JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Published: March 29th, 2021

DOI:

10.3791/62394

1Departments of Research Pathology, Genentech, 2Bioinformatics & Computational Biology, Genentech, 3Roche Sequencing Solutions, Hacienda Drive, 4Department of Medicine, Vanderbilt University Medical Center, Medical Center Drive, 5Department of Pathology, Northwestern University, Feinberg School of Medicine

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&E) annotations are used to target small tumor areas using a blade that is 250 µm2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 µm in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Next generation sequencing (NGS) is increasingly utilized for both patient care and in cancer research to help guide treatments and facilitate scientific discovery. Tissue is often limited and small specimens with variable tumor content are routinely used. Tumor adequacy and integrity, therefore, remain a barrier to obtaining meaningful data. Samples with lower tumor percentages may cause difficulty in distinguishing true variants from sequencing artifacts and are often ineligible for NGS1. Tumor enrichment of low tumor content cases, those below 20%, has been shown to help yield sufficient material in order to generate reproducible sequencing ....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Prior to initiation, obtain appropriate tissue specimens according to Institutional Review Board (IRB) protocols. All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Genentech, Inc.

1. Tissue and slide preparation

  1. Select FFPE or fresh frozen tissue blocks and utilize the corresponding processing method below.
  2. Cut tissue block sections onto positively charged glass slides at the desired thickness. Serially section the.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

FFPE and FF mouse liver sections containing metastatic colorectal cancer in xenografts were selected. Sections were H&E stained (Figure 1A,E,I) and scanned on a whole slide imager at 20x magnification. A pathologist digitally annotated tumor regions of interest and a mask was generated using commercial software and formatted as a digital png reference image (Figure 1B,F,J). Serial 10 µm and 20 µm thick unstained sa.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Presented here is a protocol for the application of digital annotation and automated dissection to dissect tumor regions from low tumor content FFPE or fresh frozen tissues for tumor enrichment and use in WES. Combining digital annotation and mask creation with automated dissection significantly reduces the required hands-on time and expertise common to classical methods of tumor enrichment inclusive of manual macrodissection and LCM. The protocol demonstrates a potentially important mid-range tumor enrichment option tha.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The authors would like to thank Carmina Espiritu and Robin E. Taylor for their support in automated dissection development as well as the Genentech Pathology Core Laboratory staff that supported this work.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Agilent SureSelectXT Agilent G9611A
AVENIO Millisect Fill Station Roche 8106533001
AVENIO Millisect Instrument, Base Roche 8106568001
AVENIO Millisect Instrument, Head Roche 8106550001
AVENIO Millisect Milling Tips Small Roche 8106509001
AVENIO Millisect PC Roche 8106495001
BioAnalyzer Agilent G2939BA
Eppendorf 5427R Eppendorf 22620700 Micro-centrifuge
Incubation Buffer Promega D920D
Leica Autostainer XL Leica ST5010 Automated stainer
Molecular Grade Mineral Oil Sigma M5904-500ML
Proteinase K Promega V302B Digestion buffer
Qiagen AllPrep DNA/RNA Mini Kit Qiagen 80284
RLT Plus buffer Qiagen 80204
Superfrost Plus positively charged microscope slides Thermo Scientific 6776214

  1. Cho, M., et al. Tissue recommendations for precision cancer therapy using next generation sequencing: a comprehensive single cancer center's experiences. Oncotarget. 8 (26), 42478-42486 (2017).
  2. Smits, A. J. J., et al. The estimation of tumor cell percentage for molecular testing by pathologists is not accurate. Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc. 27 (2), 168-174 (2014).
  3. Poole-Wilson, P. A., Langer, G. A. Effect of pH on ionic exchange and function in rat and rabbit myocardium. The American Journal of Physiology. 229 (3), 570-581 (1975).
  4. Viray, H., et al. A prospective, multi-institutional diagnostic trial to determine pathologist accuracy in estimation of percentage of malignant cells. Archives of Pathology & Laboratory Medicine. 137 (11), 1545-1549 (2013).
  5. El-Serag, H. B., et al. Gene Expression in Barrett's Esophagus: Laser capture versus whole tissue. Scandinavian Journal of Gastroenterology. 44 (7), 787-795 (2009).
  6. Harrell, J. C., Dye, W. W., Harvell, D. M. E., Sartorius, C. A., Horwitz, K. B. Contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors: a comparison of experimental breast cancers and their lymph node metastases. Clinical & Experimental Metastasis. 25 (1), 81-88 (2008).
  7. Kim, H. K., et al. Distinctions in gastric cancer gene expression signatures derived from laser capture microdissection versus histologic macrodissection. BMC Medical Genomics. 4, 48 (2011).
  8. Klee, E. W., et al. Impact of sample acquisition and linear amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-dissection cell-sampling versus bulk tissue-sampling. BMC Medical Genomics. 2, 13 (2009).
  9. Civita, P., et al. Laser capture microdissection and RNA-seq analysis: High sensitivity approaches to explain histopathological heterogeneity in human glioblastoma FFPE archived tissues. Frontiers in Oncology. 9, 482 (2019).
  10. Emmert-Buck, M. R., et al. Laser capture microdissection. Science. 274 (5289), 998-1001 (1996).
  11. Bonner, R. F., et al. Laser capture microdissection: molecular analysis of tissue. Science. 278 (5342), 1481-1483 (1997).
  12. Hunt, J. L., Finkelstein, S. D. Microdissection techniques for molecular testing in surgical pathology. Archives of Pathology & Laboratory Medicine. 128 (12), 1372-1378 (2004).
  13. Espina, V., et al. Laser-capture microdissection. Nature Protocols. 1, 586-603 (2006).
  14. Grafen, M., et al. Optimized expression-based microdissection of formalin-fixed lung cancer tissue. Laboratory Investigation; A Journal of Technical Methods and Pathology. 97 (7), 863-872 (2017).
  15. Javey, M., et al. innovative tumor tissue dissection tool for molecular oncology diagnostics. The Journal of Molecular Diagnnostics: JMD. (21), 1525-1578 (2021).
  16. Adey, N., et al. A mill based instrument and software system for dissecting slide-mounted tissue that provides digital guidance and documentation. BMC Clinical Pathology. 13 (1), 29 (2013).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved