Phase I clinical evaluation of 99mTc-labeled Affibody molecule for imaging HER2 expression in breast cancer.

The determination of tumor human epidermal growth factor receptor type 2 (HER2) status is of increasing importance with the recent approval of more efficacious HER2-targeted treatments. There is a lack of suitable methods for clinical in vivo HER2 expression assessment. Affibody molecules are small affinity proteins ideal for imaging detection of receptors, which are engineered using a small (molecular weight 6.5 kDa) nonimmunoglobulin scaffold. Labeling of Affibody molecules with positron emitters enabled the development of sensitive and specific agents for molecular imaging. The development of probes for SPECT would permit the use of Affibody-based imaging in regions where PET is not available. In this first-in-human study, we evaluated the safety, biodistribution, and dosimetry of the 99mTc-ZHER2:41071 Affibody molecule developed for SPECT/CT imaging of HER2 expression. Methods: Thirty-one patients with primary breast cancer were enrolled and divided into three cohorts (injected with 500, 1000, or 1500 µg ZHER2:41071) comprising at least five patients with high (positive) HER2 tumor expression (IHC score 3+ or 2+ and ISH positive) and five patients with low (IHC score 2+ or 1+ and ISH negative) or absent HER2 tumor expression. Patients were injected with 451 ± 71 MBq 99mTc-ZHER2:4107. Planar scintigraphy was performed after 2, 4, 6 and 24 h, and SPECT/CT imaging followed planar imaging 2, 4 and 6 h after injection. Results: Injections of 99mTc-ZHER2:41071 were well tolerated and not associated with adverse events. Normal organs with the highest accumulation were the kidney and liver. The effective dose was 0.019 ± 0.004 mSv/MBq. Injection of 1000 µg provided the best standard discrimination between HER2-positive and HER2-low or HER2-negative tumors 2 h after injection (SUVmax 16.9 ± 7.6 vs. 3.6 ± 1.4, p < 0.005). The 99mTc-ZHER2:41071 uptake in HER2-positive lymph node metastases (SUVmax 6.9 ± 2.4, n = 5) was significantly (p < 0.05) higher than that in HER2-low/negative lymph nodes (SUVmax 3.5 ± 1.2, n = 4). 99mTc-ZHER2:41071 visualized hepatic metastases in a patient with liver involvement. Conclusions: Injections of 99mTc-ZHER2:41071 appear safe and exhibit favorable dosimetry. The protein dose of 1000 µg provides the best discrimination between HER2-positive and HER2-low/negative expression of HER2 according to the definition used for current HER2-targeting drugs.

Phase I Trial of 99mTc-(HE)3-G3, a DARPin-Based Probe for Imaging of HER2 Expression in Breast Cancer.

Radionuclide molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression may enable a noninvasive discrimination between HER2-positive and HER2-negative breast cancers for stratification of patients for HER2-targeted treatments. DARPin (designed ankyrin repeat proteins) G3 is a small (molecular weight, 14 kDa) scaffold protein with picomolar affinity to HER2. The aim of this first-in-humans study was to evaluate the safety, biodistribution, and dosimetry of 99mTc-(HE)3-G3. Methods: Three cohorts of patients with primary breast cancer (each including at least 4 patients with HER2-negative and 5 patients with HER2-positive tumors) were injected with 1,000, 2,000, or 3,000 µg of 99mTc-(HE)3-G3 (287 ± 170 MBq). Whole-body planar imaging followed by SPECT was performed at 2, 4, 6, and 24 h after injection. Vital signs and possible side effects were monitored during imaging and up to 7 d after injection. Results: All injections were well tolerated. No side effects were observed. The results of blood and urine analyses did not differ before and after studies. 99mTc-(HE)3-G3 cleared rapidly from the blood. The highest uptake was detected in the kidneys and liver followed by the lungs, breasts, and small intestinal content. The hepatic uptake after injection of 2,000 or 3,000 µg was significantly (P < 0.05) lower than the uptake after injection of 1,000 µg. Effective doses did not differ significantly between cohorts (average, 0.011 ± 0.004 mSv/MBq). Tumor-to-contralateral site ratios for HER-positive tumors were significantly (P < 0.05) higher than for HER2-negative at 2 and 4 h after injection. Conclusion: Imaging of HER2 expression using 99mTc-(HE)3-G3 is safe and well tolerated and provides a low absorbed dose burden on patients. This imaging enables discernment of HER2-positive and HER2-negative breast cancer. Phase I study data justify further clinical development of 99mTc-(HE)3-G3.

