Detection of Insulitis by Pancreatic Scintigraphy with Tc-Labeled IL-2 and MRI in Patients with LADA

652 Diabetes Care Volume 38, April 2015

Alberto Signore,1,2 Gabriela Capriotti,1 Detection of Insulitis by Pancreatic Marco Chianelli,2,3 Elena Bonanno,4 99m Filippo Galli,1 Carlo Catalano,5 Scintigraphy With Tc-Labeled Ana M. Quintero,6 Giorgio De Toma,7 Silvia Manfrini,8 Paolo Pozzilli,8,9 and the IL-2 and MRI in Patients With Action LADA Group* LADA (Action LADA 10) Diabetes Care 2015;38:652–658 | DOI: 10.2337/dc14-0580

OBJECTIVE Pancreatic scintigraphy with interleukin-2 radiolabeled with 99mTc (99mTc-IL-2) is a technique used to image chronic inﬂammatory-mediated disorders. We used this method to detect a signal consistent with the presence of insulitis in patients with autoimmune diabetes. Positive and negative controls (patients with pancreatic carcinoma and type 2 diabetes, respectively) also were studied.

RESEARCH DESIGN AND METHODS 1Nuclear Medicine Unit, Faculty of Medicine and Psychology, University “Sapienza,” Rome, Italy We examined 25 patients with autoimmune diabetes (16 with recently diagnosed 2Department of Nuclear Medicine and Molecular type 1 diabetes, 9 with latent autoimmune diabetes in adults [LADA]), 6 with type Imaging, University Medical Center Groningen, Uni- 2 diabetes, and 7 with pancreatic carcinoma (the latter two groups were used as versity of Groningen, Groningen, the Netherlands 3 negative and positive controls, respectively). All patients underwent 99mTc-IL-2 Regina Apostolorum Hospital, Albano, Rome, Italy scintigraphy and contrast-enhanced MRI of the pancreas. To validate positive 4Department of Biopathology and Diagnostic Im- controls, samples were taken from patients with pancreatic carcinoma during aging, University “Tor Vergata,” Rome, Italy surgery for histological and immunohistochemical investigations. 5Department of Radiological Science, University “Sapienza,” Rome, Italy RESULTS 6Department of Radiology, Cl´ınica Colsanitas, 99m Bogota,` Colombia 7 EMERGING TECHNOLOGIES AND THERAPEUTICS Pancreatic accumulation of Tc-IL-2 was detected in patients with autoimmune Department of Surgery Science, University diabetes (61% positive) and, notably, in 6 of 9 patients with LADA; semiquantita- “Sapienza,” Rome, Italy tive evaluation of pancreatic uptake of 99mTc-IL-2 showed higher values in patients 8Department of Endocrinology and Diabetes, University Campus Bio-Medico, Rome, Italy with autoimmune diabetes (both childhood and LADA) and pancreatic carcinoma 9 ’ 6 6 6 Centre of Diabetes, St. Bartholomew s and The than in those with type 2 diabetes (4.45 1.99, 4.79 1.1, and 4.54 1.62 vs. London School of Medicine, Queen Mary, Univer- 2.81 6 0.63; P =0.06,P = 0.01, and P = 0.04, respectively). In patients with sity of London, London, U.K. pancreatic carcinoma, pancreatic interleukin-2 receptor expression correlated Corresponding author: Paolo Pozzilli, p.pozzilli@ with pancreatic 99mTc-IL-2 uptake (r =0.8;P = 0.01). In patients with LADA, unicampus.it. 99mTc-IL-2 uptake inversely correlated with duration of disease (r =0.7;P = 0.03). Received 6 March 2014 and accepted 18 Decem- ber 2014. CONCLUSIONS A.S. and G.C. contributed equally to this study. 99m Autoimmune diabetes in adults is associated with increased pancreatic Tc-IL-2 *The Action LADA Group includes R.D. Leslie, uptake, indicating the presence of insulitis, particularly within 1 year of the be- M.I. Hawa, P. Pozzilli, R. Williams, S. Brophy, ginning of insulin therapy, similar to type 1 diabetes at diagnosis. H. Beck-Nielsen, K. Yderstraede, S. Hunter, D. Hadden, R. Buzzetti, W. Scherbaum, H. Kolb, N.C. Schloot, J. Seissler, G. Schernthaner, Type 1 diabetes (T1D) is a T-cell–mediated autoimmune disease derived from the J. Tuomilehto, C. Sarti, A. de Leiva, D. Mauricio, and C. Thivolet. selective destruction of insulin-secreting b-cells. Several studies have shown that ﬁ © 2015 by the American Diabetes Association. lymphomononuclear cell in ltration of the endocrine pancreas (insulitis) is present Readers may use this article as long as the work for months or even years before the clinical diagnosis and is associated with residual is properly cited, the use is educational and not b-cell mass (1–3). for proﬁt, and the work is not altered. care.diabetesjournals.org Signore and Associates 653

