Hanneke W. M. van Laarhoven

Triple-negative breast cancer: Present challenges and new perspectives

Triple‐negative breast cancers (TNBC), characterized by absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor 2 (HER2), are typically associated with poor prognosis, due to aggressive tumor phenotype(s), only partial response to chemotherapy and present lack of clinically established targeted therapies. Advances in the design of individualized strategies for treatment of TNBC patients require further elucidation, by combined ‘omics’ approaches, of the molecular mechanisms underlying TNBC phenotypic heterogeneity, and the still poorly understood association of TNBC with BRCA1 mutations. An overview is here presented on TNBC profiling in terms of expression signatures, within the functional genomic breast tumor classification, and ongoing efforts toward identification of new therapy targets and bioimaging markers. Due to the complexity of aberrant molecular patterns involved in expression, pathological progression and biological/clinical heterogeneity, the search for novel TNBC biomarkers and therapy targets requires collection of multi‐dimensional data sets, use of robust multivariate data analysis techniques and development of innovative systems biology approaches.

Monitoring and Predicting Response to Therapy with 18F-FDG PET in Colorectal Cancer: A Systematic Review

Molecular imaging with 18F-FDG PET has been proven useful in the management of colorectal cancer. 18F-FDG PET plays a pivotal role in staging before surgical resection of recurrent colorectal cancer and metastases, in the localization of recurrence in patients with an unexplained rise in serum carcinoembryonic antigen levels, and in the assessment of residual masses after treatment. Currently, there is increasing interest in the role of 18F-FDG PET beyond staging. The technique appears to have significant potential for the characterization of tumors and for the prediction of prognosis in the context of treatment stratification and early assessment of tumor response to therapy. This systematic review provides an overview of the literature on the value of 18F-FDG PET for monitoring and predicting the response to therapy in colorectal cancer. The review covers chemotherapy response monitoring in advanced colorectal cancer, monitoring of the effects of local ablative therapies, and preoperative radiotherapy and multimodality treatment response evaluation in primary rectal cancer. Given the added value of 18F-FDG PET for these indications, implementation in clinical practice and systematic inclusion in therapeutic trials to exploit the potential of 18F-FDG PET are warranted.

ImmunoSPECT and immunoPET of IGF-1R expression with the radiolabeled antibody R1507 in a triple-negative breast cancer model.

The insulinlike growth factor 1 receptor (IGF-1R) is a new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti–IGF-1R antibodies. Therefore, the aim of the present study was to develop a noninvasive, in vivo imaging method, using radiolabeled antibodies, to visualize IGF-1R expression. Methods: R1507 is a monoclonal antibody directed against the IGF-1R. In vitro, the affinity and internalization kinetics of 111In-R1507 were determined using the IGF-1R–expressing triple-negative breast cancer cell line SUM149. In vivo, the pharmacodynamics of 111In-R1507 and 125I-R1507 were determined in mice with subcutaneous SUM149 tumors. 111In-R1507 SPECT and 89Zr-R1507 PET images of mice with subcutaneous SUM149 tumors were acquired at 1, 3, and 7 d after injection. Results: 111In-R1507 (concentration required to inhibit binding by 50%, 0.1 nM) was slowly internalized by SUM149 cells. 111In-R1507 specifically and efficiently accumulated in the SUM149 xenografts: the tumor uptake was 20 percentage injected dose per gram (%ID/g), 33 %ID/g, and 31 %ID/g at 1, 3, and 7 d after injection, respectively. 125I-R1507 accumulated in the tumor less efficiently. Small-animal SPECT and small-animal PET of mice clearly visualized the subcutaneous SUM149 xenograft, with increasing contrast at later time points. Conclusion: 111In-R1507 and 89Zr-R1507 are new tracers to noninvasively determine IGF-1R expression in vivo in breast cancer xenografts using SPECT and PET. In the future, these techniques may enable patient selection for IGF-1R–targeted therapy.

Scintigraphic Techniques for Early Detection of Cancer Treatment–Induced Cardiotoxicity

New antitumor agents have resulted in significant survival benefits for cancer patients. However, several agents may have serious cardiovascular side effects. Left ventricular ejection fraction measurement by 99mTc multigated radionuclide angiography is regarded as the gold standard to measure cardiotoxicity in adult patients. It identifies left ventricular dysfunction with high reproducibility and low interobserver variability. A decrease in left ventricular ejection fraction, however, is a relatively late manifestation of myocardial damage. Nuclear cardiologic techniques that visualize pathophysiologic processes at the tissue level could detect myocardial injury at an earlier stage. These techniques may give the opportunity for timely intervention to prevent further damage and could provide insights into the mechanisms and pathophysiology of cardiotoxicity caused by anticancer agents. This review provides an overview of past, current, and promising newly developed radiopharmaceuticals and describes the role and recent advances of scintigraphic techniques to measure cardiotoxicity. Both first-order functional imaging techniques (visualizing mechanical [pump] function), such as 99mTc multigated radionuclide angiography and 99mTc gated blood-pool SPECT, and third-order functional imaging techniques (visualizing pathophysiologic and neurophysiologic processes at the tissue level) are discussed. Third-order functional imaging techniques comprise 123I-metaiodobenzylguanidine scintigraphy, which images the efferent sympathetic nervous innervations; sympathetic neuronal PET, with its wide range of tracers; 111In-antimyosin, which is a specific marker for myocardial cell injury and necrosis; 99mTc-annexin V scintigraphy, which visualizes apoptosis and cell death; fatty-acid-use scintigraphy, which visualizes the storage of free fatty acids in the lipid pool of the cytosol (which can be impaired by cardiotoxic agents); and 111In-trastuzumab imaging, to study trastuzumab targeting to the myocardium. To define the prognostic importance and clinical value of each of these functional imaging techniques, prospective clinical trials are warranted.New antitumor agents have resulted in significant survival benefits for cancer patients. However, several agents may have serious cardiovascular side effects. Left ventricular ejection fraction measurement by (99m)Tc multigated radionuclide angiography is regarded as the gold standard to measure cardiotoxicity in adult patients. It identifies left ventricular dysfunction with high reproducibility and low interobserver variability. A decrease in left ventricular ejection fraction, however, is a relatively late manifestation of myocardial damage. Nuclear cardiologic techniques that visualize pathophysiologic processes at the tissue level could detect myocardial injury at an earlier stage. These techniques may give the opportunity for timely intervention to prevent further damage and could provide insights into the mechanisms and pathophysiology of cardiotoxicity caused by anticancer agents. This review provides an overview of past, current, and promising newly developed radiopharmaceuticals and describes the role and recent advances of scintigraphic techniques to measure cardiotoxicity. Both first-order functional imaging techniques (visualizing mechanical [pump] function), such as (99m)Tc multigated radionuclide angiography and (99m)Tc gated blood-pool SPECT, and third-order functional imaging techniques (visualizing pathophysiologic and neurophysiologic processes at the tissue level) are discussed. Third-order functional imaging techniques comprise (123)I-metaiodobenzylguanidine scintigraphy, which images the efferent sympathetic nervous innervations; sympathetic neuronal PET, with its wide range of tracers; (111)In-antimyosin, which is a specific marker for myocardial cell injury and necrosis; (99m)Tc-annexin V scintigraphy, which visualizes apoptosis and cell death; fatty-acid-use scintigraphy, which visualizes the storage of free fatty acids in the lipid pool of the cytosol (which can be impaired by cardiotoxic agents); and (111)In-trastuzumab imaging, to study trastuzumab targeting to the myocardium. To define the prognostic importance and clinical value of each of these functional imaging techniques, prospective clinical trials are warranted.

Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with 18F-Labeled Affibody Molecule ZHER2:2395 in a Mouse Model for Ovarian Cancer

Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule 111In-DOTA-ZHER2:2395 can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)–expressing tumors, indicating that radiolabeled Affibody molecules have potential for patient selection for HER2-targeted therapy. Compared with SPECT, PET with positron-emitting radionuclides, such as 18F, may improve imaging of HER2 expression because of higher sensitivity and improved quantification of PET. The aim of the present study was to determine whether the 18F-labeled NOTA-conjugated Affibody molecule ZHER2:2395 is a suitable agent for imaging of HER2 expression. The tumor-targeting properties of 18F-labeled ZHER2:2395 were compared with 111In- and 68Ga-labeled ZHER2:2395 in mice with HER2-expressing SK-OV-3 xenografts. Methods: ZHER2:2395 was conjugated with NOTA and radiolabeled with 18F, 68Ga, and 111In. Radiolabeling with 18F was based on the complexation of Al18F by NOTA. The 50% inhibitory concentration values for NOTA-ZHER2:2395 labeled with 19F, 69Ga, and 115In were determined in a competitive cell-binding assay using SK-OV-3 cells. Mice bearing subcutaneous SK-OV-3 xenografts were injected intravenously with radiolabeled NOTA-ZHER2:2395. One and 4 h after injection, PET/CT or SPECT/CT images were acquired, and the biodistribution was determined by ex vivo measurement. Results: The 50% inhibitory concentration values for 19F-, 69Ga-, and 115In-NOTA-ZHER2:2395 were 5.0, 6.3, and 5.3 nM, respectively. One hour after injection, tumor uptake was 4.4 ± 0.8 percentage injected dose per gram (%ID/g), 5.6 ± 1.6 %ID/g, and 7.1 ± 1.4 %ID/g for 18F-, 68Ga-, and 111In-NOTA-ZHER2:2395, respectively, and the respective tumor-to-blood ratios were 7.4 ± 1.8, 8.0 ± 1.3, and 4.8 ± 1.3. Tumor uptake was specific, because uptake could be blocked efficiently by coinjection of an excess of unlabeled ZHER2:2395. PET/CT and SPECT/CT images clearly visualized HER2-expressing SK-OV-3 xenografts. Conclusion: This study showed that 18F-NOTA-ZHER2:2395 is a promising new imaging agent for HER2 expression in tumors. Affibody molecules were successfully labeled with 18F within 30 min, based on the complexation of Al18F by NOTA. Further research is needed to determine whether this technique can be used for patient selection for HER2-targeted therapy.

Constitutive expression of γ-H2AX has prognostic relevance in triple negative breast cancer.

BACKGROUND AND PURPOSE Constitutive γ-H2AX expression might indicate disruption of the DNA damage repair pathway, genomic instability, or shortened telomeric ends. Here, we quantified expression of endogenous γ-H2AX and its downstream factor 53BP1 in a large number of breast cancer cell lines (n=54) and a node-negative breast cancer cohort that had not received adjuvant systemic treatment (n=122). MATERIALS AND METHODS Formalin fixed paraffin embedded breast cancer cell lines and tumors were immunohistochemically analyzed for γ-H2AX and 53BP1 expression, and related to cell line, patient and tumor characteristics and to disease progression. RESULTS In breast cancer cell lines, γ-H2AX positivity was associated with the triple negative/basal like subgroup (p=0.005), and with BRCA1 (p=0.011) or p53 (p=0.053) mutations. Specifically in triple negative breast cancer patients a high number of γ-H2AX foci indicated a significantly worse prognosis (p=0.006 for triple negative vs. p=0.417 for estrogen receptor (ER), progesterone receptor (PR) or HER2 positive patients). A similar association with disease progression was found for 53BP1. In a multivariate analysis with tumor size, grade, and triple negativity, only the interaction between triple negativity and γ-H2AX remained significant (p=0.002, Hazard Ratio=6.77, 95% CI=2.07-22.2). CONCLUSIONS Constitutive γ-H2AX and 53BP1 staining reveals a subset of patients with triple negative breast tumors that have a significantly poorer prognosis.

Tribbles homolog 3 denotes a poor prognosis in breast cancer and is involved in hypoxia response.

IntroductionHypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer.MethodsTRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed.ResultsBreast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity.ConclusionsTRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxia.

Bevacizumab reduces tumor targeting of antiepidermal growth factor and anti-insulin-like growth factor 1 receptor antibodies.

