Leveraging Tumor Stress Responses for a See and Treat Paradigm in Breast Cancer: Applications in Local and Global Health

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2018

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Abstract

With the widespread adoption of mammograms for early breast cancer detection in high income countries (HICs), modern research has principally pivoted towards a focus on reducing overtreatment of patients, particularly those with early stage breast cancer. There are numerous examples of clinical practices and regulations that reflect this shift, including changes in screening recommendations, patient monitoring, re-excision guidelines, and genetic testing, all of which seek to reduce unnecessary intervention without compromising patient outcomes.

Despite changing guidelines, there remains a distinct lack of technologies to reduce overtreatment while ensuring the best possible outcome for patients. One such example is Breast Conserving Surgery (BCS) followed by radiation therapy. There is a wide-range of re-excision rates reported in the literature, but most groups report that 20-40% of patients undergo at least one re-excision. Taking additional shavings during BCS, new guidelines dictating relationships between margin status after BCS and re-excision, and radiation therapy all strive to maximize removal of residual tumor cells with as few surgeries as possible in patients with a new breast cancer diagnosis. However, secondary cancers from radiation therapy, the potential for cancer dissemination as a result of re-excision surgeries, and the burgeoning costs of repeat visits and interventions to an already depleted health care system necessitate new and innovative solutions to improve health outcomes while reducing health expenditures.

While seeking to improve patient experience in HICs, many women in low to middle income countries (LMICs) are unable to access adequate screening and life-saving treatments. Even those who manage to receive a proper diagnostic test are all too frequently lost to follow up care, leading to a disproportionately high burden of breast cancer mortality in LMICs.

The goal of the work presented here is to reduce overtreatment in HICs while simultaneously minimizing access barriers to screening and treatment for women in LMICs through developing a rapid and low-cost molecular diagnostic platform for breast cancer. In HICs, this diagnostic platform could be deployed at two points in the breast cancer care cascade: 1) during diagnostic biopsy to ensure adequate lesion sampling at the point-of-diagnosis, and 2) during intraoperative margin assessment as a ‘see-and-treat’ paradigm that utilizes a single agent to guide surgical resection and to treat residual disease during surgery. In LMICs where limited access to tissue processing equipment and a pathologist often render histological examination of tissue impossible, the diagnostic platform could be used to cheaply and robustly diagnose tissue at the point-of-care.

Three specific aims were proposed to develop the diagnostic platform. The first aim was to demonstrate a single-agent see-and-treat paradigm in pre-clinical models of breast cancer using a fluorescent tracer across all subtypes of breast cancer. The diagnostic piece began by showing that a fluorescently-tethered Hsp90 inhibitor (HS-27), made up of an Hsp90 inhibitor previously used in clinical trials tethered to a fluorescein isothiocyanate (FITC) derivative, is taken up by breast cancer cells in vitro regardless of receptor subtype, and that blocking the ATP binding pocket of Hsp90 leads to reduced HS-27 fluorescence, confirming that fluorescence is a result of HS-27 bound to its target. The in vitro study was expanded to define the therapeutic potential of HS-27 by demonstrating degradation of Hsp90 client proteins consistent with Hsp90 inhibition, and reduction of cellular metabolism, confirming protein degradation led to downstream effects on its signaling pathway. To round out the therapy component of our ‘see-and-treat’ paradigm, HS-27 treatment was found to reduce cell proliferation rates across breast cancer receptor subtypes.

The diagnostic component was moved into animals using a dorsal skinfold window chamber model to interrogate HS-27 uptake in vivo in the context of tumors and their surrounding microenvironment. As expected, HS-27 uptake was significantly greater in tumor window chambers than in non-tumor controls. Utilizing a fluorescent glucose analog to examine glucose uptake levels in tumors as a surrogate for aggressive disease showed that HS-27 strongly correlated (R2 = 0.96) with glucose uptake, suggesting surface Hsp90 expression is upregulated in aggressive glycolytic tumors.

To finish aim 1, HS-27 staining was performed on tumors ex vivo, achieving comparable contrast to in vivo agent administration, providing a path towards translating HS-27 to ex vivo clinical use. A small ex vivo pilot clinical study in patients undergoing diagnostic biopsy revealed a significant correlation between HS-27 uptake and the percentage of tumor present in the sample, providing first proof-of-principle of our HS-27 fluorescence-based diagnostic platform in patients. HS-27 was first imaged in biopsies in order to enroll patients across different receptor subtypes rather than at surgery where the majority of patients have estrogen receptor positive (ER+) disease. To summarize, aim 1 demonstrated a ‘see-and-treat’ paradigm in pre-clinical models of breast cancer, and provided a path towards moving HS-27 into the clinic.

With proof-of-principle patient results revealing that HS-27 may be a feasible diagnostic tool, the focus of aim 2 transitioned towards optimizing the imaging system and protocol. The ex vivo imaging strategy was optimized to minimize non-specific HS-27 uptake in preclinical models. Imaging parameters were fully vetted in a clinical study designed to interrogate HS-27 uptake in patients with breast cancer or benign conditions, as well as in a disease-free population. A high-resolution microendoscope (HRME) designed to image FITC fluorescence in a pre-clinical biopsy model was used to investigate how time between tissue excision and imaging, agent incubation time, and agent dose affect the specificity of HS-27 based diagnostics. For these experiments, a modified version of HS-27 with a 100-fold reduction in Hsp90 affinity, called HS-217, was used to establish non-specific fluorophore uptake. Calculating the ratio of HS-27 fluorescence to HS-217 fluorescence provided a ‘specificity ratio’ that was maximized with a post-excision window up to 10-minutes, 1-minute incubation time, and 100 µM dose.

The optimized protocol was then tested in 37 patients undergoing ultrasound-guided core needle biopsy and in 6 disease-free patients undergoing breast reduction mammoplasty. HS-27 uptake was significantly greater in tumor samples than mammoplasty control samples. Interestingly, HS-27 uptake was similar in tumor and benign lesion samples on average, however, examining the distribution of fluorescence across the biopsy reveals different staining patterns between tumor and benign lesions. Concurrent with the finding in aim 1 that HS-27 levels are elevated in aggressive tumors, HS-27 strongly and inversely correlated with the presence of tumor infiltrating lymphocytes, a positive prognostic marker in Her2+ and triple negative breast cancers. Additionally, leveraging both intensity and spatial patterns to generate a Gaussian support vector machine classifier allowed for accurate classification of tumor, benign lesion, and mammoplasty samples. Classification of tumor vs benign lesions resulted in an area under the receiver operating characteristic curve (AUC) of 0.93 with a sensitivity of 82% and specificity of 100%. Classification of tumor vs mammoplasty samples resulted in an AUC of 0.96 with a sensitivity of 86% and specificity of 100%.

So far, HS-27 uptake has been shown to be specific to tumor over non-tumor tissues, increased HS-27 fluorescence was suggestive of an aggressive tumor phenotype, and ex vivo HS-27 imaging accurately distinguished tumor from both benign and normal breast tissue. Two limitations of the imaging system used in aims 1 and 2 were: 1) the requirement to place the HRME probe in contact with the tissue, potentially causing artificial changes in signal due to pressure differences during probe placement, and 2) the small field of view, which prohibited translation to samples larger than 1-2 cm. Thus, the goal of aim 3 was to develop a wide-field, non-contact imaging system to demonstrate feasibility of translating ex vivo HS-27 imaging to multiple points in the breast cancer care cascade.

We have previously developed a Pocket colposcope for cervical pre-cancer detection and have recently completed construction and testing of an alpha prototype. The colposcope contains a 5 MP camera and white and green light emitting diodes (LEDs) on the tip. It weighs 1 pound, and interfaces with a phone, tablet, or computer, which provides power to the device and enables image capture. The Pocket colposcope, which will now be referred to as a Pocket mammascope is well-suited for breast margin imaging with the ability to, survey breast tumor margins as large as 10 -cm2 in a few snapshots, while maintaining the ability to image a cluster of tumor cells on a length scale of several microns.

The Pocket colposcope was modified into a Pocket mammoscope to perform fluorescence imaging through the addition of a collar with excitation LEDs and a bandpass filter for fluorescence collection. A series of bench tests show that the Pocket mammoscope can perform fluorescence imaging in a wide-field mode with a diagonal field of view of 3.25 cm (compared to 750 µm with the HRME) at a resolution of 25 µm (compared to ~4 µm with the HRME), and high-resolution mode with a diagonal field of view of 1.25 cm and resolution of 12 µm. The two imaging modes are easily navigated between through the use of a simple slider mechanism. The Pocket mammoscope was next used to image HS-27 fluorescence across in vivo and ex vivo models, with comparable results to our previous imaging systems. Additionally, the optimized ex vivo imaging protocol from aim 2 was used to shown to be compatible with the Pocket mammoscope in a cohort of patients undergoing standard-of-care ultrasound-guided core needle biopsy, and that that Pocket mammoscope is capable of imaging an entire biopsy in a single snapshot. Proof-of-concept translation to intraoperative margin assessment utilizing a window chamber model, similar to aim 1, validated that the Pocket mammoscope could image HS-27 both systemically and topically delivered to a tumor.

In conclusion, this work set out to provide a theranostic tool to reduce overtreatment for patients with breast cancer in HICs, and provide a rapid diagnostic test implementable at the point-of-care in LMICs. Towards these goals, aim 1 showed that HS-27 uptake is higher in more aggressive tumors, potentially serving as a prognostic marker delineating which patients require more or less aggressive treatment regimens. Aim 2 found that a Gaussian support vector machine classification scheme based on features from ex vivo HS-27 images accurately distinguishes tumor from both benign conditions and normal breast tissue. Finally, aim 3 demonstrated the feasibility of translating HS-27 to both diagnostic biopsy and intraoperative margin assessment by creating a Pocket mammoscope capable of imaging an entire biopsy and a tumor margin in a few snapshots. Ultimately, this work demonstrates that HS-27 imaging with the Pocket mammoscope is a means for rapid, automated detection of breast cancer, regardless of subtype, which could improve breast cancer management in both HICs and LMICs.

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Crouch, Brian Thomas (2018). Leveraging Tumor Stress Responses for a See and Treat Paradigm in Breast Cancer: Applications in Local and Global Health. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/17498.

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