Understanding the Radiobiological Mechanisms Induced by 177Lu-DOTATATE in Comparison to External Beam Radiation Therapy.

Radionuclide Therapy (RNT) with 177Lu-DOTATATE targeting somatostatin receptors (SSTRs) in neuroendocrine tumours (NET) has been successfully used in routine clinical practice, mainly leading to stable disease. Radiobiology holds promise for RNT improvement but is often extrapolated from external beam radiation therapy (EBRT) studies despite differences in these two radiation-based treatment modalities. In a panel of six human cancer cell lines expressing SSTRs, common radiobiological endpoints (i.e., cell survival, cell cycle, cell death, oxidative stress and DNA damage) were evaluated over time in 177Lu-DOTATATE- and EBRT-treated cells, as well as the radiosensitizing potential of poly (ADP-ribose) polymerase inhibition (PARPi). Our study showed that common radiobiological mechanisms were induced by both 177Lu-DOTATATE and EBRT, but to a different extent and/or with variable kinetics, including in the DNA damage response. A higher radiosensitizing potential of PARPi was observed for EBRT compared to 177Lu-DOTATATE. Our data reinforce the need for dedicated RNT radiobiology studies, in order to derive its maximum therapeutic benefit.

177 Lu-DOTATATE PRRT Safety and Organ-at-Risk Dosimetry in Patients With Gastroenteropancreatic Neuroendocrine Tumors : Data From the Prospective Phase 2 LUMEN Study.

PURPOSE: The aim of this study was to assess the association among toxicity, dosimetry of organs-at-risk, and disease progression in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) treated with 177 Lu-DOTATATE. PATIENTS AND METHODS: Thirty-seven patients with GEP-NETs underwent 177 Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) in a single-arm, prospective, phase 2 study, where patients were followed up with blood tests, isotopic glomerular filtration rate (iGFR), and imaging examinations (CT/MRI and PET) every 6 months until disease progression. Adverse events (AEs) graded per CTCAEv4.03 and occurring during treatment were collected and followed up until resolution. Dosimetry, including biologically effective doses (BEDs) to kidneys, BED to bone marrow, and absorbed dose (AD) to spleen, was performed after each PRRT cycle. Statistical analyses explored associations among dosimetry, toxicity, and patient progression free-survival. RESULTS: The most common AEs were anemia and lymphopenia (65%), followed by thrombocytopenia and fatigue (each 51%), alopecia (46%), and nausea (41%). The most common grade ≥3 AE was lymphopenia (43%). There was no grade ≥3 nephrotoxicity. The median iGFR % decrease was 11% ( P < 0.001), at a median follow-up of 23 months. iGFR %decrease and renal BED did not correlate (Spearman ρ = -0.09). Similarly, no significant association was found between bone marrow BED or spleen AD and the grades of hematological toxicities. We observed no association between progression free-survival and either the decline of renal function or the occurrence of hematological toxicities during PRRT. CONCLUSIONS: This study confirms the safety profile of 177 Lu-DOTATATE PRRT in patients with GEP-NETs irrespective of the dosimetry of organs at risk. Kidney, bone marrow, and spleen dosimetry measures were not associated with renal or hematological toxicity.

Prediction of 177Lu-DOTATATE PRRT Outcome Using Multimodality Imaging in Patients with Gastroenteropancreatic Neuroendocrine Tumors: Results from a Prospective Phase II LUMEN Study.

