A smartphone pathology platform to characterize tumor cytology and HER2 expression in breast cancer

Scientists from the Department of Biomedical Engineering and the Department of Surgery, Duke University (Durham, NC, USA) have developed a multimodal mobile platform, EpiView-D4, as an economic diagnostic pathology approach for point-of-care characterization of the cellular morphology and molecular expression of clinically relevant biomarkers in fine-needle aspiration specimens from breast tumors.

In a proof-of-concept study, EpiView-D4 could accurately and reliably distinguish between human epidermal growth factor receptor-2 (HER2)-positive and HER2-negative cell lines and tumor specimens, while the assay required less than 1 hour. The findings of this study were published in njp Breast Cancer on July 2, 2021.

Cellular and molecular profiles of breast tumors

Pathology plays a critical role in breast cancer diagnosis and clinical management. Typically, surgically excised breast tissues are immunohistochemically stained for Ki67, HER2, estrogen receptor (ER), and progesterone receptor (PR). The expression levels of these biomarkers form the basis of tumor subtyping and play a fundamental role in determining the best treatment for each patient.1

Personalized treatment based on biomarker expression can significantly improve treatment outcomes in patients with breast cancer. However, accurate cellular and molecular characterization of breast tumors requires well-equipped centralized diagnostic facilities and trained laboratory staff—all of which are often scarce in countries and areas with limited resources.2

“Pathology analysis of breast tumor tissue is the cornerstone of breast cancer management, as it provides a cellular diagnosis of cancer and also profiles the expression of key molecular biomarkers which guide selection of medicines to which the tumors are most likely to respond,” said Dr Ashutosh Chilkoti, Alan L. Kaganov Distinguished Professor and Chair of Biomedical Engineering at Duke University and corresponding author of the study. “Unfortunately, clinical pathology laboratory services are resource-intensive and not widely available in low resource settings.”

EpiView-D4: a point-of-care smartphone diagnostic platform

“We have developed a handheld pathology platform (the so-called “EpiView-D4”) based on a cell phone that can accomplish both cellular and molecular evaluation of breast tumors in a low-cost, user- and resource-friendly fashion,” said Dr Chilkoti.

The multimodal mobile phone-based platform EpiView-D4 consists of two main components. The first component of the platform, D4, is a self-contained sandwich immunodiagnostic chip consisting of an antibody pair onto a glass substrate and coated with a “non-fouling” polymer brush film. The chip can directly determine the expression levels of protein biomarkers, such as HER2, directly in cell lysates.3

The second key component of EpiView-D4 is a custom mobile phone-based optical microscope, which can perform imaging using the mobile phone camera. EpiView is a modular device consisting of two switchable imaging units: one for brightfield imaging and one for fluorescence imaging. Hence, the system can perform both brightfield imaging (to analyze imaging cytology samples) and fluorescence imaging (to analyze biomarker data acquired from chip D4).3

Fine-needle aspiration specimens from breast tumors are smeared on a glass slide and processed with rapid staining kits for cytology evaluation. Stained samples are then subjected to brightfield imaging using the smartphone camera.

For biomarker evaluation, fine-needle aspirates are lysed, and crude lysates are immunoassayed using the D4 chip. Subsequently, samples are imaged using the fluorescence imaging system, and the levels of HER2—or a different analyte of interest—are determined based on fluorescence signal intensity.3

“The mainstay approach for specimen collection in low resource settings is fine-needle aspiration,” said Dr Chilkoti. “The cellphone-based mobile microscope allows a user to perform bright field imaging of fine-needle aspiration cytology specimens for cellular morphology evaluation, and then the device is switched to fluorescence mode to read out the results of a disposable point-of-care chip that analyzes molecular biomarker expression levels in the aspirate specimen—in this case, HER2.”


Source: Joh, D.Y., Heggestad, J.T., Zhang, S. et al. Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates. npj Breast Cancer 7, 85 (2021). https://doi.org/10.1038/s41523-021-00290-0

Source: Joh, D.Y., Heggestad, J.T., Zhang, S. et al. Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates. npj Breast Cancer 7, 85 (2021). https://doi.org/10.1038/s41523-021-00290-0

Molecular specificity of the D4 immunodiagnostic chip

To determine the dynamic range of the D4 chip in quantifying different analytes, scientists used the system to quantify HER2 levels in samples containing various amounts of recombinant HER2. The system could detect as little as ~16 pM of recombinant protein, with a dynamic range spanning more than three orders of magnitude.3

Additionally, scientists used the chip D4 to analyze HER2 expression in multiple established cancer cell lines and patient-derived tumor cells. They found that the immunodiagnostic chip could reliably distinguish between HER2-positive and HER2-negative samples. Additionally, the results of the HER2 D4 assay were highly consistent with those of immunoblotting, confirming the specificity of the assay.3

Proof-of-concept: Using EpiView-D4 to evaluate cellularity and HER2 expression in breast cancer

To validate the performance of EpiView-D4, researchers used the mobile platform to assess cellular morphology and HER2 expression levels in various animal models of breast cancer. In xenografts from mice engrafted with human breast cancer cell lines, EpiView-D4 could reliably distinguish between HER2-positive and HER2-negative samples. HER2 levels determined by EpiView-D4 assay were highly consistent with those of immunoblotting, ELISA, and immunohistochemistry.3

EpiView-D4 was also used to analyze human tissues from 19 patients with breast cancer. The use of EpiView-D4 to analyze fine-needle aspiration cytology specimens of breast tumors provided accurate and reliable characterization of lesional cellularity and tumor content.

In keeping with the ability of EpiView-D4 to reliably characterize the expression levels of HER2 in cell lines and animal xenografts, EpiView-D4 accurately distinguished between HER2-negative and HER2-positive breast tumors from human subjects. Notably, fine-needle aspiration specimens were prepared in less than 5 minutes with rapid staining kits, and the total duration of the assay did not exceed 1 hour.3

Future perspectives

Limited resources are a significant factor hindering the successful treatment of breast cancer and contributing to inequalities in breast cancer mortality between developed and developing countries. This association between limited-resource settings and poor breast cancer outcomes can be attributed, to some extent, to late-stage diagnosis of breast tumors due to the lack of adequate diagnostic pathology services.

The findings of this study suggest that EpiView-D4 may represent a low-cost, simple, user-friendly, fast, and logistically sustainable molecular and cellular diagnostic pathology approach to guide clinical decision making for patients with breast cancer and improve breast cancer mortality—especially in countries with insufficient healthcare infrastructure and diagnostic pathology services.

“Although modern breast cancer care that is “personalized” based on biomarker expression has led to dramatically improved survival rates in abundant resource settings in the last several decades, the lack of adequate pathology services continues to be a major contributor to the wide mortality gap between underdeveloped versus developed countries,” said Dr Chilkoti. “This study provides proof-of-concept demonstration that our technology can accomplish both cellular and molecular pathology assessment of breast tumors on a single low-cost device in an hour or less. We think that the EpiView-D4 can be a valuable diagnostic adjunct to breast cancer services in low resource settings and has the potential to democratize access to effective breast cancer treatment worldwide.”

However, certain limitations need to be addressed to improve the diagnostic and analytical performance of this smartphone pathology platform. For instance, EpiView has a relatively low analytical sensitivity, with a limit of detection (LOD) of 77 pM. This analytical sensitivity is ~4-fold lower than that of a tabletop scanner. In addition, as EpiView-D4 uses fine-needle aspirates, the analysis does not take into account tissue morphology and structural information.

Future large-scale studies are warranted to evaluate the performance of EpiView-D4 in characterizing the expression of HER2 and other clinically relevant biomarkers in various tumor specimens. Future studies are also required to compare the diagnostic and analytical performance of EpiView-D4 to that of standard cell-block pathology methods.


To learn more about the use of EpiView to characterize tumor cytology and HER2 expression in breast cancer, read the article by Joh D, Heggestad J, Zhang S et al., “Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates,” npj Breast Cancer 7, 85 (2021).


References

  1. Taneja P, Maglic D, Kai F, et al. Classical and Novel Prognostic Markers for Breast Cancer and their Clinical Significance. Clin Med Insights Oncol. 2010;4:15-34. doi:10.4137/cmo.s4773
  2. Horgan D, Ciliberto G, Conte P, et al. Bringing Greater Accuracy to Europe’s Healthcare Systems: The Unexploited Potential of Biomarker Testing in Oncology. Biomed Hub. 2020;5(3):1-42. doi:10.1159/000511209
  3. Joh DY, Heggestad JT, Zhang S, et al. Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates. npj Breast Cancer. 2021;7(1):85. doi:10.1038/s41523-021-00290-0

 

Christos received his Masters in Cancer Biology from Heidelberg University and PhD from the University of Manchester.  After working as a scientist in cancer research for ten years, Christos decided to switch gears and start a career as a medical writer and editor. He is passionate about communicating science and translating complex science into clear messages for the scientific community and the wider public.

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