Radioimmunotherapy of PANC-1 Human Pancreatic Cancer Xenografts in NRG Mice with Panitumumab Modified with Metal-Chelating Polymers Complexed to 177Lu.

Our aim was to evaluate the effectiveness and normal tissue toxicity of radioimmunotherapy (RIT) of s.c. PANC-1 human pancreatic cancer (PnCa) xenografts in NRG mice using anti-EGFR panitumumab linked to metal-chelating polymers (MCPs) that present 13 DOTA chelators to complex the ß-emitter, 177Lu. The clonogenic survival (CS) of PANC-1 cells treated in vitro with panitumumab-MCP-177Lu (0.3-1.2 MBq) and DNA double-strand breaks (DSBs) in the nucleus of these cells were measured by confocal immunofluorescence microscopy for γ-H2AX. Subcellular distribution of radioactivity for panitumumab-MCP-177Lu was measured, and absorbed doses to the cell nucleus were calculated. Normal tissue toxicity was assessed in non tumor-bearing NRG mice by monitoring body weight, complete blood cell counts (CBC), serum alanine aminotransferase (ALT), and creatinine (Cr) after i.v. injection of 6 MBq (10 µg) of panitumumab-MCP-177Lu. RIT was performed in NRG mice with s.c. PANC-1 tumors injected i.v. with 6 MBq (10 µg) of panitumumab-MCP-177Lu. Control mice received nonspecific human IgG-MCP-177Lu (6 MBq; 10 µg), unlabeled panitumumab (10 µg), or normal saline. The tumor growth index (TGI) was compared. Tumor and normal organ doses were estimated based on biodistribution studies. Panitumumab-MCP-177Lu reduced the CS of PANC-1 cells in vitro by 7.7-fold at the highest amount tested (1.2 MBq). Unlabeled panitumumab had no effect on the CS of PANC-1 cells. γ-H2AX foci were increased by 3.8-fold by panitumumab-MCP-177Lu. Panitumumab-MCP-177Lu deposited 3.84 Gy in the nucleus of PANC-1 cells. Administration of panitumumab-MCP-177Lu (6 MBq; 10 µg) to NRG mice caused no change in body weight, CBC, or ALT and only a slight increase in Cr compared to NRG mice treated with normal saline. Panitumumab-MCP-177Lu strongly inhibited tumor growth in NRG mice (TGI = 2.3 ± 0.2) compared to normal saline-treated mice (TGI = 5.8 ± 0.5; P < 0.01). Unlabeled panitumumab had no effect on tumor growth (TGI = 6.0 ± 1.6; P > 0.05). The absorbed dose of PANC-1 tumors was 12.3 Gy. The highest normal organ doses were absorbed by the pancreas, liver, spleen, and kidneys. We conclude that EGFR-targeted RIT with panitumumab-MCP-177Lu was able to overcome resistance to panitumumab in KRAS mutant PANC-1 tumors in NRG mice and may be a promising approach to treatment of PnCa in humans.

MicroSPECT/CT Imaging of Cell-Line and Patient-Derived EGFR-Positive Tumor Xenografts in Mice with Panitumumab Fab Modified with Hexahistidine Peptides To Enable Labeling with 99mTc(I) Tricarbonyl Complex.

We aimed to investigate the feasibility of conjugating synthetic hexahistidine peptides (His6) peptides to panitumumab Fab (PmFab) to enable labeling with [99mTc(H2O)3(CO)3]+ complex and study these radioimmunoconjugates for imaging EGFR-overexpressing tumor xenografts in mice by microSPECT/CT. Fab were reacted with a 10-fold excess of sulfo-SMCC to introduce maleimide functional groups for reaction with the terminal thiol on peptides [CGYGGHHHHHH] that harbored the His6 motif. Modification of Fab with His6 peptides was assessed by SDS-PAGE/Western blot, and the number of His6 peptides introduced was quantified by a radiometric assay incorporating 123I-labeled peptides into the conjugation reaction. Radiolabeling was achieved by incubation of PmFab-His6 in PBS, pH 7.0, with [99mTc(H2O)3(CO)3]+ in a 1.4 MBq/µg ratio. The complex was prepared by adding [99mTcO4]- to an Isolink kit (Paul Scherrer Institute). Immunoreactivity was assessed in a direct (saturation) binding assay using MDA-MB-468 human triple-negative breast cancer (TNBC) cells. Tumor and normal tissue uptake and imaging properties of 99mTc-PmFab-His6 (70 µg; 35-40 MBq) injected i.v. (tail vein) were compared to irrelevant 99mTc-Fab 3913 in NOD/SCID mice engrafted subcutaneously (s.c.) with EGFR-overexpressing MDA-MB-468 or PANC-1 human pancreatic ductal carcinoma (PDCa) cell-line derived xenografts (CLX) at 4 and 24 h post injection (p.i.). In addition, tumor imaging studies were performed with 99mTc-PmFab-His6 in mice with patient-derived tumor xenografts (PDX) of TNBC, PDCa, and head and neck squamous cell carcinoma (HNSCC). Biodistribution studies in nontumor bearing Balb/c mice were performed to project the radiation absorbed doses for imaging studies in humans with 99mTc-PmFab-His6. PmFab was derivatized with 0.80 ± 0.03 His6 peptides. Western blot and SDS-PAGE confirmed the presence of His6 peptides. 99mTc-PmFab-His6 was labeled to high radiochemical purity (≥95%), and the Kd for binding to EGFR on MDA-MB-468 cells was 5.5 ± 0.4 × 10-8 mol/L. Tumor uptake of 99mTc-PmFab-His6 at 24 h p.i. was significantly (P < 0.05) higher than irrelevant 99mTc-Fab 3913 in mice with MDA-MB-468 tumors (14.9 ± 3.1%ID/g vs 3.0 ± 0.9%ID/g) and in mice with PANC-1 tumors (5.6 ± 0.6 vs 0.5 ± 0.1%ID/g). In mice implanted orthotopically in the pancreas with the same PDCa PDX, tumor uptake at 24 h p.i. was 4.2 ± 0.2%ID/g. Locoregional metastases of these PDCa tumors in the peritoneum exhibited slightly and significantly lower uptake than the primary tumors (3.1 ± 0.3 vs 4.2 ± 0.3%ID/g; P = 0.02). In mice implanted with different TNBC or HNSCC PDX, tumor uptake at 24 h p.i. was variable and ranged from 3.7 to 11.4%ID/g and 3.8-14.5%ID/g, respectively. MicroSPECT/CT visualized all CLX and PDX tumor xenografts at 4 and 24 h p.i. Dosimetry estimates revealed that in humans, the whole body dose from administration of 740-1110 MBq of 99mTc-PmFab-His6 would be 2-3 mSv, which is less than for a 99mTc-medronate bone scan (4 mSv).

Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 111In and 177Lu-An EGFR-Targeted Theranostic for Pancreatic Cancer.

A metal-chelating polymer (MCP) with a polyglutamide (PGlu) backbone presenting on average 13 DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators for complexing 111In or 177Lu and 10 polyethylene glycol (PEG) chains to minimize liver and spleen uptake was conjugated to antiepidermal growth factor receptor (EGFR) monoclonal antibody (mAb), panitumumab. Because panitumumab-MCP may be dual-labeled with 111In and 177Lu for SPECT, or radioimmunotherapy (RIT) exploiting the Auger electrons or ß-particle emissions, respectively, we propose that panitumumab-MCP could be a useful theranostic agent for EGFR-positive PnCa. Bioconjugation was achieved by reaction of a hydrazine nicotinamide (HyNIC) group on the MCP with an aryl aromatic aldehyde introduced into panitumumab by reaction with succinimidyl-4-formylbenzamide (S-4FB). The conjugation reaction was monitored by measurement of the chromophoric bis-aryl hydrazone bond formed (ε350 nm = 24 500 M-1 cm-1) to achieve two MCPs/panitumumab. Labeling of panitumumab-MCP with 111In or 177Lu demonstrated that masses as small as 0.1 µg were labeled to >90% labeling efficiency (L.E.) and a specific activity (SA) of >70 MBq/µg. Panitumumab-DOTA incorporating two DOTA per mAb was labeled with 111In or 177Lu to a maximum SA of 65 MBq/µg and 46 MBq/µg, respectively. Panitumumab-MCP-177Lu exhibited saturable binding to EGFR-overexpressing MDA-MB-468 human breast cancer cells. The Kd for binding of panitumumab-MCP-177Lu to EGFR (2.2 ± 0.6 nmol/L) was not significantly different than panitumumab-DOTA-177Lu (1.0 ± 0.4 nmol/L). 111In and 177Lu were stably complexed to panitumumab-MCP. Panitumumab-MCP-111In exhibited similar whole body retention (55-60%) as panitumumab-DOTA-111In in NOD-scid mice up to 72 h postinjection (p.i.) and equivalent excretion of radioactivity into the urine and feces. The uptake of panitumumab-MCP-111In in most normal tissues in NOD-scid mice with EGFR-positive PANC-1 human pancreatic cancer (PnCa) xenografts at 72 h p.i. was not significantly different than panitumumab-DOTA-111In, except for the liver which was 3-fold greater for panitumumab-MCP-111In. Tumor uptake of panitumumab-MCP-111In (6.9 ± 1.3%ID/g) was not significantly different than panitumumab-DOTA-11In (6.6 ± 3.3%ID/g). Tumor uptake of panitumumab-MCP-111In and panitumumab-DOTA-111In were reduced by preadministration of excess panitumumab, suggesting EGFR-mediated uptake. Tumor uptake of nonspecific IgG-MCP (5.4 ± 0.3%ID/g) was unexpectedly similar to panitumumab-MCP-111In. An increased hydrodynamic radius of IgG when conjugated to an MCP may encourage tumor uptake via the enhanced permeability and retention (EPR) effect. Tumor uptake of panitumumab-DOTA-111In was 3.5-fold significantly higher than IgG-DOTA-111In. PANC-1 tumors were imaged by microSPECT/CT at 72 h p.i. of panitumumab-MCP-111In or panitumumab-DOTA-111In. Tumors were not visualized with preadministration of excess panitumumab to block EGFR, or with nonspecific IgG radioimmunoconjugates. We conclude that linking panitumumab to an MCP enabled higher SA labeling with 111In and 177Lu than DOTA-conjugated panitumumab, with preserved EGFR binding in vitro and comparable tumor localization in vivo in mice with s.c. PANC-1 human PnCa xenografts. Normal tissue distribution was similar except for the liver which was higher for the polymer radioimmunoconjugates.

Formulation of a kit under Good Manufacturing Practices (GMP) for preparing [111In]In-BnDTPA-trastuzumab-NLS injection: a theranostic agent for imaging and Meitner-Auger Electron (MAE) radioimmunotherapy of HER2-positive breast cancer.

BACKGROUND: 111In[In]-BnDTPA-trastuzumab-NLS is a radiopharmaceutical with theranostic applications for imaging and Meitner-Auger electron (MAE) radioimmunotherapy (RIT) of HER2-positive breast cancer (BC). Nuclear localization sequence (NLS) peptides route the radiopharmaceutical to the nucleus of HER2-positive BC cells following receptor-mediated internalization for RIT with subcellular range MAEs. The γ-photons emitted by 111In permit tumour imaging by SPECT. Our aim was to formulate a kit under Good Manufacturing Practices conditions to prepare 111In[In]-BnDTPA-trastuzumab-NLS injection for a first-in-human clinical trial. RESULTS: Trastuzumab was derivatized with p-SCN-BnDTPA to introduce Bn-DTPA for complexing 111In, then modified with maleimide groups for conjugation to the thiol on cysteine in NLS peptides [CGYGPKKKRKVGG]. BnDTPA-trastuzumab-NLS (5 mg in 1.0 mL of 0.05 M ammonium acetate buffer, pH 5.5) was dispensed into unit dose sterile glass vials to produce kits for labeling with 100-165 MBq of 111In[In]Cl3. The kits met specifications for protein concentration (4.5-5.5 mg/mL), volume (0.95-1.05 mL), pH (5.5-6.0), appearance (clear, pale-yellow, particulate-free), BnDTPA substitution level (2.0-7.0 BnDTPA/trastuzumab), purity and homogeneity (SDS-PAGE and SE-HPLC), 111In labeling efficiency (> 90%), binding to HER2-positive SK-BR-3 human breast cancer cells (Ka = 1-8 × 108 L/mmol; Bmax = 0.5-2 × 106 sites/cell), NLS peptide conjugation (upward band shift on SDS-PAGE), sterility (USP Sterility Test) and endotoxins (USP Bacterial Endotoxins Test). 111In-BnDTPA-trastuzumab-NLS injection met specifications for pH (5.5-6.5), radiochemical purity (≥ 90%), radionuclide purity (≥ 99%), appearance (clear, colourless, particle-free) and sterility (retrospective USP Sterility Test). Kits were stable stored at 2-8 °C for up to 661 days (d) meeting all key specifications. Protein concentration remained within or just slightly greater than the specification for up to 139 d. 111In[In]-BnDTPA-trastuzumab-NLS injection was stable for up to 24 h. An expiry of 180 d was assigned for the kits and 8 h for the final radiopharmaceutical. CONCLUSION: A kit was formulated under GMP conditions for preparing 111In[In]-BnDTPA-trastuzumab-NLS injection. This radiopharmaceutical was safely administered to 4 patients with HER2-positive BC to trace the uptake of trastuzumab into brain metastases before and after MRI-guided focused ultrasound (MRIg-FUS) by SPECT imaging.