With the exception of pancreatic bi- tissue specimens revealed that jejunal (T2D). The IL-2 scan was evaluated fur- opsy, which is an invasive and risky pro- accumulation of 99mTc-IL-2, as mea- ther in a control group of patients with cedure, to date no diagnostic markers sured in vivo by single photon emission pancreatic carcinoma undergoing pan- are available to detect insulitis and iden- computerized tomography (SPECT), cor- createctomy because this type of can- tify responders to immunotherapy, relates with the number of infiltrating cerisusuallyinfiltrated by activated which is currently being evaluated in tri- activated mononuclear cells per milli- Tcells. als of patients with recent-onset T1D (4). meter of mucosa (9). Similar results Lymphocytes and monocytes are respon- were achieved in patients with active RESEARCH DESIGN AND METHODS 123 sible for the destruction of b-cells, and Crohn’s disease in whom I-IL-2 scin- Patients the “in vivo” visualization of islets infil- tigraphy was used to monitor the effi- Sixteen T1D patients (8 males and 8 fe- trated by mononuclear cells by means cacy of therapy with corticosteroids males; mean age 16.6 6 8.4 years; me- of a noninvasive technique could also be (10). 99mTc-IL-2 scintigraphy was able dian age 13 years; age range 8–33 years; an important tool for the identification of to predict time to disease relapse in pa- all but one positive for anti-glutamic acid prediabetic patients in whom the islet cell tients with inactive Crohn’s disease (11). decarboxylase autoantibodies [GAD+] destructive process is potentially treat- Radiolabeled IL-2 is also able to evaluate or anti–insulinoma-associated protein able and may be halted. the degree of lymphocytic infiltration of [IA]) within 4 weeks from clinical diag- Therefore, the possibility of evaluat- carotid atherosclerotic plaques, thus nosis and nine patients with LADA (6 ing in vivo changes in insulitis could be providing useful information for the se- receiving insulin therapy, 3 still receiv- useful for monitoring the disease pro- lection of infiltrated vulnerable plaques ing oral antidiabetic therapy; 4 males and cess and the efficacy of an immune at risk for rupture (12). In patients af- 5 females; mean age 44 6 9.1 years; all intervention. For this purpose, we devel- fected by cutaneous melanoma the up- positive for anti-GAD or anti-IA2 or insulin oped an interleukin-2 (IL-2) radiolabeled take of radiolabeled IL-2 correlates with autoantibodies [IAA]) (Table 1) were en- with 99mTc (99mTc-IL-2), a cytokine that IL-2 receptor expression on activated rolled in this study. We selected patients binds to a specific receptor expressed T-lymphocytes in tumor infiltration. with LADA according to the criteria estab- on activated lymphocytes (5). This ra- Thus, a positive response to 99mTc-IL-2 lished by the Action LADA project (17): dioactive probe has optimal character- scintigraphy could represent a prognos- basically a diagnosis of diabetes when istics for in vivo detection of activated tic marker to predict the efficacy of IL-2 more than 30 years old, no insulin re- T cells in infiltrated tissues. In previous immunotherapy in patients with mela- quired for at least 6 months, and positivity studies, 99mTc-IL-2 allowed the suc- noma (13). Our first study of 99mTc-IL-2 for at least one autoantibody islet cell– cessful demonstration of insulitis in scintigraphy in human patients with related autoantibody, including GAD, IA2, two animal models of autoimmune dia- T1D, published in 2008, reported that or IAA. These autoantibodies were mea- betes (6–8). Indeed, one of the most rel- 65% of patients .15 years of age with sured centrally in a Diabetes Autoantibody evant characteristics of islet-infiltrating newly diagnosed T1D showed a pancre- Standardization Program–recognized lab- lymphocytes is their activation