Cetuximab PET delineated changes in cellular distribution of EGFR upon dasatinib treatment in triple negative breast cancer.

BACKGROUND: At least 50% of triple negative breast cancer (TNBC) overexpress the epidermal growth factor receptor, EGFR, which paved the way for clinical trials investigating its blockade. Outcomes remained dismal stemming from mechanisms of resistance particularly the nuclear cycling of EGFR, which is enhanced by Src activation. Attenuation of Src reversed nuclear translocation, restoring EGFR to the cell surface. Herein, we hypothesize that changes in cellular distribution of EGFR upon Src inhibition with dasatinib can be annotated through the EGFR immunopositron emission tomography (immunoPET) radiotracer, [89Zr]Zr-cetuximab. METHODS: Nuclear and non-nuclear EGFR levels of dasatinib-treated vs. untreated MDA-MB-231 and MDA-MB-468 cells were analyzed via immunoblots. Both treated and untreated cells were exposed to [89Zr]Zr-cetuximab to assess binding at 4 °C and 37 °C. EGFR-positive MDA-MB-231, MDA-MB-468, and a patient-derived xenograft were treated with dasatinib or vehicle followed by cetuximab PET imaging to compare EGFR levels. After imaging, the treated mice were separated into two groups: one cohort continued with dasatinib with the addition of cetuximab while the other cohort received dasatinib alone. Correlations between the radiotracer uptake vs. changes in tumor growth and EGFR expression from immunoblots were analyzed. RESULTS: Treated cells displayed higher binding of [89Zr]Zr-cetuximab to the cell membrane at 4 °C and with greater internalized activity at 37 °C vs. untreated cells. In all tumor models, higher accumulation of the radiotracer in dasatinib-treated groups was observed compared to untreated tumors. Treated tumors displayed significantly decreased pSrc (Y416) with retained total Src levels compared to control. In MDA-MB-468 and PDX tumors, the analysis of cetuximab PET vs. changes in tumor volume showed an inverse relationship where high tracer uptake in the tumor demonstrated minimal tumor volume progression. Furthermore, combined cetuximab and dasatinib treatment showed better tumor regression compared to control and dasatinib-only-treated groups. No benefit was achieved in MDA-MB-231 xenografts with the addition of cetuximab, likely due to its KRAS-mutated status. CONCLUSIONS: Cetuximab PET can monitor effects of dasatinib on EGFR cellular distribution and potentially inform treatment response in wild-type KRAS TNBC.

Progesterone receptor A promotes invasiveness and metastasis of luminal breast cancer by suppressing regulation of critical microRNAs by estrogen.

Distal metastasis of luminal breast cancer is frequent and incurable, yet the metastasis mechanisms are poorly understood. Estrogen, even at postmenopausal concentrations, suppresses invasiveness of luminal breast cancer cells through the estrogen receptor (ER). Invasive tumors overexpress the short progesterone receptor A (PR-A) isoform. Even at postmenopausal concentrations, progesterone activates PR-A, inducing invasiveness by counteracting estrogen's effects, particularly when cells are hypersensitized to progesterone by PR-A overexpression. To interrogate the role of this cross-talk in metastasis, we investigated selective cross-talk mechanisms of PR-A with ER. We developed a quantitative PCR-based lymph node infiltration assay to address the slowness of metastasis of tumor xenografts. We found that 15 microRNAs (miRNAs) are regulated by progesterone via PR-A, but not the longer PR-B isoform, with increased progesterone sensitivity when PR-A was overexpressed. Two of these miRNAs whose induction (miR-92a-3p) or repression (miR-26b-5p) by estrogen was suppressed by progesterone plus PR-A were critical for the PR-A-ER cross-talk causing a gene-regulatory pattern of invasiveness and metastasis and complete rescue of invasiveness in vitro Constitutive expression of miR-92a-3p or inhibition of miR-26b-5p profoundly suppressed metastasis. Finally, in primary breast tumors, PR-A expression was correlated negatively with miR-92a-3p expression and positively with miR-26b-5p expression. Therefore, hormonal cross-talk of PR-A with ER is probably a fundamental mechanism that enables metastasis of luminal breast cancer. Moreover, miRNA biomarkers of hyperactive PR-A may help predict metastatic potential of luminal breast tumors. Further, miR-92a-3p and miR-26b-5p may reveal target pathways for selective intervention to suppress hormone-regulated metastasis, both pre- and postmenopause.