Amplifications of Stemness Gene Loci-New Markers for the Determination of the Need for Neoadjuvant Chemotherapy for Patients with Breast Cancer. A Prospective Study.

In this prospective study, a new strategy for the prescription of neoadjuvant chemotherapy (NAC) was prospectively tested and depended on the presence of stemness gene amplifications in the tumor before treatment, which in our early studies showed a connection with metastasis. The study included 92 patients with grade IIA-IIIB luminal B breast cancer. Patients underwent a biopsy before treatment, and with the use of a CytoScan HD Array microarray (Affymetrix, Santa Clara, CA, USA), the presence of stemness gene amplifications (3q, 5p, 6p, 7q, 8q, 13q, 9p, 9q, 10p, 10q21.1, 16p, 18chr, 19p) in the tumor was determined. In group 1 (n = 41), in the presence of two or more amplifications, patients were prescribed a personalized NAC regimen. In group 2 (n = 21), if there was no amplification of stemness genes in the tumor, then patients were not prescribed NAC, and treatment began with surgery. Group 3 (n = 30) served as a historical control. The frequency of an objective response to NAC in groups 1 and 3 was 79%. Nonmetastatic survival was found in 100% of patients in group 2, who did not undergo NAC. In patients in group 1, the frequency of metastasis was 10% (4/41). At the same time, in patients in group 3, who received NAC, the rate of metastasis was 47% (14/30). The differences between group 1 and group 3 and between group 2 and group 3 were statistically significant, both by Fisher's criterion and a log-rank test. The appointment of NAC was most feasible in patients with clones with stemness gene amplifications in the primary tumor, while in the absence of amplifications, preoperative chemotherapy led to a sharp decrease in metastasis-free survival. This strategy of NAC prescription allowed us to achieve 93% metastatic survival in patients with breast cancer.

Neoadjuvant Chemotherapy Induces the Appearance of New Copy Number Aberrations in Breast Tumor and is Associated with Metastasis.

BACKGROUND: In this study, we examined the CNA-genetic landscape (CNA - copy number aberration) of breast cancer prior to and following neoadjuvant chemotherapy (NAC) and correlated changes in the tumor landscape with chemotherapy efficiency as well as metastasis-free survival. OBJECTIVE: Breast cancer patients (n = 30) with luminal B molecular subtypes were treated with anthracycline- based therapy. METHODS: To study CNAs in breast tumors, microarray analysis was performed. RESULTS: Three effects of NAC on tumor CNA landscape were identified: 1 - the number of CNAbearing tumor clones decreased following NAC; 2 - there were no alterations in the number of CNAcontaining clones after NAC; 3 - the treatment with NAC increased the number of CNA-bearing clones (new clones appeared). All NAC-treated patients who had new tumor clones with amplification (20%) had a 100% likelihood of metastasis formation. In these cases, NAC contributed to the emergence of potential metastatic clones. Our study identified the following loci - 5p, 6p, 7q, 8q, 9p, 10p, 10q22.1, 13q, 16p, 18Chr and 19p - that were amplified during the treatment with NAC and maybe the markers of potential metastatic clones. In other patients who showed total or partial elimination of CNA-bearing cell clones, no new amplification clones were observed after NAC, and no evidence of metastases was found with follow-up for 5 years (р = 0.00000). CONCLUSION: Our data suggest that the main therapeutic result from NAC is the elimination of potential metastatic clones present in the tumor before treatment. The results showed the necessity of an intelligent approach to NAC to avoid metastasis stimulation.