Pre-clinical evaluation of immunoPET imaging using agonist CD40 monoclonal antibody in pancreatic tumor-bearing mice.

BACKGROUND: A novel [64Cu]Cu-NOTA-aCD40 immunoPET tracer was developed to image a CD40+ pancreatic tumor model in C57BL/6 mice and to study the biodistribution profile of the agonist CD40 (aCD40) monoclonal antibody (mAb) alone or combined with other mAbs. PROCEDURES: Copper-64 ([64Cu]Cu) labeled NOTA-aCD40 and NOTA-IgG (10 µg; 7 MBq) were injected intravenously into C57BL/6 mice with subcutaneous mT4 tumors to assess specificity 48 h post injection (p.i.) through positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies (n = 5). [64Cu]Cu-NOTA-aCD40 was injected alone or simultaneously in combination with a therapeutic mass of cold aCD40 (100 µg), aPD-1 (200 µg) and aCTLA-4 (200 µg) mAbs. A group of mice with or without tumor received the second round of injections 1 or 3 weeks apart, respectively. PET/CT imaging and biodistribution studies were performed at 48 h p.i. The organ dose for [64Cu]Cu was estimated based on biodistribution studies with 2 µg [64Cu]Cu-NOTA-aCD40 (corresponds to 5 mg patient dose) in non-tumor bearing mice. RESULTS: [64Cu]Cu-NOTA-aCD40 accumulation was 2.3- and 7.8-fold higher than [64Cu]Cu-NOTA-IgG in tumors and spleen, respectively, indicating the specificity of aCD40 mAb in a mouse pancreatic tumor model. Tumor accumulation of [64Cu]Cu-NOTA-aCD40 was 21.2 ± 7.3%ID/g at 48 h after injection. Co-injection of [64Cu]Cu-NOTA-aCD40 with cold aCD40 mAb alone or with PD-1 and CTLA-4 mAbs reduced both spleen and tumor uptake, whereas liver uptake was increased. With the second round of injections, the liver was the only organ with substantial uptake. With a 2 µg administered dose of [64Cu]Cu-NOTA-aCD40 in a dosimetry study, the liver to spleen ratio was greater compared to the 10 µg dose (2.8 vs 0.37; respectively). The human equivalent for the highest dose organ (liver) was 198 ± 28.7 µSv/MBq. CONCLUSIONS: A CD40-immunoreactive [64Cu]Cu-NOTA-aCD40 probe was developed. The ratio of spleen to liver accumulation exceeded that of the IgG isotype and was greatest with a single small, injected mass. The safety of human patient imaging with [64Cu]Cu was established based on extrapolation of the organ specificity to human imaging.

Radioimmunotherapy of PANC-1 human pancreatic cancer xenografts in NOD/SCID or NRG mice with Panitumumab labeled with Auger electron emitting, 111In or ß-particle emitting, 177Lu.