Monitoring Src status after dasatinib treatment in HER2+ breast cancer with 89Zr-trastuzumab PET imaging.

BACKGROUND: De novo or acquired resistance in breast cancer leads to treatment failures and disease progression. In human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer, Src, a non-receptor tyrosine kinase, is identified as a major mechanism of trastuzumab resistance, with its activation stabilizing aberrant HER2 signaling, thus making it an attractive target for inhibition. Here, we explored the causal relationship between Src and HER2 by examining the potential of 89Zr-trastuzumab as a surrogate imaging marker of Src activity upon inhibition with dasatinib in HER2+ breast cancer. METHODS: HER2+ primary breast cancer cell lines BT-474 and trastuzumab-resistant JIMT-1 were treated with dasatinib and assessed for expression and localization of HER2, Src, and phosphorylated Src (pSrc) (Y416) through western blots and binding assays. Mice bearing BT-474 or JIMT-1 tumors were treated for 7 or 14 days with dasatinib. At the end of each treatment, tumors were imaged with 89Zr-trastuzumab. The results of 89Zr-trastuzumab positron emission tomography (PET) was compared against tumor uptake of fluorodeoxyglucose (18F-FDG) obtained the day before in the same group of mice. Ex vivo western blots and immunohistochemical staining (IHC) were performed for validation. RESULTS: In BT-474 and JIMT-1 cells, treatment with dasatinib resulted in a decrease in internalized 89Zr-trastuzumab. Confirmation with immunoblots displayed abrogation of pSrc (Y416) signaling; binding assays in both cell lines demonstrated a decrease in cell surface and internalized HER2-bound tracer. In xenograft models, dasatinib treatment for 7 days (BT-474, 11.05 ± 2.10 % injected dose per gram of tissue %(ID)/g; JIMT-1, 3.88 ± 1.47 %ID/g)) or 14 days (BT-474, 9.20 ± 1.85 %ID/g; JIMT-1, 4.45 ± 1.23 %ID/g) resulted in a significant decrease in 89Zr-trastuzumab uptake on PET compared to untreated control (BT-474, 17.88 ± 2.18 %ID/g; JIMT-1, 8.04 ± 1.47 %ID/g). No difference in 18F-FDG uptake was observed between control and treated cohorts. A parallel decrease in membranous HER2 and pSrc (Y416) staining was observed in tumors post treatment on IHC. Immunoblots further validated the 89Zr-trastuzumab-PET readout. Positive correlation was established between 89Zr-trastuzumab tumor uptake versus tumor regression, pSrc and pHER2 expression. CONCLUSIONS: 89Zr-trastuzumab can potentially assess tumor response to dasatinib in HER2+ breast cancer and could be used as a surrogate tool to monitor early changes in Src signaling downstream of HER2.

89 Zr-ImmunoPET companion diagnostics and their impact in clinical drug development.

Therapeutic monoclonal antibodies have been used in cancer treatment for 30 years, with around 24 mAb and mAb:drug conjugates approved by the FDA to date. Despite their specificity, efficacy has remained limited, which, in part, derails nascent initiatives towards precision medicine. An image-guided approach to reinforce treatment decisions using immune positron emission tomography (immunoPET) companion diagnostic is warranted. This review provides a general overview of current translational research using Zr-89 immunoPET and opportunities for utilizing and harnessing this tool to its full potential. Patient case studies are cited to illustrate immunoPET probes as tools for profiling molecular signatures. Discussions on its utility in reinforcing clinical decisions as it relates to histopathological tumor assessment and standard diagnostic methods, and its potential as predictive biomarkers, are presented. We finally conclude with an overview of practical considerations to its utility in the clinic.