status, atic accumulation of 99mTc-IL-2 (14). oratory. Patients with LADA were investi- with surface expression of several re- Most important, patients with positive gated within 12 months of diagnosis of ceptors. Among these, the IL-2 receptor, scintigraphy at diagnosis showed better LADA and recent introduction of insulin an heterotrimeric transmembrane G long-term metabolic control if treated therapy. protein–coupled receptor (a-chain CD25, with nicotinamide compared with pa- As negative controls we studied six b-chain CD122, g-chain CD132). The tients with negative scintigraphy, sug- patients with long-standing T2D (four a2b (CD25–CD122) heterodimer is re- gesting that positive patients had men and two women; mean age 54 6 sponsible for IL-2 binding with high affin- persistent pancreatic inflammation at 6.8 years; negative for islet cell auto- ity. Large numbers of these receptors diagnosis with residual b-cell function antibodies and GAD; diabetes duration are expressed on the surface of activated and that nicotinamide was effective in 5 6 4.1 years) and, as positive controls, T cells; a lesser amount is expressed on protecting b-cells. seven patients with pancreatic adeno- monocytes and other cells. Early at- Since then, we continued investigat- carcinoma (ADK) (four men and three tempts to image activated T cells with ing the use of radiolabeled IL-2 in several women; mean age 59 6 8.7 years) in 123I-IL-2 followed by 99mTc-IL-2 have diseases, including T1D, and we whom the neoplastic lesion was infil- been made. developed a simple kit for easy and re- trated by activated (CD25+) T cells. In humans, scintigraphy with either producible production of this radiophar- Patients were recruited at the Endocri- 123I-IL-2 or 99mTc-IL-2 has been used to maceutical (15,16). nology and Surgery Departments of image chronic inflammation-mediated The main aim of the current study Universities “Campus Bio-Medico” disorders and lymphocyte homing in was to evaluate the ability of 99mTc-IL-2 and “Sapienza” in Rome, Italy. cancer. In particular, in patients with ce- scintigraphy to detect the presence of All patients underwent IL-2 scintig- liac disease it was possible to detect je- activated lymphocytes in the pancreas raphy and contrast-enhanced MRI of junal infiltration and to monitor the of different types of diabetic patients, thepancreas.Atissuesamplewascol- effect of diet on the immune process in particular patients with T1D with clinical lected from patients with ADK during (9). Specific binding of radiolabeled onset at an adult age (latent autoimmune surgery for histological and immuno- IL-2 to activated lymphocytes was also diabetes in adults [LADA]) compared histochemical investigations. This clearly demonstrated by ex vivo micro- with children with T1D and patients type of cancer is usually infiltrated by autoradiography and immunostaining; with long-standing type 2 diabetes activated T cells (18) and was used as a 654 99m Tc-IL-2 and MRI Detection of Insulitis Diabetes Care Volume 38, April 2015

Table 1—Immunological and scintigraphic data of patients studied Disease Pancreas (counts/pixels) Background (counts/pixels) T:B* Age (years)† Anti-GAD Anti-IA2 Therapy‡ T2D 21.66 10.65 2.03 64 22Diet 11.91 3.49 3.42 60 22OA 8.67 2.53 3.43 51 22OA 8.83 3.44 2.57 46 22OA 19.57 5.78 3.39 49 22OA 9.46 4.65 2.04 55 22OA Mean 2.81 54 SD 0.63 6.8 Mean +2 SD 4.06 ADK 29.6 4.51 6.56 69 22Surgery 17.36 3.12 5.57 55 22Surgery 14 11.59 1.21 66 22Surgery 19.57 5.78 3.39 48 22Surgery 16.59 3.49 4.76 48 22Surgery 12.58 2.42 5.19 66 22Surgery 15.06 2.97 5.08 59 22Surgery Mean 4.54 59 SD 1.62 8.7 T1D 14.21 4.41 3.22 11 220.3 16.1 4.53 3.55 12 + + 0.22 12.78 6.19 2.06 14 + 2 0.7 18.8 4.23 4.44 10 + + 0.4 24.15 3.92 6.16 15 + + 0.65 12.38 4.61 2.69 12 + 2 0.65 22.76 3.93 5.79 25 + 2 0.25 10.81 3.27 3.31 12 + + 0.42 24.62 4.22 5.84 14 + 2 0.82 11.58 4.86 2.38 8 + + 0.52 28.3 3.42 8.29 17 + 2 0.4 32.17 3.89 8.27 30 + 2 0.1 26.73 6.32 4.23 9 + + 0.33 13.88 4.73 2.93 11 + + 0.32 16.52 6.44 2.57 32 + 2 0.4 38.49 7.1 5.42 33 + 2 0.22 Mean 4.45 16.6 0.43 SD 1.99 8.4 0.21 LADA 13.71 