Our objective was to predict the outcome of peptide receptor radionuclide therapy (PRRT) using multimodality imaging and tumor dosimetry on gastroenteropancreatic neuroendocrine tumor (GEP-NET) lesions and patients. Methods: This prospective study included patients with progressive GEP-NETs. Treatment consisted of 4 cycles of 7.4 GBq of 177Lu-DOTATATE. Imaging parameters were measured on 68Ga-DOTATATE PET/CT (SUVmax/mean, somatostatin receptor [SSTR] tumor volume [TV], total lesion SSTR expression, and tumor-to-blood and tumor-to-spleen ratios), 18F-FDG PET/CT (SUVmax/mean, metabolically active TV, and total lesion glycolysis), and diffusion-weighted MRI (apparent diffusion coefficient) in a maximum of 5 target lesions per patient at approximately 10 wk after each injection. Tumor dosimetry was performed using SPECT/CT at 3 time points for every cycle. Baseline imaging parameters, their relative changes after PRRT cycle 1 (C1), and the tumor-absorbed dose at C1 were correlated with lesion morphologic outcome. The average values of the imaging parameters and the minimal, maximal, and mean C1 tumor-absorbed dose in each patient were tested for association with progression-free survival (PFS) and best objective response (RECIST 1.1). Results: In the 37 patients, the median PFS was 28 mo. Eleven of the 37 (30%) achieved a partial response (RECIST 1.1). After a median follow-up of 57 mo, the median time to lesion progression had not been reached in 84 morphologically evaluable lesions, with only 12 (14%) progressing (size increase ≥ 20% from baseline). Patients receiving a minimal C1 dose of 35 Gy in all target lesions exhibited a significantly longer PFS (48.1 vs. 26.2 mo; hazard ratio, 0.37; 95% CI, 0.17-0.82; P = 0.02). Volumetric 68Ga-DOTATATE PET parameters correlated with lesion and patient outcome: patients with an SSTR TV decrease of more than 10% after C1 had a longer PFS (51.3 vs. 22.8 mo; hazard ratio, 0.35; 95% CI, 0.16-0.75; P = 0.003). There was no statistical evidence of an association between other dosimetric or imaging parameters and the lesion or patient outcome. Conclusion: Minimal tumor-absorbed dose at C1 is predictive of outcome in patients with GEP-NETs treated with PRRT, providing a basis for personalized dosimetry-guided treatment strategies. An SSTR TV decrease after C1 could be used for early therapy response assessment as a predictor of PRRT outcome.

Molecular imaging predicts lack of T-DM1 response in advanced HER2-positive breast cancer (final results of ZEPHIR trial).

Efficacy of the human epidermal growth factor receptor (HER)2-targeting trastuzumab emtansine (T-DM1) in breast cancer (BC) relies on HER2 status determined by immunohistochemistry or fluorescence in-situ hybridization. Heterogeneity in HER2 expression, however, generates interest in "whole-body" assessment of HER2 status using molecular imaging. We evaluated the role of HER2-targeted molecular imaging in detecting HER2-positive BC lesions and patients unlikely to respond to T-DM1. Patients underwent zirconium-89 (89Zr) trastuzumab (HER2) PET/CT and [18F]-2-fluoro-2-deoxy-D-glucose (FDG) PET/CT before T-DM1 initiation. Based on 89Zr-trastuzumab uptake, lesions were visually classified as HER2-positive (visible/high uptake) or HER2-negative (background/close to background activity). According to proportion of FDG-avid tumor load showing 89Zr-trastuzumab uptake (entire/dominant part or minor/no part), patients were classified as HER2-positive and HER2-negative, respectively. Out of 265 measurable lesions, 93 (35%) were HER2-negative, distributed among 42 of the 90 included patients. Of these, 18 (19%) lesions belonging to 11 patients responded anatomically (>30% decrease in axial diameter from baseline) after three T-DM1 cycles, resulting in an 81% negative predictive value (NPV) of the HER2 PET/CT. In combination with early metabolic response assessment on FDG PET/CT performed before the second T-DM1 cycle, NPVs of 91% and 100% were reached in predicting lesion-based and patient-based (RECIST1.1) response, respectively. Therefore, HER2 PET/CT, alone or in combination with early FDG PET/CT, can successfully identify BC lesions and patients with a low probability of clinical benefit from T-DM1.

Disturbing the Redox Balance Using Buthionine Sulfoximine Radiosensitized Somatostatin Receptor-2 Expressing Pre-Clinical Models to Peptide Receptor Radionuclide Therapy with 177Lu-DOTATATE.

Peptide receptor radionuclide therapy with 177Lu-DOTATATE improves the outcome of patients with somatostatin receptor (SSTR)-expressing neuroendocrine tumours. Nevertheless, stable disease has been the main response pattern observed, with some rare complete responses. Lu-177 exerts about two-thirds of its biological effects via the indirect effects of ionizing radiation that generate reactive oxygen species, eventually leading to oxidative damage and cell death. This provides a rationale for targeting the antioxidant defence system in combination with 177Lu-DOTATATE. In the present study, the radiosensitizing potential and the safety of depleting glutathione (GSH) levels using buthionine sulfoximine (BSO) during 177Lu-DOTATATE therapy were assessed in vitro and in vivo using a xenograft mouse model. In vitro, the combination resulted in a synergistic effect in cell lines exhibiting a BSO-mediated GSH decrease. In vivo, BSO neither influenced 177Lu-DOTATATE biodistribution nor induced liver, kidney or bone marrow toxicity. In terms of efficacy, the combination resulted in reduced tumour growth and metabolic activity. Our results showed that disturbing the cell redox balance using a GSH synthesis inhibitor increased 177Lu-DOTATATE efficacy without additional toxicity. Targeting the antioxidant defence system opens new safe treatment combination opportunities with 177Lu-DOTATATE.

