Angiogenesis Imaging Study Using [18F] RGD-K5 PET/CT in Patients with Lymphoma Under Chemotherapy.

Background : Our aim was to measure the impact of two cycles of standard chemotherapy on tumoral neoangiogenesis by [ 18 F] Fluorine arginine-glycine-aspartic (RGD-K5) Positron Emission Tomography – Computed Tomography (PET) on patients presenting with lymphoma. Twenty one patients were prospectively included in Rouen’s Henri Becquerel Cancer Center. Fluorodeoxyglucose (FDG) and RGD-K5 PET were performed before (C0) and after (C2) two cycles of chemotherapy. End of treatment FDG PET was performed for nal staging. Maximum Standardized Uptake Value (SUVmax), Mean SUV and Metabolic Tumor Volume (MTV) were measured for all lesions. RGD SUVmax and meanSUV were also analysed in 13 normal organs at C0 and C2. Patient’s treatment response was considered using Deauville score (DS) on end of treatment FDG PET. (DS 1 to 3 were considered as responders, DS 4 and 5 non-responders). Results : Nineteen patients had both C0 FDG and C0 RGD PET. Twelve patients had both C2 FDG and C2 RGD, completed treatment protocol and were included for end of treatment analysis. No statistical difference was found on normal organs RGD uptake before and after chemotherapy for SUVmax and SUVmean. On C0 RGD, apart from Classical Hodgkin lymphoma (cHL)and Grey Zone Lymphoma (GZL), other lymphoma sub-types had low RGD uptake (p<0.001). Regarding FDG, there was no signicant difference for SUVmax, SUVmean and MTV on C0 and C2 between patients with cHL and non Hodgkin lymphoma (NHL)... On C2 RGD, non-responder patients had higher SUVmax, and SUVmean compared to responder patients (p<0.001). There was no signicant difference for RGD MTV between responder and non-responder patients. Conclusions : Our study showed signicant higher initial RGD uptake in patients presenting of standard chemotherapy on tumoral neoangiogenesis assessed by RGD-K5 PET/CT on patients presenting with lymphoma. The secondary objective of this study was to analyze RGD uptake according to patient’s lymphoma subtype.

response assessment, especially in patients with lymphoma (2). Integrin αvβ3 is a heterodimeric transmembrane glycoproteins consisting of an α-and β-subunit which plays an important role in angiogenesis (3). They interact in cell-cell-and cell-matrix-interactions, easing endothelial growth and cell migration, and therefore paving the way to angiogenesis (4). In tumors, integrins assist the progress of tumor development and tumor metastasis by facilitating endothelial and tumor cell migration. An important phenomenon that depends on cell-extracellular matrix (ECM) interactions is the growth or sprouting of new blood vessels from a pre-existing vascular bed (4). It has been found that several ECM proteins like vitronectin, brinogen and bronectin interact with integrins via the amino acid sequence arginine-glycine-aspartic acid or RGD in the single letter code (3). Based on these ndings, monomeric, multimeric and cyclic peptides including the RGD sequence have been introduced to allow integrin αvβ3 imaging, and to picture pathological angiogenesis. Targeting speci c angiogenesis molecular markers by PET imaging, like the integrin αvβ3, might be used for angiogenesis imaging. Also, L. Li et al., recently showed that high RGD uptake on pre-treatment PET predicted antiangiogenic response in refractory patients presenting with solid cancer (5). Patients presenting with solid tumors which had low RGD uptake did not bene t from antiangiogenic effect. Patients presenting why high RGD uptake bene ted from antiangiogenic effect. Therefore, RGD imaging could also be used for response assessment of antiangiogenic therapies.
RGD-K5 PET/CT has been used in clinical studies on patients presenting with head and neck cancer (6), breast cancer (7) and lung cancer (8). To the best of our knowledge, no previous clinical imaging study of angiogenesis in patients presenting with lymphoma has been performed.
The main objective of this study was to measure the impact of two cycles of standard chemotherapy on tumoral neoangiogenesis assessed by RGD-K5 PET/CT on patients presenting with lymphoma.
The secondary objective of this study was to analyze RGD uptake according to patient's lymphoma subtype.