A diagnosis of discernment: Identifying a novel ATRX mutation in myelodysplastic syndrome with acquired α-thalassemia.

Myelodysplastic syndromes (MDS) are a heterogeneous category of myeloid neoplasms that represent the most common class of acquired bone marrow failure syndromes in adults. MDS is typically associated with a hypoproliferative macrocytic anemia, but atypical findings on initial diagnostic evaluations can raise concern for a distinct pathophysiological process and lead to the investigation of alternative etiologies. Here, we report a case of MDS with a concomitant hypoproliferative microcytic and hypochromic anemia that led to the identification of acquired hemoglobin H due to a novel somatic ATRX mutation.

IFNγ PET Imaging as a Predictive Tool for Monitoring Response to Tumor Immunotherapy.

IFNγ is an attractive target for imaging active antitumor immunity due to its function in the T-cell signaling axis. Here, we test an IFNγ immuno-PET (immunoPET) probe for its capacity to identify adaptive immunotherapy response after HER2/neu vaccination in both spontaneous salivary and orthotopic neu+ mouse mammary tumors. IFNγ immunoPET detected elevated cytokine levels in situ after vaccination, which inversely correlated with tumor growth rate, an indicator of response to therapy. In a model of induced T-cell anergy where CD8 T cells infiltrate the tumor, but upregulate PD-1, IFNγ tracer uptake was equivalent to isotype control, illustrating a lack of antitumor T-cell activity. The IFNγ immunoPET tracer detected IFNγ protein sequestered on the surface of tumor cells, likely in complex with the IFNγ receptor, which may explain imaging localization of this soluble factor in vivo Collectively, we find that the activation status of cytotoxic T cells is annotated by IFNγ immunoPET, with reduced off-target binding to secondary lymphoid tissues compared with imaging total CD3+ tumor-infiltrating lymphocytes. Targeting of soluble cytokines such as IFNγ by PET imaging may provide valuable noninvasive insight into the function of immune cells in situ Significance: This study presents a novel approach to monitor therapeutic outcomes via IFNγ-targeted positron emission tomography. Cancer Res; 78(19); 5706-17. ©2018 AACR.

Imaging EGFR and HER3 through 89Zr-labeled MEHD7945A (Duligotuzumab).

Tumor resistance to treatment paved the way toward the development of single agent drugs that target multiple molecular signatures amplified within the malignancy. The discovered crosstalk between EGFR and HER3 as well as the role of HER3 in mediating EGFR resistance made these two receptor tyrosine kinases attractive targets. MEHD7945A or duligotuzumab is a single immunotherapy agent that dually targets both molecular signatures. In this study, a positron emission tomography (PET) companion diagnostic to MEHD7945A is reported and evaluated in pancreatic cancer. Tumor accretion and whole body pharmacokinetics of 89Zr-MEHD7945A were established. Specificity of the probe for EGFR and/or HER3 was further examined.

SEC23B is required for pancreatic acinar cell function in adult mice.

Mice with germline absence of SEC23B die perinatally, exhibiting massive pancreatic degeneration. We generated mice with tamoxifen-inducible, pancreatic acinar cell-specific Sec23b deletion. Inactivation of Sec23b exclusively in the pancreatic acinar cells of adult mice results in decreased overall pancreatic weights from pancreatic cell loss (decreased pancreatic DNA, RNA, and total protein content), as well as degeneration of exocrine cells, decreased zymogen granules, and alterations in the endoplasmic reticulum (ER), ranging from vesicular ER to markedly expanded cisternae with accumulation of moderate-density content or intracisternal granules. Acinar Sec23b deletion results in induction of ER stress and increased apoptosis in the pancreas, potentially explaining the loss of pancreatic cells and decreased pancreatic weight. These findings demonstrate that SEC23B is required for normal function of pancreatic acinar cells in adult mice.

Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice.

In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes.