Reliability and Variability of Ki-67 Digital Image Analysis Methods for Clinical Diagnostics in Breast Cancer.

Ki-67 is a nuclear protein associated with proliferation, and a strong potential biomarker in breast cancer, but is not routinely measured in current clinical management owing to a lack of standardization. Digital image analysis (DIA) is a promising technology that could allow high-throughput analysis and standardization. There is a dearth of data on the clinical reliability as well as intra- and interalgorithmic variability of different DIA methods. In this study, we scored and compared a set of breast cancer cases in which manually counted Ki-67 has already been demonstrated to have prognostic value (n = 278) to 5 DIA methods, namely Aperio ePathology (Lieca Biosystems), Definiens Tissue Studio (Definiens AG), Qupath, an unsupervised immunohistochemical color histogram algorithm, and a deep-learning pipeline piNET. The piNET system achieved high agreement (interclass correlation coefficient: 0.850) and correlation (R = 0.85) with the reference score. The Qupath algorithm exhibited a high degree of reproducibility among all rater instances (interclass correlation coefficient: 0.889). Although piNET performed well against absolute manual counts, none of the tested DIA methods classified common Ki-67 cutoffs with high agreement or reached the clinically relevant Cohen's κ of at least 0.8. The highest agreement achieved was a Cohen's κ statistic of 0.73 for cutoffs 20% and 25% by the piNET system. The main contributors to interalgorithmic variation and poor cutoff characterization included heterogeneous tumor biology, varying algorithm implementation, and setting assignments. It appears that image segmentation is the primary explanation for semiautomated intra-algorithmic variation, which involves significant manual intervention to correct. Automated pipelines, such as piNET, may be crucial in developing robust and reproducible unbiased DIA approaches to accurately quantify Ki-67 for clinical diagnosis in the future.

Panitumumab-DOTA-111In: An Epidermal Growth Factor Receptor Targeted Theranostic for SPECT/CT Imaging and Meitner-Auger Electron Radioimmunotherapy of Triple-Negative Breast Cancer.

Epidermal growth factor receptors (EGFR) are overexpressed in triple-negative breast cancer (TNBC) and are an attractive target for the development of theranostic radiopharmaceuticals. We studied anti-EGFR panitumumab labeled with 111In (panitumumab-DOTA-111In) for SPECT/CT imaging and Meitner-Auger electron (MAE) radioimmunotherapy (RIT) of TNBC. Panitumumab-DOTA-111In was bound, internalized, and routed to the nucleus in MCF7, MDA-MB-231/Luc, and MDA-MB-468 human breast cancer (BC) cells dependent on the EGFR expression level (1.5 × 104, 1.7 × 105, or 1.3 × 106 EGFR/cell, respectively). The absorbed dose in the nuclei of MCF7, MDA-MB-231/Luc, and MDA-MB-468 cells incubated with 4.4 MBq of panitumumab-DOTA-111In (20 nM) was 1.20 ± 0.02, 2.2 ± 0.1, and 25 ± 2 Gy, respectively. The surviving fraction (SF) of MDA-MB-231/Luc cells treated with panitumumab-DOTA-111In (10-300 nM; 1.5 MBq/µg) was reduced as the absorbed dose in the cell increased, with clonogenic survival reduced to an SF = 0.12 ± 0.05 at 300 nM corresponding to 12.7 Gy. The SFs of MDA-MB-468, MDA-MB-231/Luc, and MCF7 cells treated with panitumumab-DOTA-111In (20 nM; 1.7 MBq/µg) were <0.01, 0.56 ± 0.05, and 0.67 ± 0.04, respectively. Unlabeled panitumumab had no effect on SF, and irrelevant IgG-DOTA-111In only modestly reduced the SF of MDA-MB-231/Luc cells but not MCF7 or MDA-MB-468 cells. The cytotoxicity of panitumumab-DOTA-111In was mediated by increased DNA double-strand breaks (DSB), cell cycle arrest at G2/M-phase and apoptosis measured by immunofluorescence detection by flow cytometry. MDA-MB-231/Luc tumors in the mammary fat pad (MFP) of NRG mice were clearly imaged with panitumumab-DOTA-111In by microSPECT/CT at 4 days postinjection (p.i.), and biodistribution studies revealed high tumor uptake [18 ± 2% injected dose/g (% ID/g] and lower normal tissue uptake (<10% ID/g). Administration of up to 24 MBq (15 µg) of panitumumab-DOTA-111In to healthy NRG mice caused no major hematological, renal, or hepatic toxicity with no decrease in body weight. Treatment of NOD SCID mice with MDA-MB-231 tumors with panitumumab-DOTA-111In (22 MBq; 15 µg) slowed tumor growth. The mean time for tumors to reach a volume of ≥500 mm3 was 61 ± 5 days for RIT with panitumumab-DOTA-111In compared to 42 ± 6 days for mice treated with irrelevant IgG2-DOTA-111In (P < 0.0001) and 35 ± 3 days for mice receiving 0.9% NaCl (P < 0.0001). However, tumors regrew at later time points. The median survival of mice treated with panitumumab-DOTA-111In was 70 days versus 46 days for IgG2-DOTA-111In (P < 0.0001) or 40 days for 0.9% NaCl (P < 0.0001). We conclude that panitumumab-DOTA-111In is a promising theranostic agent for TNBC. Increasing the administered amount of panitumumab-DOTA-111In and/or combination with radiosensitizing PARP inhibitors used for treatment of patients with TNBC may provide a more durable response to RIT.