2-Methods a) Population and treatment
This prospective study included 21 patients between July 2016 and November 2019 in Rouen's Henri Becquerel Cancer Centre presenting with biopsy-proven lymphoma. To avoid partial volume effect on RGD PET, only patients with a target lesion of at least 3 cm on CT were included. All patients were treated according to standard routine practice and national guidelines and to their histological subtype. Patients with indolent lymphomas and no treatment criteria may not receive treatment. All patients were staged using FDG PET/CT (FDG). Additional RGD-K5 PET/CT (RGD) was performed before or after initial and intermediate FDG (median +7 days, range -4; +22 for initial FDG and median -0.5 days, range -5; +1 for intermediate FDG). Intermediate imaging using FDG and RGD was performed after two cycles of chemotherapy for patients receiving treatment. No additional treatment was performed between FDG and RGD imaging. All patients had nal FDG for nal Deauville score staging at end of treatment. Patients with nal Deauville score of 4 or 5 were considered non-responders and nal Deauville score of 1, 2 or 3 as responders (9). All participants were followed at least 12 months after end of treatment. The study was conducted in accordance with the precepts of the Helsinki declaration and received approval by the Ethical Committee. All patients gave written consent for the study. Sixty minutes later (±10 min), the acquisition began with non-injected CT in the cephalocaudal direction on a General Electric 710 PET/CT (Buc, FRANCE). The images were acquired with the patients' arms positioned over the head while breathing freely. The PET data were then acquired in the caudocephalic direction using a whole-body protocol (2 min per bed position). The delay between injection and acquisition was standardized to 60 minutes in order to obtain a normalized counting rate for all patients.
RGD was performed using Siemens® PETnet's RGD-K5. Patients were not at fast. Standard dose of 4 MBq/kg (Maximum of 450 MBq) was administrated after 60 minutes at rest. Images were acquired on same PET machine as for FDG, using the same bed protocol, but with 100kV and 80 mAs CT parameters to avoid unnecessary irradiation. Estimated additional estimated dose delivered for RGD was 10 mSv. The tracer uptake was quanti ed using the standardized uptake value (SUV) calculated as tissue concentration (Bq/g)/[injected dose (Bq)/body weight (g)].
c) PET analysis All FDG and RGD PET were analysed by one junior and one senior nuclear physicians. On FDG, all lesions (primary tumor and involved lymph nodes) with signi cant uptake were considered allowing Maximal Standardized Uptake Value (SUVmax), mean SUV (SUVmean), and Metabolic Tumor Volume (MTV) using a 41% of SUVmax threshold.
On RGD, on addition to SUVmax and SUVmean on all lesions, SUVmax and SUVmean were also considered in 13 organs: occipital cortex, thyroid, ascending aorta, inside left ventricular, lung, liver, spleen, gall bladder, kidney cortex, iliopsoas muscle, femoral head, T12 vertebra and inferior vena cava. Organs were excluded of normal uptake analysis if pathological on FDG PET or CT. For RGD's MTV, threshold was determined by adaptive method and if necessary, by expert's visual sampling. d) Treatment and follow-up All patients were treated according to national guidelines according to their histological subtype. Chemotherapy was performed using standard protocols. The patients were followed up by physical examination and FDG imaging. Biopsy was performed for any suspicious residual/recurrent tumors whenever possible. e) Histopathological analysis All patients had a biopsy proven lymphoma. All tumor tissues were routinely xed in 4% buffered formaldehyde and processed by standard methods into para n blocks. Four micrometer slides were prepared and stained with hematoxylin and eosin.

3-Results
Patient's clinical data are summarized in Table 1. Nineteen patients (7 women and 12 men) were included. Mean age at diagnostic was 55 ± 13 years. ECOG Performance Status was inferior or equal to 1 in 18 patients (95%). Nine patients had stage IV lymphoma (47%). The median follow-up time was 17.8 months (range 12-28). No patient died during or after treatment. One patient had synchronous lymphoma, and thus both primary lesions were analysed.  Figure 1 for visual comfort) was due to radiotracer's elimination Ex vivo analysis was performed on 10 patient's initial biopsy. No correlation was found between the endothelial cell marking via the ERGmarker, the number of vascular sections and C0 and C2 RGD uptake (data not shown).
RGD uptakes were analysed according to histological subtype ( Figure 2). Apart from classical Hodgkin lymphoma (HL), and grey zone lymphoma (GZL) other lymphoma sub-types had low RGD uptake. RGD uptakes were therefore compared between cHL and non-Hodgkin lymphoma (NHL) -(including grey zone lymphoma).
To illustrate these results is displayed on gure 3 a patient who had synchronous follicular and classical hodgkin lymphoma. Patient had a history of cHL in 2014 which was considered in full remission after 8 ABVD cycles, with early bone relapse in 2015. Bone relapse was successfully treated with 4 ICE cycles.
Patient could not undergo autologous bone marrow transplant due to stem cell collection failure. In september 2017, patient seeked her hematologist for cervical swelling. Cervical biopsy revealed grade II follicular lymphoma. Initial FDG revealed lymph node and left sacrum wing involvement. However, RGD uptake was only signi cant for the left sacrum wing. Dissimilarity between FDG and RGD uptake questioned on patient's histopathological sub-type. Bone biopsy was performed and con rmed concomitant bone cHL relapse.
On table 2 are summarized FDG and RGD uptakes on C0 and C2 according to patients presenting with HL and NHL. On C0 and C2 FDG, there was no signi cant difference for SUVmax, SUVmean and MTV between patients with HL and NHL. On C0 RGD, patients with HL had higher SUVmax, SUVmean and MTV than NHL. On C2 RGD, there was no signi cant difference for SUVmax, SUVmean and MTV between HL and NHL. An example is presented on gure 4.
On table 3 are summarized FDG and RGD uptakes on C0 and C2 according to nal Deauville score. On C0 and C2 FDG, and on C0 RGD there was no signi cant difference for SUVmax, SUVmean and MTV between responder and non-responder patients. On C2 RGD, non-responder patients had higher SUVmax, and SUVmean compared to responder patients ( Figure 5). There was no signi cant difference for RGD MTV between responder and non-responder patients.

