[68Ga]Pentixafor-PET/CT for imaging of chemokine receptor 4 expression in small cell lung cancer - initial experience

Chemokine receptor CXCR4 is a key factor for tumor growth and metastasis in several types of human cancer. This study investigated the feasibility of CXCR4-directed imaging of small cell lung cancer (SCLC) with positron emission tomography/computed tomography (PET/CT) using the radiolabelled chemokine ligand [68Ga]Pentixafor. 10 patients with primarily diagnosed (n=3) or pre-treated (n=7) SCLC (n=9) or large cell neuroendocrine carcinoma of the lung (LCNEC, n=1) underwent [68Ga]Pentixafor-PET/CT. 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG, n=6) and/or somatostatin receptor (SSTR)-directed PET/CT with [68Ga]DOTATOC (n=5) and immunohistochemistry (n=10) served as standards of reference. CXCR4-PET was positive in 8/10 patients and revealed more lesions with significantly higher tumor-to-background ratios than SSTR-PET. Two patients who were positive on [18F]FDG-PET were missed by CXCR4-PET, in the remainder [68Ga]Pentixafor detected an equal (n=2) or higher (n=2) number of lesions. CXCR4 expression of tumor lesions could be confirmed by immunohistochemistry. Non-invasive imaging of CXCR4 expression in SCLC is feasible. [68Ga]Pentixafor as a novel PET tracer might serve as readout for confirmation of CXCR4 expression as prerequisite for potential CXCR4-directed treatment including receptor-radio(drug)peptide therapy.


INTRODUCTION
Small cell lung cancer (SCLC) is a neuroendocrine tumor that represents 15% of all lung cancers [1]. It occurs predominantly in smokers as almost all patients have a history of tobacco use.
SCLC is distinguished clinically from most other types of non-small cell lung cancer by its rapid doubling time, high growth fraction, and the early development of metastases. Only one-third of patients are diagnosed with localized disease, and treatment strategies have focused on systemic therapy [2]. Although SCLC is highly responsive to both chemotherapy and radiotherapy, it commonly relapses within months despite treatment [3,4]. Response rates to second-line treatments have been reported to range between 10-20% [5]. Thus, new treatment options www.impactjournals.com/oncotarget including personalized medicine targeting specific molecular markers are urgently needed.
Chemokine receptor CXCR4 has been described to play a pivotal role in tumor growth and progression, tumor invasiveness and metastasis [6]. Overexpression of the receptor has been reported in more than 30 different types of cancer, including lymphoma, breast, pancreatic, ovarian and lung cancer [7][8][9][10]. In SCLC, almost ubiquitous CXCR4 overexpression has been shown to correlate with negative outcome [11].
This is the first report of non-invasive detection of CXCR4-expression in SCLC patients. CXCR4 may serve as a promising new target for both diagnostic and therapeutic interventions, especially in selecting potential candidates for endoradiotherapy.
In comparison to [ 18  On semi-quantitative analysis, the median SUV mean of the primary tumors was 6.9 (range, 2.6-11.3) and the median SUV max was 8

DISCUSSION
This is the first report of in vivo imaging of CXCR4 expression in humans with both newly diagnosed as well as pre-treated, recurrent SCLC. A recent report evaluating biopsy samples of bronchopulmonary neuroendocrine tumors demonstrated a high intensity of CXCR4 receptor expression in SCLC. Additionally, chemokine receptor expression was a predictor of poor overall survival [11]. In concordance, moderate to high receptor density on the cell surface was visualized by PET/CT in our cohort in the vast majority of cases.
Of note, in comparison to [ 18 F]FDG-PET/CT as reference, almost all tumor lesions proved to be CXCR4-positive with high tumor-to-background ratios, thus rendering CXCR4 a promising target for endoradiotherapy. Given the high relapse rate of SCLC after 1 st line chemotherapy as well as the modest response rates to subsequent treatments, a new approach to the patient with relapsed/refractory SCLC is urgently needed. In addition to conventional chemotherapeutic agents including anthracycline-based regimes, topotecan or amrubicin [19], peptide receptor radionuclide therapy using radiolabelled somatostatin receptor ligands has been performed with rather modest success rates [20,21]. Important prerequisites to receptor-targeted therapies are a robust expression of the target receptor as well as a specific receptor binding. In our patient cohort, CXCR4-PET/CT clearly outperformed SSTR-PET on a patient as well as a lesion basis, underlining the potential superiority of CXCR4 as a therapeutic target compared to SSTR.
Recently, a derivative of the diagnostic compound allowing labelling with various α-and β-emitters called Pentixather has been developed. Proof-of-concept for endoradiotherapy could be demonstrated in patients with advanced multiple myeloma with partial and complete metabolic responses [22]. Thus, further assessment of this promising tool in a theranostic approach is warranted.
This pilot study has several limitations. First, only a limited number of patients could be included in the study. Second, biopsies were not always obtained on a short-term period compared to the time point of PET imaging and re-biopsies could not always be acquired.
[ 68 Ga]Pentixafor uptake did not seem to correlate with histological receptor expression, maybe due to receptor kinetics and internalization. Of note, receptor surface expression of CXCR4 seems to be a dynamic process which is influenced by a number of factors including therapeutic agents. The patient demonstrating the highest CXCR4 expression in her sample analyzed (patient #6) presented with a negative [ 68 Ga]Pentixafor PET after concurrent 1 st line chemotherapy. In addition, another patient (patient #2) with recently diagnosed SCLC also did not exhibit high receptor expression on PET after initiation of combined radio-chemotherapy two weeks earlier. Therefore, one might speculate that surface expression of CXCR4 is downregulated as a response to treatment in a time and dose dependent manner. In line with this observation is the fact that another subject (patient #5) showed intermediate SUV and no immunohistochemical CXCR4-positivity after a nine-day-duration of chemotherapy. While high expression of CXCR4 in SCLC has recently been demonstrated [11], future studies to further investigate therapy-induced down-and -preferablyup-regulation of CXCR4 are warranted. Potentially a sequential combination with chemotherapeutic agents with might lead to improved efficacy of CXCR4-directed endoradiotherapy. All patients with relapsed disease had undergone 1 st -and/or 2 nd -line treatment (patients #1, #3, #9). 1 st -line treatment included 2-6 cycles of platinum/etoposide-containing chemotherapy (patient #1: 2 cycles, patients #3 and #9: 4 cycles, patients #4, #6, #7 and #8: 6 cycles) (+radiotherapy), 2 nd -line therapy had topotecan as the mainstay (all three patients [#1, #3 and #9] received 6 cycles). Bx = biopsy; CTx = chemotherapy; IRS = immunoreactive score; LCNEC = large cell neuroendocrine carcinoma of the lung; LN = lymph node; N/A = not assessed; PD= primary diagnosis; RCTx = radio-chemotherapy; SCLC= small cell lung cancer; Tx = treatment.
In conclusion, our data demonstrate the feasibility of [ 68 Ga]Pentixafor for PET imaging of CXCR4 chemokine receptor expression in SCLC patients. This novel PET tracer might serve as an innovative imaging agent for in vivo biomarker identification that could result in patient selection for CXCR4-directed treatment, and, eventually, for receptor-radio(drug)peptide therapy. Patients` characteristics are given in more detail in Table  1.

Subjects and study design
[ 68 Ga]Pentixafor was administered on a compassionate use basis in compliance with §37 of the Declaration of Helsinki and The German Medicinal Products Act, AMG §13 2b and in accordance with the responsible regulatory body (Regierung von Oberfranken). The study adhered to the standards established in the declaration of Helsinki. All patients gave written informed consent prior to imaging.

Imaging
[ 68 Ga]Pentixafor was prepared as previously described [23]. In short, all syntheses were performed in a fully automated, GMP-compliant procedure using a GRP ® module (SCINTOMICS GmbH, Germany) equipped with a disposable single-use cassette kit (ABX, Germany). The eluate ( 68 Ga 3+ in 0.6 M HCl) of a 68 Ge/ 68 Ga-generator (iThemba Labs, Faure, South Africa) was transferred to a cation exchange cartridge, eluted with 5 N NaCl, added to a solution of 40 µg Pentixafor (Scintomics, Fürstenfeldbruck, Germany) in HEPES-buffer and heated for 6 minutes at 125°C. The product was immobilized on a SepPak C18-cartridge, washed with water und eluted with ethanol/water 50/50. The eluate was passed through a sterile filter (0.22 µm) into a sterile vial und diluted with phosphate buffer solution to a total volume of 15 mL. Radiochemical purity was determined by gradient high performance liquid chromatography and thin-layer chromatography. Additionally, the product was also tested for ethanol content, pH, radionuclide purity, sterility, and

Image analysis
All PET scans were first visually rated by a boardcertified nuclear medicine physician in a binary visual fashion as positive for disease or negative for disease. Semi-quantitative analysis was performed for the primary as well as the hottest metastatic lesion. The axial PET image slice displaying the maximum tumor uptake was selected by drawing a 3D-volume of interest (VOI) around the whole tumor area. Tumor regions of interest (ROIs) were defined in 2 ways. First, a standardized 10mm circular region was placed over the area with the peak activity. This first ROI was used to derive maximum (SUV max ) and mean standardized uptake values (SUV mean ). A reference region was defined by drawing a ROI (diameter of 50 mm) involving normal liver parenchyma to derive tumor-to-background ratios. Both primary-tobackground (P/B) as well as metastasis-to-background (M/B) ratios for SUV max and SUV mean were calculated. The radiotracer concentration in the ROIs was normalized to the injected dose per kilogram of patient's body weight to derive the SUVs.

Immunohistochemistry
Immunohistochemistry was carried out on 10% formalin fixed paraffin embedded tissue sections (3µm) according to established protocols and scored as described previously (13). CXCR4-immunohistochemistry was performed using an anti-CXCR4 rabbit polyclonal antibody (ab2074; Abcam, Cambridge, UK) followed by detection with the DAKO en vision system according to the manufacturer′s protocol.

Statistical analysis
Most of the data are descriptive. Statistical analyses were performed using IBM SPSS (version 22.0; SPSS, Inc. Chicago, IL). Quantitative values were expressed as mean ± standard deviation or median and range as appropriate. Comparisons of related metric measurements were performed using Mann-Whitney-U test. A p-value < 0.05 was considered to indicate statistical significance.