Innovative Tumor Tissue Dissection Tool for Molecular Oncology Diagnostics

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-μm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation. Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-μm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation. Molecular diagnostic tests that use tumor tissue specimens are an integral part of today's oncology practice and patient care.1Nangalia J. Campbell P.J. Genome sequencing during a patient's journey through cancer.N Engl J Med. 2019; 381: 2145-2156Crossref PubMed Scopus (24) Google Scholar Such testing is largely performed on formalin-fixed, paraffin-embedded (FFPE) tissue from patient biopsy specimens or surgical procedures to provide critical information for diagnosis, treatment guidance, and prognosis. It is imperative to meet pre-analytic tissue quality requirements to obtain optimal results in downstream applications, especially if tissue samples are limited. In addition to many other quality parameters, somatic mutation testing heavily depends on the percentage of non-necrotic tumor cells in patient specimens. However, the tumor percentage can be confounded by the tumor tissue size in a section and has a different meaning across specimen types, such as larger surgical specimens versus small core needle biopsy specimens.2Goswami R.S. Luthra R. Singh R.R. Patel K.P. Routbort M.J. Aldape K.D. Yao H. Dang H.D. Barkoh B.A. Manekia J. Medeiros L.J. Roy-Chowdhuri S. Stewart J. Broaddus R.R. Chen H. Identification of factors affecting the success of next-generation sequencing testing in solid tumors.Am J Clin Pathol. 2016; 145: 222-237Crossref PubMed Google Scholar Hence, the sample requirements (such as tissue dimensions, thickness of sections, tumor content, and necrosis) for downstream analytical processes may vary across the specimen types and require macrodissection or microdissection for tumor enrichment. Different molecular testing approaches require different input material thresholds; for example, Sanger sequencing requires >40% tumor content and may yield false-negative results if the tested sample tumor cellularity is low.3Janne P.A. Borras A.M. Kuang Y. Rogers A.M. Joshi V.A. Liyanage H. Lindeman N. Lee J.C. Halmos B. Maher E.A. Distel R.J. Meyerson M. Johnson B.E. A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening.Clin Cancer Res. 2006; 12: 751-758Crossref PubMed Scopus (192) Google Scholar, 4Liu X. Lu Y. Zhu G. Lei Y. Zheng L. Qin H. Tang C. Ellison G. McCormack R. Ji Q. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.J Clin Pathol. 2013; 66: 1065-1069Crossref PubMed Scopus (122) Google Scholar, 5Zhu C.Q. da Cunha Santos G. Ding K. Sakurada A. Cutz J.C. Liu N. Zhang T. Marrano P. Whitehead M. Squire J.A. Kamel-Reid S. Seymour L. Shepherd F.A. Tsao M.S. National Cancer Institute of Canada Clinical Trials Group Study BR.21Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21.J Clin Oncol. 2008; 26: 4268-4275Crossref PubMed Scopus (636) Google Scholar Although next-generation sequencing (NGS)–based assays can detect mutations in samples with approximately 20% tumor content,6Singh R.R. Patel K.P. Routbort M.J. Aldape K. Lu X. Manekia J. Abraham R. Reddy N.G. Barkoh B.A. Veliyathu J. Medeiros L.J. Luthra R. Clinical massively parallel next generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours.Br J Cancer. 2014; 111: 2014-2023Crossref PubMed Scopus (66) Google Scholar to prevent false-negative results, especially for low allele frequency variants, enrichment methods are recommended. Hence, tumor enrichment, which entails tumor dissection and selective exclusion of the adjacent nontumor tissue, is a critical preanalytic step to ensure sufficient tumor-derived nucleic acids and protein representation in the sample for downstream molecular testing. The current standardized workflow across molecular diagnostic laboratories for tumor tissue enrichment is not very efficient and entails manual dissection on tissue sections derived from FFPE tumor tissue blocks. The procedure involves hematoxylin and eosin (H&E)–stained slide annotation by a pathologist, with the annotated slide guiding the technician to manually scrape the tissue with a scalpel or razor blade from a consecutive, unstained slide. This process is time-consuming and laborious when multiple slides need to be processed simultaneously to produce enough input material for downstream processes. In addition, manual dissection can be inaccurate7Smits A.J. Kummer J.A. de Bruin P.C. Bol M. van den Tweel J.G. Seldenrijk K.A. Willems S.M. Offerhaus G.J. de Weger R.A. van Diest P.J. Vink A. The estimation of tumor cell percentage for molecular testing by pathologists is not accurate.Mod Pathol. 2014; 27: 168-174Crossref PubMed Scopus (113) Google Scholar, 8Viray H. Li K. Long T.A. Vasalos P. Bridge J.A. Jennings L.J. Halling K.C. Hameed M. Rimm D.L. A prospective, multi-institutional diagnostic trial to determine pathologist accuracy in estimation of percentage of malignant cells.Arch Pathol Lab Med. 2013; 137: 1545-1549Crossref PubMed Scopus (66) Google Scholar, 9Hipp J.D. Johann D.J. Chen Y. Madabhushi A. Monaco J. Cheng J. Rodriguez-Canales J. Stumpe M.C. Riedlinger G. Rosenberg A.Z. Hanson J.C. Kunju L.P. Emmert-Buck M.R. Balis U.J. Tangrea M.A. Computer-aided laser dissection: a microdissection workflow leveraging image analysis tools.J Pathol Inform. 