BACKGROUND: Epidermal growth factor receptors (EGFR) are overexpressed on > 90% of pancreatic cancers (PnCa) and represent an attractive target for the development of novel therapies, including radioimmunotherapy (RIT). Our aim was to study RIT of subcutaneous (s.c.) PANC-1 human PnCa xenografts in mice using the anti-EGFR monoclonal antibody, panitumumab labeled with Auger electron (AE)-emitting, 111In or ß-particle emitting, 177Lu at amounts that were non-toxic to normal tissues. RESULTS: Panitumumab was conjugated to DOTA chelators for complexing 111In or 177Lu (panitumumab-DOTA-[111In]In and panitumumab-DOTA-[177Lu]Lu) or to a metal-chelating polymer (MCP) with multiple DOTA to bind 111In (panitumumab-MCP-[111In]In). Panitumumab-DOTA-[177Lu]Lu was more effective per MBq exposure at reducing the clonogenic survival in vitro of PANC-1 cells than panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In. Panitumumab-DOTA-[177Lu]Lu caused the greatest density of DNA double-strand breaks (DSBs) in the nucleus measured by immunofluorescence for γ-H2AX. The absorbed dose in the nucleus was 3.9-fold higher for panitumumab-DOTA-[177Lu]Lu than panitumumab-DOTA-[111In]In and 7.7-fold greater than panitumumab-MCP-[111In]In. No normal tissue toxicity was observed in NOD/SCID mice injected intravenously (i.v.) with 10.0 MBq (10 µg; ~ 0.07 nmoles) of panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In or in NRG mice injected i.v. with 6.0 MBq (10 µg; ~ 0.07 nmoles) of panitumumab-DOTA-[177Lu]Lu. There was no decrease in complete blood cell counts (CBC) or increased serum alanine aminotransferase (ALT) or creatinine (Cr) or decreased body weight. RIT inhibited the growth of PANC-1 tumours but a 5-fold greater total amount of panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In (30 MBq; 30 µg; ~ 0.21 nmoles) administered in three fractionated amounts every three weeks was required to achieve greater or equivalent tumour growth inhibition, respectively, compared to a single amount of panitumumab-DOTA-[177Lu]Lu (6 MBq; 10 µg; ~ 0.07 nmoles). The tumour doubling time (TDT) for NOD/SCID mice with s.c. PANC-1 tumours treated with panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In was 51.8 days and 28.1 days, respectively. Panitumumab was ineffective yielding a TDT of 15.3 days vs. 15.6 days for normal saline treated mice. RIT of NRG mice with s.c. PANC-1 tumours with 6.0 MBq (10 µg; ~ 0.07 nmoles) of panitumumab-DOTA-[177Lu]Lu increased the TDT to 20.9 days vs. 11.5 days for panitumumab and 9.1 days for normal saline. The absorbed doses in PANC-1 tumours were 8.8 ± 3.0 Gy and 2.6 ± 0.3 Gy for panitumumab-DOTA-[111In]In and panitumumab-MCP-[111In]In, respectively, and 11.6 ± 4.9 Gy for panitumumab-DOTA-[177Lu]Lu. CONCLUSION: RIT with panitumumab labeled with Auger electron-emitting, 111In or ß-particle-emitting, 177Lu inhibited the growth of s.c. PANC-1 tumours in NOD/SCID or NRG mice, at administered amounts that caused no normal tissue toxicity. We conclude that EGFR-targeted RIT is a promising approach to treatment of PnCa.

Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation.

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.

Radioimmunotherapy of cancer with high linear energy transfer (LET) radiation delivered by radionuclides emitting α-particles or Auger electrons.

Radioimmunotherapy (RIT) aims to selectively deliver radionuclides emitting α-particles, ß-particles or Auger electrons to tumors by conjugation to monoclonal antibodies (mAbs) that recognize tumor-associated antigens/receptors. The approach has been most successful for treatment of non-Hodgkin's B-cell lymphoma but challenges have been encountered in extending these promising results to the treatment of solid malignancies. These challenges include the low potency of ß-particle emitters such as 131I, 177Lu or 90Y which have been commonly conjugated to the mAbs, due to their low linear energy transfer (LET=0.1-1.0keV/µm). Furthermore, since the ß-particles have a 2-10mm range, there has been dose-limiting non-specific toxicity to hematopoietic stem cells in the bone marrow (BM) due to the cross-fire effect. Conjugation of mAbs to α-particle-emitters (e.g. 225Ac, 213Bi, 212Pb or 211At) or Auger electron-emitters (e.g. 111In, 67Ga, 123I or 125I) would increase the potency of RIT due to their high LET (50-230keV/µm and 4 to 26keV/µm, respectively). In addition, α-particles have a range in tissues of 28-100µm and Auger electrons are nanometer in range which greatly reduces or eliminates the cross-fire effect compared to ß-particles, potentially reducing their non-specific toxicity to the BM. In this review, we describe the results of preclinical and clinical studies of RIT of cancer using radioimmunoconjugates emitting α-particles or Auger electrons, and discuss the potential of these high LET forms of radiation to improve the outcome of cancer patients.