3.56 3.85 59 + + 0 19.68 3.15 6.25 46 + + 0.30 33.24 6.54 5.08 39 + + 0.23 16.49 2.89 5.71 30 + + 0 17.58 3.30 5.33 43 + + 0.24 19.69 4.35 4.53 44 + + 0.42 11.24 2.36 4.77 55 + + 0 16.55 6.00 2.76 45 + + 0.31 7.38 3.34 2.21 34 + + 0.25 Mean 4.79 44 0.19 SD 1.1 9.1 0.16 Values in bold are +2 SD higher than the mean of T2D patients. OA, oral antidiabetic therapy; 1, indicates presence of antibodies; 2, indicates absence of autoantibodies. *Two-tailed test for unpaired data of T:B: type 1 vs. type 2: P = 0.06; type 1 vs. LADA: P = 0.67; type 1 vs. ADK: P = 0.92; LADA vs. type 2: P = 0.01; LADA vs. ADK: P = 0.96; ADK vs. type 2: P =0.04.†Two-tailed test for unpaired data of age: type 1 vs. type 2: P = 0.0001; type 1 vs. LADA: P = 0.0001; type 1 vs. ADK: P = 0.0001; LADA vs. type 2: P = 0.03; LADA vs. ADK: P = 0.006; ADK vs. type 2: P = 0.32. ‡Numeric values are presented as international units per kilogram.

positive control and to correlate IL-2 up- Scintigraphic Studies uptake of free 99mTc originating from take in the lesion with the histological Radiolabeled IL-2 was prepared, as pre- the metabolism of 99mTc-IL-2. The dose ﬁndings. viously described (5), according to in children was calculated by weight, ac- This study was carried out in accordance the principles of Good Manufacturing cording to international guidelines of the with the principles of the Declaration of Practice. Subjects were intravenously Pediatric Group of the European Associ- Helsinki and was approved by the Ethics injected with 74–185 MBq of 99mTc-IL-2 ation of Nuclear Medicine. Committee at Sapienza University. All pa- (20–50 mg IL-2) and orally treated with Patients were positioned under a com- tients or parents gave written informed KClO4 (400 mg) 20 minutes before the puterized gamma camera, and SPECT im- consent before entering the study. study to prevent thyroid and stomach ages of the abdomen were acquired care.diabetesjournals.org Signore and Associates 655

45 to 60 min after injection using a 64 3 64 pixel matrix. The obtained transaxial abdominal section (3 cm thick), including the body/tail of the pancreas (or the pancreatic tumor in the head or body of the pancreas), was selected by image fusion with the corresponding section obtained by MRI (Figs. 1 and 2). For data analysis, pancreatic radioactivity (or tumor radioactivity) was calculated by drawing an irregular region of interest over the pancreatic region (body/tail of the pancreas in diabetic patients or the pancreatic tumor mass in patients with ADK) in the most representative transaxial section. On the same section, a circular region of interest was drawn over the spine; this was considered background radioactivity. Pancreatic or tumor uptake of 99mTc-IL-2 was calculated as the pancreas (target)-to-background radioactivity ratio (T:B), as described in a previous study (14). The effective dose equivalent (EDE) of a diagnostic dose of 99mTc-IL-2 (3 mCi, 110 MBq) is approximately 0.9 mSv, which is signiﬁ- cantly lower than the EDE of most radiological investigations (the EDE of a computed tomography [CT] scan of the abdomen is 4–5 mSv) and falls into cate- gory IIa of the International Committee of Radiation Protection. The amount of IL-2 injected (20–50 mg) is nontoxic and not immunogenic. Figure 1—Transaxial sections of contrast-enhanced MRI showing the body and tail of the Pancreatic scintigraphy was consid- pancreas (A and B). Fusion images between MRI and SPECT for drawing the pancreatic and ered positive if the T:B was higher than bone regions of interest (ROI) for the calculation of the T:B (C and D). Transaxial sections of the average of that in T2D patients +2 SD. SPECT corresponding to the MRI slide in which the pancreatic and bone ROI are shown (irreg- Scintigraphies were evaluated by ular ROI for pancreas as target [T] and circle ROI for bone as background [B]) for calculation of the T:B. The images show an example of a T2D patient with a T:B of 3.42 (A, C,andE)andan two independent nuclear medicine example of a T2D patient with LADA with a T:B of 5.33 (B, D,andF). physicians (A.S. and G.C.).

MRI Study were fixed in formalin and embedded magnification of 1,7003 to quantify Contrast-enhanced MRI of the pancreas in paraffin for further histological ex- the number of positive cells in at least was performed using 1.5T Siemens Vi- amination. Paraffin sections were five randomly selected fields. sion Plus (gradient power 25 mT/m; stained for CD25 antigen expression gradient rise time 600 msec) using fat- by an initial incubation with a normal suppressed sequences. Slices (3 mm) Statistical Analysis goat serum blocking reagent followed Data are presented as mean 6 SD. To were acquired for better anatomic de- by a primary monoclonal antibody evaluate the differences of T:B between tail to determine signal and enhance- and were revealed with a peroxidase- studied populations we used the Stu- ment differences within the pancreas conjugated secondary antibody using dent t test for unpaired data. and between patient groups. Volumet- diaminobenzidine as a chromogen. Cells Regression analysis between 99mTc-IL-2 ric slices (3 cm thick) were acquired for were counted using a Quantimet 920 Im- pancreatic uptake and other parame- image fusion with SPECT images. MRI age Analyzer with a monochrome charge- ters was performed. Pearson correla- images were evaluated by two indepen- coupled device camera (Hamamatsu tion for normally distributed data was dent radiologists (C.C. and A.M.Q.). C3077). Using an off-peak intensity of used to calculate statistical signifi- Pancreatic Surgical Specimens 546 nm with a Reichert Polyvar micro- cance. We used Excel version 14.4.5 Specimens were collected from the scope connected to the Hamamatsu andAcaStatversion8.3.10tocalculate edgesofthetumormassinpatients camera, the diaminobenzidine immu- means and SDs, t tests, and correlations with pancreatic ADK. Tissue samples nostaining was evaluated at a final and to create graphs. SPSS software 656 99m Tc-IL-2 and MRI Detection of Insulitis Diabetes Care Volume 38, April 2015

version 22.0 was used for Pearson correlations.

RESULTS No adverse effects were observed fol- lowing the administration of 99mTc-IL-2. The procedure to radiolabel IL-2 was carried out under sterile conditions, and all labelings satisfied the criteria for biological and radiochemical quality controls (radiochemical purity .95% by instant thin-layer chromatography). All patients completed the study. MRIs clearly showed the pancreas in all patients, with no significant differences between patient groups. Both T2-weighted and fat-suppressed images showed homogenous intensity of the body and tail of the pancreas in patients with T1D, LADA, and T2D. No significant differences between con- Figure 2—Pancreatic uptake of 99mTc-IL-2 in diabetic patients, patients with LADA, and patients trast enhancement of MRI within the with pancreatic ADK. Data are mean 6 SD. pancreas and between diabetic patients were detected. We found differ- with IL-2 receptor expression in cancer- persist for some years after clinical diag- ences in focal intensity only in patients infiltrating T-lymphocytes (r =0.8;P = nosis, and progressively decrease as the with ADK. There was always agreement 0.01) (Fig. 4). b-cell mass declines (19,20). Although between the reports from the two radi- Pancreatic uptake of radiolabeled IL-2 both genetic and autoantibody screening ologists. did not correlate in all patients with age, canhelptopredictautoimmunediabetes Good correlation also was observed body weight, or autoantibody titer in some cases, the identification and evo- between the two nuclear medicine (GAD, IA2, and IAA), but in patients lution of insulitis can offer new clues for , readers (r =0.91;P 0.001); the mean with LADA it correlated with disease du- disease pathogenesis and follow-up ther- results of reading of the two operators ration (r =0.7;P =0.03). apy after an immune intervention. were used for statistical analysis. 99m Performing multiple pancreatic biop- Pancreatic Tc-IL-2 uptake in T2D pa- CONCLUSIONS sies would be the ideal method to study 6 tients (negative controls) was 2.81 0.63. Autoantibodies to islet cell antigens and the presence and the severity of insulitis; Thus, using a positive threshold of 4.06 mononuclear cell infiltration in the islet, as an invasive technique, however, (mean + 2 SD), 8 of 16 T1D patients the pathological hallmark of T1D, appear biopsies are routinely impracticable. (50%) and 6 of 9 patients with LADA long before the onset of the disease, The goal of imaging modalities is to (66.6%) showed a significant accumulation of IL-2 in the pancreas. In T1D patients, the T:B did not correlate with age or body weight (T:B vs. age, r = 0.16; T:B vs. body weight, r = 0.31) but was higher than in T2D patients, although it did not reach statistical significance (4.45 6 1.99 vs. 2.81 6 0.63; P = 0.06). Mean pancreatic 99mTc-IL-2 uptake in patients with LADA and pancreatic ADK was significantly higher than in patients with T2D (4.79 6 1.1 and 4.54 6 1.62 vs. 2.81 6 0.63; P =0.01andP =0.04,respectively) (Fig. 3). No significant differences between patients with T1D and LADA, T1D and pancreatic carcinoma, or LADA and pancreatic carcinoma were observed. Pancreatic tissue specimens were obtained in five of seven patients studied. Two patients were nonoperable. Pan- 99m creatic accumulation of Tc-IL-2 in pa- Figure 3—Correlation between uptake of 99mTc-IL-2 by tumors and CD25 expression in tumor- tients with pancreatic ADK correlated infiltrating cells, as evaluated by immunohistochemistry (r =0.876;P = 0.01). care.diabetesjournals.org Signore and Associates 657

specimens from only five patients, these results confirm those previously obtained in other diseases (9–13) and support the feasibility of imaging insulitis in autoimmune diabetes. In a previously published study, 123I-IL-2 accumulation also was observed in subjects at risk of developing autoimmune diabetes. Patients with a positive scan developed diabetes within 1 year (25), suggesting that this technique may be useful for predicting disease. In thiscontextitisofinterestthatourpa- tients with LADA were investigated in the early phases of the autoimmune process (i.e., within 1 year from diagnosis) and showed abnormal 99mTc-IL-2 uptake in the pancreas. These results open new avenues in the management of diabetic patients. Thus, it will be possible to identify in Figure 4—Correlation between pancreatic 99mTc-IL-2 uptake (T:B) and disease duration the preclinical phase the presence of in- (months) in patients with LADA (r = 20.703; P = 0.03). White circles represent patients receiving sulitis so that patients could be treated insulin therapy and black circles are patients not yet taking insulin. The type of treatment does with immunotherapies at an early stage not influence the presence of insulitis as much as disease duration from the first episode of and followed up. IL-2 scintigraphy can hyperglycemia. be used at time of diagnosis to identify a subgroup of patients with integrate diagnostic information with diagnosis and in 66.6% of patients with persistent insulitis (and therefore re- anatomic and functional data to charac- LADA (a sign of the presence of activated sidual b-cell mass) in whom the efficacy terize the insulitis process. The relatively mononuclear cells), particularly if they of treatment can modulate the autoim- small inflammatory process of insulitis is are diagnosed within 1 year of the first mune process. difficult to study by CT and MRI. Further- episode of hyperglycemia. The differences Other groups used different radio- more, these radiological techniques are in the percentage of positivity can be re- pharmaceuticals to image b-cells, but not able to supply functional informa- lated to different stages of the disease. All the results of SPECT were not promising. tion about b-cells. T1D patients were insulin dependent, and Better results have been achieved using Nuclear medicine provides a wide in some of these there probably was no positron emission tomography (PET) ra- spectrum of radiopharmaceuticals to residual inflammation in pancreatic diopharmaceuticals to evaluate residual image chronic inflammation. Pancreatic b-cells (23). Patients with LADA were all b-cell mass (26). insulitis in T1D patients was first detected newly diagnosed and three of them were After clinical diagnosis during the first 30 years ago by 111In-labeled lymphocytes noninsulin dependent at the time of year of disease, a small proportion of but without convincing and conclusive re- study; therefore they can be considered T1D patients undergo spontaneous clin- sults (21). Other authors showed that as being in a preclinical phase of ical remission. This finding has encour- 111In-labeled lymphocytes do not image the disease in which we expect to find aged the beginning of clinical trials with