Bevacizumab (antivascular endothelial growth factor [anti‐VEGF]) and cetuximab (antiepidermal growth factor receptor [anti‐EGFR]) are approved antibodies for treatment of cancer. However, in advanced colorectal cancer, the combination fails to improve survival. As the reason for the lack of activity is unknown, our study aims to determine the effect of bevacizumab on targeting of anti‐EGFR and insulin‐like growth factor 1 receptor (IGF‐1R) antibodies in tumors with single‐photon emission computed tomography (SPECT)/CT imaging. Mice with subcutaneous EGFR and IGF‐1R‐expressing SUM149 xenografts received a single dose of bevacizumab (10 mg/kg) or saline. After 4 days, mice were injected with radiolabeled cetuximab or R1507, an anti‐IGF‐1R antibody. A control group received a radiolabeled irrelevant IgG (hLL2). Three days later, SPECT/CT images were acquired and mice were dissected to determine the concentration of antibodies in the tissues. Tumors were analyzed immunohistochemically to determine vascular density (CD34), VEGF, EGFR and IGF‐1R expression. SPECT/CT imaging revealed that bevacizumab treatment significantly reduced tumor targeting of radiolabeled cetuximab by 40% from 33.1 ± 1.1 %ID/g to 19.8 ± 5.7 %ID/g (p = 0.009) for untreated and bevacizumab‐treated tumors, respectively. A similar effect was found for 111In‐R1507: tumor targeting of R1507 decreased by 35%. No significant differences in tumor uptake were observed in mice that received an irrelevant IgG. Uptake in normal organs was not altered by bevacizumab. Immunohistochemical analysis showed that vascular density decreased with 43%, whereas EGFR and IGF‐1R expression was unaltered. In conclusion, bevacizumab treatment significantly reduces tumor targeting of anti‐EGFR and anti‐IGF‐1R antibodies. This emphasizes the importance of timing and sequencing of bevacizumab in combination with other antibodies.

Tumour response prediction by diffusion-weighted MR imaging: Ready for clinical use?

BACKGROUND The efficacy of anticancer therapy is usually evaluated by anatomical imaging. However, this method may be suboptimal for the evaluation of novel treatment modalities, such as targeted therapy. Theoretically, functional assessment of tumour response by diffusion weighted imaging (DWI) is an attractive tool for this purpose and may allow an early prediction of response. The optimal use of this method has still to be determined. METHOD We reviewed the published literature on clinical DWI in the prediction of response to anticancer therapy, especially targeted therapy. Studies investigating the role of DWI in patients with cancer either for response prediction and/or response monitoring were selected for this analysis. RESULTS We identified 24 studies that met our criteria. Most studies showed a significant correlation between (changes in) apparent diffusion coefficient (ADC) values and treatment response. However, in different tumours and studies, both high and low pretreatment ADC were found to be associated with response rate. In the course of treatment, an increase in ADC was associated with response in most cases. CONCLUSION The potential of DWI for (early) response monitoring of anticancer therapies has been demonstrated. However, validation is hampered by the lack of reproducibility and standardisation. We recommend that these issues should be properly addressed prior to further testing the clinical use of DWI in the assessment of treatments.

The CYP2C19*2 genotype predicts tamoxifen treatment outcome in advanced breast cancer patients

AIMS Tamoxifen is metabolized by cytochrome P450s, with an important role for CYP2D6. Recently, we demonstrated in 80 patients that CYP2C19*2 is associated with increased survival in breast cancer patients using tamoxifen. Here, we aimed to confirm this in a large group of 499 patients. MATERIALS & METHODS A total of 499 estrogen receptor-positive primary breast tumor specimens of advanced disease patients treated with first-line tamoxifen were genotyped for CYP2C19*2 and *17 variant alleles, with primary end point time-to-treatment failure (TTF). Effects of CYP2C19, independent of treatment, were analyzed in 243 primary systematic untreated patients. RESULTS CYP2C19*2 hetero- and homozygote patients combined showed significantly longer TTFs (hazard ratio [HR]: 0.72; 95% CI: 0.57-0.90; p = 0.004). In multivariate analysis, including CYP2D6*4 status, CYP2C19*2 remained independently associated with TTF (HR: 0.73; 95% CI: 0.58-0.91; p = 0.007). In untreated patients, the CYP2C19*17 allele was significantly associated with a longer disease-free interval (HR: 0.66; 95%CI: 0.46-0.95; p = 0.025). CONCLUSION CYP2C19 genotyping is potentially important for tamoxifen therapy for advanced disease and for breast cancer prognosis.