The anticancer activity and HSA binding properties of the structurally related platinum (II) complexes.

The development of resistance and unwanted harmful interaction with other biomolecules instead of DNA are the major drawbacks for application of platinum (Pt) complexes in cancer chemotherapy. To conquer these problems, much works have been done so far to discover innovative Pt complexes. The objective of the current study was to evaluate the anti cancer activities of a series of four and five-coordinated Pt(II) complexes, having deprotonated 2-phenyl pyridine (abbreviated as C^N), biphosphine moieties, i.e., dppm = bis(diphenylphosphino) methane (Ph(2)PCH(2)PPh(2)) and dppa = bis(diphenylphosphino)amine (Ph(2)PNHPPh(2)), as the non-leaving carrier groups. The growth inhibitory effect of the Pt complexes [Pt(C^N)(dppm)]PF(6): C(1), [Pt(C^N)(dppa)]PF(6): C(2), and [Pt(C^N)I(dppa)]: C(3), toward the cancer cell lines was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. In addition, the florescence quenching experiments of the interaction between human serum albumin (HSA) and the Pt complexes were performed in order to obtain the binding parameters and to evaluate the denaturing properties of these complexes upon binding to the general carrier protein of blood stream. The structure-activity relationship studies reveal that four-coordinated Pt complexes C(1) and C(2) with both significant hydrophobic and charge characteristics, not only exhibit strong antiproliferation activity toward the cancer cell lines, but also they display lower denaturing effect against carrier protein HSA. On the other hand, five-coordinated C(3) complex with the unusual intermolecular NH…Pt hydrogen binding and the intrinsic ability for oligomerization, exhibits poor anticancer activity and strong denaturing property. The current study reveals that the balance between charge and hydrophobicity of the Pt complexes, also their hydrogen binding abilities and coordination mode are important for their anticancer activities. Moreover, this study may suggest C(1) and C(2) as the potential template structures for synthesis of new generation of four-coordinated Pt complexes with strong anticancer activities and weak denaturing effects against proteins.

Radiation dose to neonates undergoing X-ray imaging in special care baby units in Iran.

Radiographic imaging has a significant role in the timely diagnosis of the diseases of neonates in intensive care units. The estimation of the dose received by the infants undergoing radiographic examination is of great importance, due to greater more radiosensitivity and longer life expectancy of the neonates and premature babies. In this study, the values of entrance skin dose (ESD), dose area products (DAPs), energy imparted (EI), whole-body dose, effective dose and risk of childhood cancer were estimated using three methods including direct method [using thermoluminescence dosimetry (TLD) chips], indirect method (using tube output) and Monte Carlo (MC) method (using MCNP4C code). In the first step, the ESD of the neonates was directly measured using TLD-100 chips. Fifty neonates, mostly premature, with different weights and gestational ages in five hospitals mostly suffering from respiratory distress syndrome and pneumonia were involved in this study. In the second step, the values of ESD to neonates were indirectly obtained from the tube output in different imaging techniques. The imaging room, incubator, neonates and other components were then simulated in order to obtain the ESD values using the MCNP4C code. Finally, the values of ESD assessed by the three methods were used for calculation of DAP, EI, whole-body dose, effective dose and risk of childhood cancer. The results indicate that the mean ESD per radiograph estimated by the direct, indirect and MC methods are 56.6±4.1, 50.1±3.1 and 54.5±3.3 µGy, respectively. The mean risk of childhood cancer estimated in this study varied between 4.21×10(-7) and 2.72×10(-6).