the infiltrated pancreas in biobreeding insulitis. Different degrees of radiola- several adjuvant therapies to improve rats (22). Instead, radiolabeled IL-2 has beled IL-2 uptake in the pancreas have the rate of clinical remission at the optimal characteristics for in vivo detec- alreadybeenreportedinnewlydiag- time of diabetes onset. Patients with tion of activated T cells in infiltrated nosed T1D patients, concluding that it active, ongoing pancreatic inflamma- tissues. It allowed detection of insulitis may reflect a different pattern of infiltration may benefit from immunotherapy. in two animal models of autoimmune tion at the time of diagnosis (24). Thus, Therefore, using a receptor ligand such diabetes, and a positive correlation be- because it is able to identify the degree as radiolabeled IL-2 for mononuclear tween 123I-IL-2 scintigraphic uptake and of lymphocytic infiltration, IL-2 could be cells is the ideal method to quantify in thedegreeoflymphocyticinfiltration used as a marker to highlight the rela- vivo the severity of the inflammatory was observed by gamma camera imaging tionship between the autoimmune pro- process. One of the principal difficulties and proved histologically (9–13). cess and progression of the disease in in studying the insulitis process in vivo In the current study we used 99mTc-IL-2 patients with LADA. by scintigraphic techniques, however, is to attempt evaluation of ongoing inflam- In this study we also demonstrated related to the low resolution and the mation in the endocrine pancreas. Our re- that 99mTc-IL-2 uptake in patients with poor anatomical definition of system sults showed that 99mTc-IL-2 accumulates pancreatic carcinoma correlates with detection. in the pancreas of approximately 50% IL-2 receptor expression, as evaluated In our study MRI was performed in of T1D patients at the time of clinical by immunostaining. Although we had each patient, and scintigraphic images 658 99m Tc-IL-2 and MRI Detection of Insulitis Diabetes Care Volume 38, April 2015

of the pancreatic region were fused with pancreatic adenocarcinoma and performed sur- in melanoma lesions: a validation study. J Nucl MRIs to anatomically localize the pan- gery. S.M. selected diabetic patients and those Med 2004;45:1647–1652 creas. Although this fusion method is with LADA. P.P. recruited patients, analyzed 14. Chianelli M, Parisella MG, Visalli N, et al.; data, and wrote the manuscript. A.S. is the fi IMDIAB study group. Pancreatic scintigraphy ef cacious, young patients had a poor guarantor of this work and, as such, had full with 99mTc-interleukin-2 at diagnosis of type 1 compliance with MRI, and this technique access to all the data in the study and takes diabetes and after 1 year of nicotinamide ther- may not be used routinely, particularly in responsibility for the integrity of the data and apy. Diabetes Metab Res Rev 2008;24:115–122 children. This problem could be overcome the accuracy of the data analysis. 15. Karczmarczyk U, Garnuszek P, Maurin M, by the use of hybrid SPECT–CT or PET–CT et al. Investigation of 99mTc-labelling of re- References cameras that simultaneously provide a combinant human interleukin-2 via hydrazino- 1. Lo B, Swafford AD, Shafer-Weaver KA, et al. nicotinamide. Nucl Med Biol 2010;37:795–803 multislice CT scan and a tomographic scin- Antibodies against insulin measured by electro- 16. D’Alessandria C, di Gialleonardo V, Chianelli tigraphic scan of the target organ with chemiluminescence predicts insulitis severity M, et al. Synthesis and optimization of the la- image fusion and high anatomic and func- and disease onset in non-obese diabetic mice beling procedure of 99mTc-HYNIC-interleukin-2 tional definition of the target. Indeed, and can distinguish human type 1 diabetes sta- for in vivo imaging of activated T lymphocytes. 18F-labeled IL-2 has recently been synthe- tus. J Transl Med 2011;9:203 Mol Imaging Biol 2010;12:539–546 2. 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Bottazzo GF, Dean BM, McNally JM, MacKay lem related to these aspects is the poor labelled interleukin-2: a new radiopharmaceuti- EH, Swift PG, Gamble DR. In situ characterization cal for the in vivo detection of mononuclear cell of autoimmune phenomena and expression of availability of markers to evaluate the effi HLA molecules in the pancreas in diabetic insulitis. fi in ltrates in immune-mediated diseases. Nucl cacy of treatment and to select respond- Med Biol 1997;24:579–586 N Engl J Med 1985;313:353–360 ers to different therapies. Our data 6. Signore A, Parman A, Pozzilli P, Andreani D, 21. Kaldany A, Hill T, Wentworth S, et al. Trapping demonstrate that 99mTc-IL-2 scintigraphy, Beverley PC. Detection of activated lympho- of peripheral blood lymphocytes in the pancreas as a noninvasive technique, allows the in cytes in endocrine pancreas of BB/W rats by of patients with acute-onset insulin-dependent di- – vivo detection of CD25+ cells in tissues injection of 123I-interleukin-2: an early sign of abetes mellitus. Diabetes 1982;31:463 466 type 1 diabetes. Lancet 1987;2:537–540 22. Gallina DL, Pelletier D, Doherty P, et al. and may be a suitable tool for monitoring 7. Signore A, Chianelli M, Toscano A, et al. A 111Indium-labelled lymphocytes do not image immune intervention in patients with au- radiopharmaceutical for imaging areas of lym- or label the pancreas of the BB/W rat. Diabeto- toimmune diabetes, although large inter- phocytic infiltration: 123I-interleukin-2. Label- logia 1985;28:143–147 vention studies are needed to confirm the ling procedure and animal studies. Nucl Med 23. Kolb H. Benign versus destructive insulitis. – clinical role of this technique. Commun 1992;13:713 722 Diabetes Metab Rev 1997;13:139–146 8. Signore A, Chianelli M, Ferretti E, et al. New 24. Signore A, Chianelli M, Ronga G, Pozzilli P, approach for in vivo detection of insulitis in type Beverley PC. In vivo labelling of activated T lym- I diabetes: activated lymphocyte targeting with phocytes by i.v. injection of 123I-IL2 for detec- Acknowledgments. The authors acknowledge 123I-labelled interleukin 2. Eur J Endocrinol tion of insulitis in type 1 diabetes. Prog Clin Biol – the Association of Nuclear Medicine Discovery 1994;131:431 437 Res 1990;355:229–238 (Nu.Me.D.) for help and support during this 9. Signore A, Chianelli M, Annovazzi A, et al. 25. Signore A, Picarelli A, Chianelli M, et al. I- fi study and Dr. E. Maddaloni, Department of Imaging active lymphocytic in ltration in coeliac interleukin-2 scintigraphy: a new approach to disease with iodine-123-interleukin-2 and the Endocrinology and Diabetes, University Campus assess disease activity in autoimmunity. J Pediatr response to diet. Eur J Nucl Med 2000;27:18–24 Bio-Medico, Rome, Italy, for statistical analysis. Endocrinol Metab. 1996;9(Suppl. 1):139–144 10. Signore A, Chianelli M, Annovazzi A, et al. Funding. This research was supported by the 26. Sweet IR, Cook DL, Lernmark A, et al. Systematic 123I-interleukin-2 scintigraphy for in vivo assess- Italian Ministry of Education, University and screening of potential beta-cell imaging agents. Bio- Research (MIUR) and the Action LADA Group ment of intestinal mononuclear cell infiltration chem Biophys Res Commun 2004;314:976–983 from the European Union (contract no. QLG1- in Crohn’s disease. J Nucl Med 2000;41:242–249 27. Di Gialleonardo V, Signore A, Glaudemans CT-2002-01886). 11. Annovazzi A, Biancone L, Caviglia R, et al. 99m AW,DierckxRA,DeVriesEFN.N-(4-18F- No potential conflicts of Tc-interleukin-2 and (99m)Tc-HMPAO gran- Duality of Interest. fl interest relevant to this article were reported. ulocyte scintigraphy in patients with inactive uorobenzoyl)interleukin-2 for PET of human- Author Contributions. A.S. received funding Crohn’s disease. Eur J Nucl Med Mol Imaging activated T lymphocytes. J Nucl Med 2012;53: – for the study, performed scans, analyzed data, 2003;30:374–382 679 686 and wrote the manuscript. G.C. performed 12. Signore A, Bonanno E, De Toma G, et al. 28. Di Gialleonardo V, Signore A, Willemsen scans, analyzed data, and wrote the manuscript. 99mTc-Interleukin-2 scintigraphy for imaging un- AT, Sijbesma JW, Dierckx RA, de Vries M.C. performed scans and analyzed data. E.B. stable atherosclerotic plaques. Eur J Nucl Med EF. Pharmacokinetic modelling of N-(4- performed histological analysis. F.G. performed Mol Imaging 2004;31(Suppl. 2):S203 [(18)F]fluorobenzoyl)interleukin-2 binding to all labeling procedures. C.C. and A.M.Q. 13. Signore A, Annovazzi A, Barone R, et al. activated lymphocytes in an xenograft model performed nuclear magnetic resonance scans 99mTc-interleukin-2 scintigraphy as a potential of inflammation. Eur J Nucl Med Mol Imaging and analyzed data. G.D.T. selected patients with tool for evaluating tumor-infiltrating lymphocytes 2012;39:1551–1560