4-Discussion
To the best of our knowledge, this is the rst clinical study using RGD in patients with lymphoma. Patients presenting with cHL had signi cant higher RGD uptake on baseline RGD than NHL. Our prospective study showed that non responder patients had higher RGD uptake on C2 RGD compared to responder patients.
RGD-K5 is an expensive (around 1000€) and di cult product to synthesize. Due to RGD-K5 production technical problems, two patients were lost of sight, two RGD PET were delayed and ten potentially includable patients were withdrawn. No patients had their treatment delayed because of RGD.
Our study allows to measure RGD uptake in normal organs before and after chemotherapy.
Preliminary histopathological results performed on initial biopsy showed no correlation between endothelial cell marking via the ERGmarker, the number of vascular sections and C0 and C2 RGD uptake. Endothelial cell marking via the ERGmarker is an indirect expression of neovascularization. The number of vascular sections were counted per 3.15 cm² frame. Further histopathological biopsies are necessary to con rm these results.
Patients with cHL had higher RGD uptake. cHL is mainly characterized by the presence of Reed Sternberg cells (RSC) which are multinucleated neoplastic cells. RSC are derived from B germinal centre and produce their own grown factors, Th2 cytokines and chemokines which creates high in ammatory in ltrate (12). The RSC constitute only a minor component of the tumor (usually 1-3%), whereas the majority of the malignancy is composed of a mixed in ammatory in ltrate variably composed of lymphocytes, eosinophils, broblasts, macrophages, and plasma cells (13). RSC also expresses high Vascular Endothelial Growth Factor (VEGF) which facilitates tumor progression (14). High RGD uptake in cHL compared to NHL could be due to αvβ3 overexpression in neovessels. An alternative explanation could be the presence of αvβ3 receptors on tumoral in ammatory cells. Complementary histopathological studies are being considered to verify these hypotheses.
SUVmax FDG was non-signi cant between responder and non-responder patients because two patients presenting with DLBCL had intermediate Deauville score at 4 and nal Deauville score at 2.
One of the major issues which is raised by RGD uptake is it's ability to predict antiangiogenic drug response. On the one hand, patients with DLBCL had in our study low RGD uptake. Seymour et al., (16) showed lack of Bevacizumab (anti VEGF) e cacy in DLBCL when added to standard chemotherapy compared to chemotherapy alone. Low RGD uptake in DLBCL could therefore be linked to the lack of antiangiogenic treatment e ciency. On the other hand, patients with HL had in our study high RGD uptake. Pre-clinical study on xenografter Hodgkin, showed e ciency of anti-angiogenic treatments (17). No clinical data of antiangiogenic drugs in HL in human is available. However, that with high RGD uptake in HL found in our study, interrogates on testing anti-angiogenic treatments in refractory patients presenting with HL.
Non responder patients had higher RGD uptake than responders on C2 PET ( Figure 6). Further studies are necessary to understand if uptake is due to neoangiogenesis or in ammatory process. Integrins are a class of heterodimeric cell surface adhesion receptors that are overexpressed on tumor endothelial cells during tumor angiogenesis, resulting in cancers that are more invasive, more migratory, and better able to survive in different microenvironments. Among integrins, αvβ3 is highly expressed in tumors such as osteosarcomas, neuroblastomas, glioblastomas, malignant melanomas, breast, lung and prostate carcinomas but its expression is weak in most healthy organ systems (34).
Limits of our study are the small number of patients but patients did present with heterogeneous histological lymphoma subtypes.

Conclusion
Our study showed signi cant higher initial RGD uptake in patients presenting with cHL compared to NHL. Non responder patients also had higher post chemotherapy RGD uptake compared to responder patients.
Issues raised by RGD uptake, particularly in cHL, are yet to be explored and need to be con rmed in a larger population. EudraCT number was 2015-000757-20 and the study National Clinical Trial identi er was NCT02490891. All patients signed written consent prior to inclusion Consent for publication Not applicable

List Of Abbreviations
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
All authors declare that they have no competing interests Funding This study was funded by a Ligue Contre le Cancer grant.
Authors' contributions DT was involved in data acquisition, literature research and manuscript writing. PB was involded in study design, pharmaceutical preparation and theoretical support. SB, PD and ST were involved in revise of the manuscript and theoretical support. VC, HT and FJ were involved in study design and review. PV was involved in data acquisition, theoretical support and revise of the manuscript. All the authors agreed on the content of the nal manuscript. All authors read and approved the nal manuscript. Bone marrow biopsy involvement (n) . Negative 13  SUV max uptake in organs before (C0) and after (C2) chemotherapy.