Everolimus-induced somatostatin receptor overexpression in a rectal neuroendocrine tumor patient may promote somatostatin receptor-guided radionuclide therapy (peptide receptor radiotherapy) as an additional treatment option.

We present a case of Grade II, well-differentiated rectal neuroendocrine tumor in a 39-year-old patient. Following different sequential treatment modalities, the disease progressed both on metabolic (18F-fluorodeoxyglucose positron emission tomography-computed tomography [18F-FDG PET/CT]) and somatostatin receptor (SSTR)-imaging (68Ga-DOTA-Tyr3-octreotate [68Ga-DOTATATE] PET/CT), and the patient received three cycles of peptide receptor radiotherapy (PRRT). Two years later, upon new progression due to the appearance of metabolically active, 68Ga-DOTATATE PET/CT-negative liver lesions, targeted treatment with everolimus was introduced. Further morphologic and metabolic progression occurred 4 months after everolimus initiation, however, this time liver lesions demonstrated increased SSTR-expression on68Ga-DOTATATE PET/CT. Thus, the patient became eligible for a second PRRT course.

Investigating intrinsic radiosensitivity biomarkers to peptide receptor radionuclide therapy with [177Lu]Lu-DOTATATE in a panel of cancer cell lines.

INTRODUCTION: [177Lu]Lu-DOTATATE is an effective systemic targeted radionuclide therapy for somatostatin receptor (SSTR) positive metastatic or inoperable neuroendocrine tumours (NET). However, for a given injected activity, tumour responses are variable. Our aim was to investigate whether SSTR expression/functionality and known characteristics of intrinsic radiosensitivity, namely proliferation rate, glucose metabolism, cell cycle phase, DNA repair and antioxidant defences were predictors of sensitivity to [177Lu]Lu-DOTATATE in SSTR expressing human cancer cell lines. METHODS: In six human cancer cell lines and under basal condition, SSTR expression was assessed by qRT-PCR and immunocytochemistry. Its functionality was evaluated by binding/uptake assays with [68Ga]Ga- and [177Lu]Lu-DOTATATE. The radiosensitivity parameters were evaluated as follows: proliferation rate (cell counting), glucose metabolism ([18F]FDG uptake), antioxidant defences (qRT-PCR, colorimetric assay, flow cytometry), DNA repair (qRT-PCR) and cell cycle (flow cytometry). Effect of [177Lu]Lu-DOTATATE on cell viability was assessed 3, 7 and 10 days after 4 h incubation with [177Lu]Lu-DOTATATE using crystal violet. RESULTS: Based on cell survival at day 10, cell lines were classified into two groups of sensitivity to [177Lu]Lu-DOTATATE. One group with <20% of survival decrease (-14 to -1%) and one group with >20% of survival decrease (-22 to -33%) compared to the untreated control cell lines. The latter had significantly lower total antioxidant capacity, glutathione (GSH) levels and glucose metabolism (p < 0.05) compared to the first group. SSTR (p = 0.64), proliferation rate (p = 0.74), cell cycle phase (p = 0.55), DNA repair (p > 0.22), combined catalase and GSH peroxidase expression (p = 0.42) and superoxide dismutase (SOD) activity (p = 0.41) were not significantly different between the two groups. CONCLUSION: Antioxidant defences may be major determinants in [177Lu]Lu-DOTATATE radiosensitivity.

Downregulation of the FTO m6A RNA demethylase promotes EMT-mediated progression of epithelial tumors and sensitivity to Wnt inhibitors.

Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), has been shown to play a role in cancer, but its contribution to tumor progression and the underlying mechanisms remain unclear. Here, we report widespread FTO downregulation in epithelial cancers associated with increased invasion, metastasis and worse clinical outcome. Both in vitro and in vivo, FTO silencing promotes cancer growth, cell motility and invasion. In human-derived tumor xenografts (PDXs), FTO pharmacological inhibition favors tumorigenesis. Mechanistically, we demonstrate that FTO depletion elicits an epithelial-to-mesenchymal transition (EMT) program through increased m6A and altered 3'-end processing of key mRNAs along the Wnt signaling cascade. Accordingly, FTO knockdown acts via EMT to sensitize mouse xenografts to Wnt inhibition. We thus identify FTO as a key regulator, across epithelial cancers, of Wnt-triggered EMT and tumor progression and reveal a therapeutically exploitable vulnerability of FTO-low tumors.

Tumor Drug Penetration Measurements Could Be the Neglected Piece of the Personalized Cancer Treatment Puzzle.

Precision medicine aims to use patient genomic, epigenomic, specific drug dose, and other data to define disease patterns that may potentially lead to an improved treatment outcome. Personalized dosing regimens based on tumor drug penetration can play a critical role in this approach. State-of-the-art techniques to measure tumor drug penetration focus on systemic exposure, tissue penetration, cellular or molecular engagement, and expression of pharmacological activity. Using in silico methods, this information can be integrated to bridge the gap between the therapeutic regimen and the pharmacological link with clinical outcome. These methodologies are described, and challenges ahead are discussed. Supported by many examples, this review shows how the combination of these techniques provides enhanced patient-specific information on drug accessibility at the tumor tissue level, target binding, and downstream pharmacology. Our vision of how to apply tumor drug penetration measurements offers a roadmap for the clinical implementation of precision dosing.

A dosimetry procedure for organs-at-risk in 177Lu peptide receptor radionuclide therapy of patients with neuroendocrine tumours.

PURPOSE: Peptide receptor radionuclide therapy with 177Lu-DOTATATE has become a standard treatment modality in neuroendocrine tumours (NETs). No consensus has yet been reached however regarding the absorbed dose threshold for lesion response, the absorbed dose limit to organs-at-risk, and the optimal fractionation and activity to be administered. This is partly due to a lack of uniform and comparable dosimetry protocols. The present article details the development of an organ-at-risk dosimetry procedure, which could be implemented and used routinely in a clinical context. METHODS: Forty-seven patients with NETs underwent 177Lu-DOTATATE therapy. Three SPECT/CT images were acquired at 4, 24 and 144-192 h post-injection. Three blood samples were obtained together with the SPECT/CT acquisitions and 2 additional samples were obtained around 30 min and 1 h post-injection. A bi-exponential fit was used to compute the source organ time-integrated activity coefficients. Coefficients were introduced into OLINDA/EXM software to compute organ-at-risk absorbed doses. Median values for all patients were computed for absorbed dose coefficient D/A0 and for late effective half-life T1/2eff for kidneys, spleen and red marrow. RESULTS: Dosimetry resulted in a median[interquartile range] of 0.78[0.35], 1.07[0.58] and 0.028[0.010] Gy/GBq for D/A0 and of 55[9], 71[9] and 52[18] h for T1/2eff for kidneys, spleen and red marrow respectively. CONCLUSIONS: A dosimetry procedure for organs-at-risk in 177Lu-DOTATATE therapy based on serial SPECT/CT images and blood samples can be implemented routinely in a clinical context with limited patient burden. The results obtained were in accordance with those of other centres.

Accuracy and precision assessment for activity quantification in individualized dosimetry of 177Lu-DOTATATE therapy.

BACKGROUND: In order to obtain a reliable 177Lu-DOTATATE therapy dosimetry, it is crucial to acquire accurate and precise activity measurements with the radionuclide calibrator, the SPECT/CT camera, and the NaI(Tl) well counter. The aim of this study was to determine, in a clinical context, the accuracy and the precision of their activity quantification over a range of activities and time. Ninety-three 177Lu sources from the manufacturer were measured in the radionuclide calibrator over 2.5 years to evaluate its calibration accuracy and precision compared to the manufacturer's value. A NEMA 2012/IEC 2008 phantom was filled with a 177Lu activity concentration sphere-to-background ratio of five. It was acquired with the SPECT/CT camera to determine the reconstruction parameters offering the best compromise between partial volume effect and signal-to-noise ratio. The calibration factor was computed accordingly. The calibration quality was monitored over 2.5 years with 33 phantom acquisitions with activities ranging from 7040 to 0.6 MBq. Home-made sources were used to calibrate the well counter. Its reliability was evaluated with activities ranging from 150 to 0.2 kBq measured 34 times over 2.5 years. RESULTS: For the radionuclide calibrator, median [interquartile range] for the error on activity measurement was -0.99 [1.31] %. The optimal SPECT reconstruction parameters were obtained with 16 iterations, 16 subsets and a 12-mm Gaussian post-filter. The calibration factor was 9.87 cps/MBq with an error of -1.05 [2.12] %. The well counter was calibrated with 31.5 cps/kBq, and the error was evaluated to -12.89 [16.55] %. CONCLUSIONS: The accuracy and the precision of activity quantification using dedicated quality control were found to be sufficient for use in dosimetry implemented in clinical routine. The proposed methodology could be implemented in other centres to obtain reproducible 177Lu-based treatment dosimetry.

N-Acetylcysteine breaks resistance to trastuzumab caused by MUC4 overexpression in human HER2 positive BC-bearing nude mice monitored by 89Zr-Trastuzumab and 18F-FDG PET imaging.

