemokine receptor profile observed in-vitro when compared to in-vivo findings provided by [⁶⁸ Ga]Ga-pentixafor PET. A recent analysis enrolling 19 subjects with different solid cancers also showed that [⁶⁸ Ga]Ga-pentixafor provided fluctuating uptake depending on the analyzed subtype [11]. Last, a recent study investigated a larger number of subjects, but only focused on a “hottest lesion” analysis (i.e., the target lesion [TL] with most intense uptake), while other PET-positive TL were not included [6].

In addition to investigations focusing on diagnosis, CXCR4-targeted PET/CT can be conducted to assess patient’s suitability for CXCR4-targeted therapies in a theranostic approach, e.g., radioligand therapy (RLT) or “cold” inhibitory drugs [12, 13]. CXCR4-directed RLT has already been applied to various hematological tumor entities [14‐16]. Of note, a recent study demonstrated, for the first time, that this approach is also feasible in solid cancers by treating patients with desmoplastic small round cell tumor (DSRCT) [17]. Nevertheless, CXCR4-RLT may be limited to aggressive disease with virtually no other treatment options, given that CXCR4-directed “hot” treatment is associated with myeloablative effects due to CXCR4 expression on hematopoietic stem cells [18].

In the present investigation, we aimed to provide a comprehensive overview of intensity of uptake in 142 patients with 23 different kinds of solid cancers on 152 [⁶⁸ Ga]Ga-pentixafor PET/CTs. For this purpose, we manually segmented the whole-body, chemokine receptor-avid tumor burden, which then allowed to sort tumor entities based on intensity of uptake. Regarding myeloablative effects of CXCR4-directed RLT, we additionally performed quantitative analyses of bone marrow (BM) receptor expression for the different entities. Furthermore, we assessed previous lines of therapy and concurrent diseases to examine their influence on tumor uptake and BM CXCR4-expression. We also conducted a correlation analysis between radiotracer accumulation and ex-vivo findings, including immunohistochemical staining of CXCR4-expression. Moreover, we aimed to determine patients eligible for a therapeutic approach based on the number of PET-positive TL and intensity of [⁶⁸ Ga]Ga-pentixafor uptake in sites of disease.

Investigating the largest cohort of patients with solid cancers imaged with [⁶⁸ Ga]Ga-pentixafor PET/CT to date, CXCR4-positive tumor burden was identified in more than two-thirds of the patients, along with high image contrast indicative for good read-out capabilities. Conducting a comprehensive tumor assessment, most intense uptake was observed in SCLC, DSRCT, and ACC, when investigating entities with a substantial number of examined lesions in our study. Assessing BM uptake in our cohort, no significant difference based on tumor subtypes could be identified, although those results may be potentially hampered for some of the entities due to the small number of subjects. When comparing chemokine receptor expression between primary lesions and metastases, no relevant differences in uptake could be determined (except for lung lesions). Furthermore, we observed no significant difference in tumor uptake when comparing patients with different therapies or concurrent diseases, whereas CXCR4 expression in the BM was significantly lower in patients having received prior CTx or RTx. Correlating multiple SUV parameters with immunohistochemically acquired IRS, a respective link between intensity of uptake and histopathologic findings was recorded, supporting the notion that [⁶⁸ Ga]Ga-pentixafor PET achieves high specificity for the target. Last, more than 40% revealed a minimum of five target lesions positive on molecular imaging. Among such subjects with widespread disease, more than 28% also exhibited substantially high uptake levels, thereby potentially rendering those individuals eligible for “hot” or “cold” CXCR4 treatment.

Previous studies have already provided evidence that CXCR4 emerges as a suitable target for imaging and therapy in multiple solid tumors, including ACC, ovarian cancer, neuroendocrine tumors, or SCLC [9, 30‐32]. Of note, those ex-vivo findings were further corroborated by our in-vivo imaging approach. First, our comprehensive tumor assessment yielded a substantially high median TBR > 4 when regarding TL from all tumor entities, indicative for an excellent image contrast when compared to physiological background activity. Second, we observed uniform and high receptor density on the primary and distant sites of disease in varying different cancer entities, except for lung lesions, although this exception could conceivably be explained by the limited spatial resolution of small nodules. Altogether, this finding could potentially indicate that CXCR4 RLT or “cold” chemokine receptor-mediated therapeutic approaches would most likely exert anti-tumor effects in all sites of disease throughout the body [12, 13]. Third, we also observed a correlation between different SUV parameters and IRS, thereby indicating that the PET signal provided by [⁶⁸ Ga]Ga-pentixafor accurately reflects the chemokine receptor density on targeted tumor cells. In this regard, a previous analysis including a limited number of patients has also reported on an association between ex- and in-vivo findings after injection of this radiopharmaceutical [11]. In this study, we further elaborated on those preliminary findings by investigating more than 50 subjects with available histology. Of note, the observed association applied to both SUV_peak and SUV_max, which are both independent from lesion size [33].

CXCR4-targeted RLT has already achieved durable response including partial or complete remission in patients with hematological malignancies [14, 15]. Our results, which revealed substantial tracer uptake in several entities and no significant difference in tumor uptake after different lines of therapy, support the notion that CXCR4-directed RLT could represent a viable option for extensively pre-treated patients in solid tumor entities. Nonetheless, such an approach would require stem cell backup because of its myeloablative effects, as CXCR4 is also part of the hematopoietic stem cell niche, thereby limiting this theranostic concept to patients that per se are in need of hematopoietic stem cell transplantation [34]. In patients with solid cancers, however, this would then be seen as major side effect, i.e., only suitable in a salvage setting in highly aggressive disease [34]. Additionally, the overall rate of patients suitable for RLT according to the applied criteria in our study was overall only 12/152 (8%). Despite these two rather unfavorable aspects regarding the practicability of CXCR4-RLT in solid tumors, one has to consider that a substantial portion of patients were diagnosed with difficult-to-treat tumor entities, including ACC. This tumor entity has a dismal prognosis [35] and even widely used therapeutic regimen has recently demonstrated heterogeneous outcome in selected cases, thereby emphasizing the urgent need for novel therapeutic approaches in those patients [36]. In this regard, a previous immunohistochemical-centered publication has already reported on substantial CXCR4 expression in half of the subjects, along with a relevant association of chemokine receptor upregulation with proliferation indices [37]. In line with those ex-vivo findings, another report investigating uptake on chemokine receptor PET in patients with ACC has also shown that up to 57% of the patients would have been eligible for CXCR4-RLT [21]. The present study indicates a lower number of patients suitable for treatment. As a possible explanation, the predefined criteria for (hypothetical) eligibility were rather strict in our study and less rigid predefinitions such as lower SUV_max may have led to a larger number of individuals suitable for CXCR4-targeted therapy. Furthermore, current clinical trials are investigating the appropriate amount of radiotherapeutic activity to achieve anti-tumor efficacy without exerting myeloablation (PTT101, EudraCT No. 2021–002364-43). Our finding of reduced BM tracer uptake in patients who had previously undergone CTx or RTx could also be of relevance in this regard. However, further investigation of the connection between pre-therapy uptake of the diagnostic PET tracer [⁶⁸ Ga]Ga-pentixafor and myeloablative effects of CXCR4-directed RLT is needed. Nevertheless, once a desired activity range has been identified, the current study may provide a rationale to expand chemokine receptor theranostics beyond hematological malignancies toward solid tumors, which are currently not adequately covered by nuclear oncology. In this regard, based on our preliminary imaging findings, solid tumor entities including ACC, neuroendocrine neoplasms, and SCLC exhibited increased uptake with a relevant number of TL, potentially rendering those tumor entities eligible for RLT. Pleural mesothelioma, mediastinal tumor, Ewing-sarcoma, or osteosarcoma, however, may then rather not be well suited for treatment.

This study has limitations, including its retrospective design and limited number of patients for some of the examined tumor entities. Furthermore, we observed a high per-tumor variability in our study. Nonetheless, we herein report on the largest cohort of patients with solid tumors imaged with CXCR4-directed PET, along with substantial correlative indices between ex- and in-vivo findings. As such, the herein provided overview of subjects with solid cancers may therefore provide a roadmap to identify subjects eligible for CXCR4-directed imaging and therapy.

In the largest cohort of subjects affected with solid cancers and imaged with [⁶⁸ Ga]Ga-pentixafor PET to date, we observed elevated CXCR4 expression (provided by SUV_max) and good image contrast (indicated by TBR) in two-thirds of the patients. In addition, ex-vivo chemokine receptor levels derived from IHC were linked to in-vivo uptake, suggesting that the PET signal indeed provides a read-out of the receptor density on the tumor cell surface. Of note, in-vivo chemokine receptor levels seem to be uniformly expressed, as no relevant uptake differences in the primary and metastases (with the exception of lung metastases) were observed, indicating that CXCR4-directed RLT would then exert anti-tumor effects in all lesions attributable to the underlying disease. As such, our preliminary findings may therefore provide a roadmap to identify subjects affected with solid tumors eligible for CXCR4-directed imaging and therapy.