Trastuzumab remains an important drug in the management of human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer (BC). Several studies reported resistance mechanisms to trastuzumab, including impaired HER2-accessibility caused by mucin 4 (MUC4). Previously, we demonstrated an increase of Zirconium-89-radiolabeled-trastuzumab (89Zr-Trastuzumab) accumulation when MUC4-overexpressing BC-cells were challenged with the mucolytic drug N-Acetylcysteine (NAC). Hereby, using the same approach we investigated whether tumor exposure to NAC would also enhance trastuzumab-efficacy. Dual SKBr3 (HER2+/MUC4-, sensitive to trastuzumab) and JIMT1 (HER2+/MUC4+, resistant to trastuzumab) HER2-BC-bearing-xenografts were treated with trastuzumab and NAC. Treatment was monitored by molecular imaging evaluating HER2-accessibility/activity (89Zr-Trastuzumab HER2-immunoPET) and glucose metabolism (18F-FDG-PET/CT), as well as tumor volume and the expression of key proteins. In the MUC4-positive JIMT1-tumors, the NAC-trastuzumab combination resulted in improved tumor-growth control compared to trastuzumab alone; with smaller tumor volume/weight, lower 18F-FDG uptake, lower %Ki67 and pAkt-expression. NAC reduced MUC4-expression, but did not affect HER2-expression or the trastuzumab-sensitivity of the MUC4-negative SKBr3-tumors. These findings suggest that improving HER2-accessibility by reducing MUC4-masking with the mucolytic drug NAC, results in a higher anti-tumor effect of trastuzumab. This provides a rationale for the potential benefit of this approach to possibly treat a subset of HER2-positive BC overexpressing MUC4.

Imaging Diagnostic and Therapeutic Targets: Human Epidermal Growth Factor Receptor 2.

Since the approval of trastuzumab, a humanized monoclonal antibody against the extracellular domain of human epidermal growth factor receptor 2 (HER2), 3 other HER2-targeting agents have gained regulatory approval: lapatinib, pertuzumab, and trastuzumab-emtansine. These agents have revolutionized the management of HER2-positive breast cancer, highlighting the concept that targeted therapies are successful when patients exhibit tumor-selective expression of a molecular target-in this case, HER2. However, response prediction and innate or acquired resistance remain serious concerns. Predictive biomarkers of a response-which could help in the selection of patients who might benefit from a selected targeted therapy-are currently lacking. Molecular imaging with anti-HER2 probes allows the noninvasive, whole-body assessment of HER2 tumor burden and has the potential to improve patient selection, optimize the dose and schedule, and rationalize assessment of the response to anti-HER2 therapies. Furthermore, unlike biopsy-based HER2 assessment, this approach can reveal inter- or intratumoral heterogeneity as well as variations in HER2 expression over time. This review summarizes the available literature and the current status of molecular imaging as a tool for the assessment of HER2 (target) expression or the prediction of an early treatment response in early and advanced HER2-positive breast cancer.

Mucolytic Agents Can Enhance HER2 Receptor Accessibility for [(89)Zr]Trastuzumab, Improving HER2 Imaging in a Mucin-Overexpressing Breast Cancer Xenograft Mouse Model.

PURPOSE: Binding of trastuzumab to HER2 receptors can be impaired by steric hindrance caused by mucin MUC4. As mucolytic drugs can breakdown disulfide bonds of mucoproteins, we checked if this approach could positively affect zirconium-89-labeled trastuzumab ([(89)Zr]T) binding/uptake. PROCEDURES: The effect of N-acetylcysteine (NAC) and MUC4 knockdown/stimulation on [(89)Zr]T binding/uptake were evaluated in MCF7(HER2-), BT474 and SKBr3(HER2+/MUC4-), and JIMT1(HER2+/MUC4+) cell lines. The results were then validated in SKBR3 and JIMT1 tumor-bearing nude mice with a microPET-CT and ex vivo analysis. RESULTS: Significant increases in [(89)Zr]T binding/uptake were observed in JIMT1 cells following MUC4 knockdown (62.4 ± 6.5%) and exposure to NAC (62.8 ± 19.4%). Compared to controls, mice treated with NAC showed a significant increase in [(89)Zr]T uptake in MUC4 tumors on microPET-CT (SUVmean (18.3 ± 4.7%), SUVmax (41.7 ± 8.4%)) and individual organ counting (37.3 ± 18.3%). In contrast, no significant differences were observed in SKBr3. CONCLUSION: NAC can enhance [(89)Zr]T accumulation and improve the HER2 imaging of MUC4-overexpressing tumors. The potential positive impact on trastuzumab-based treatment deserves further investigation.