2018; 9: 45Crossref PubMed Scopus (5) Google Scholar because of imprecise alignment of unstained and H&E-stained slides, potentially leading to false-negative results.10Geiersbach K. Adey N. Welker N. Elsberry D. Malmberg E. Edwards S. Downs-Kelly E. Salama M. Bronner M. Digitally guided microdissection aids somatic mutation detection in difficult to dissect tumors.Cancer Genet. 2016; 209: 42-49Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Accurate morphologic visualization is essential in microdissection of small biopsy specimens and low tumor content excisional specimens. Although there have been efforts to stain slides for various tissue-specific markers with immunohistochemistry for accurate annotation, this exercise requires additional steps and delays subsequent dissection.11Rosenberg A.Z. Armani M.D. Fetsch P.A. Xi L. Pham T.T. Raffeld M. Chen Y. O'Flaherty N. Stussman R. Blackler A.R. Du Q. Hanson J.C. Roth M.J. Filie A.C. Roh M.H. Emmert-Buck M.R. Hipp J.D. Tangrea M.A. High-throughput microdissection for next-generation sequencing.PLoS One. 2016; 11: e0151775Crossref PubMed Scopus (15) Google Scholar Laser capture microdissection can offer an alternative option to manual dissection12Emmert-Buck M.R. Bonner R.F. Smith P.D. Chuaqui R.F. Zhuang Z. Goldstein S.R. Weiss R.A. Liotta L.A. Laser capture microdissection.Science. 1996; 5289: 998-1001Crossref Scopus (2060) Google Scholar,13Magel L. Bartels S. Lehmann U. Next-generation sequencing analysis of lasermicrodissected formalin-fixed and paraffin-embedded (FFPE) tissue specimens.Methods Mol Biol. 2018; 1723: 111-118Crossref PubMed Scopus (3) Google Scholar; however, the equipment is technically complex to operate, carries a significant startup investment, and can be time-consuming when a large set of slides needs to be dissected. Hence, there is an unmet need for a tool that allows molecular laboratories to easily automate the tumor enrichment step and ensure high-quality input material generation for both nucleic acid and protein extraction. Here, we report on the AVENIO Millisect System (Roche Sequencing Solutions, Branchburg, NJ), an automated instrument that is intended for FFPE tumor tissue dissection for tumor enrichment that is compatible with commercially available nucleic acid and protein isolation kits for downstream applications, such as PCR and NGS. The AVENIO Millisect System is a compact, multislide, high-performance tissue dissection system that enables tumor enrichment for FFPE tumor tissue specimens. The instrument is composed of a milling module, a stage, and a base with an optical system (Supplemental Figure S1). The workflow (Figure 1) starts with alignment and annotation using a previously annotated H&E slide to generate a reference image. The stage (9000 mm2) accommodates four glass slides at a time and digitally aligns the reference image to unstained tissue sections (camera resolution, 14 MP; optical zoom, 5×; digital zoom, 4×). Milling of the area of interest (AOI) uses the milling module, which is composed of a plunger, a blade, and internal fluidics for 5- to 10-μm-thick tissue sections. Depending on the size of the AOI, different sizes of milling tip blades (small, approximately 250 μm; medium, approximately 525 μm; and large, approximately 725 μm) can be used. The milled tissue is collected in dissection buffer (10 mmol/L Tris hydrochloride, pH 8.0, 1 mmol/L EDTA, 5% SDS) and is ready for downstream extraction of nucleic acids and protein. The software provides digital assistance to determine and document the estimated tissue volume before dissection and the actual tissue volume after dissection. To evaluate dissection accuracy by AVENIO Millisect System, six glass slides were created artificially by covering with a 5-μm or a 10-μm paraffin layer, with a printed dissection pattern affixed to the slides underneath the paraffin. The dissection pattern was created to include 30 shapes to evaluate the dissection accuracy when using the spot, path, draw, and color picker tools (Supplemental Figure S2). After the dissection was performed, the location of the actual dissection was compared with the printed shape on the slides to assess the dissection accuracy using a Dino-Lite Pro Digital Microscope and software accuracy was by the on the printed dissection pattern and on the actual dissection A of was a To evaluate dissection six of medium, and large milling tip sizes were dissected on of six slides, for a of for milling tip After the of the dissected area was The of dissection was by the of the dissected area and by The that were a were to for to for medium, and to for large tip To evaluate the were assessed across Millisect by different A was as a dissection with a tip to four tissue The a milling of and A if or of the were a software or the such that not be the sample was not the or the tip was in the The for was a success across multiple and multiple volume accuracy was assessed for samples across and six The sample was by the from the of the the The sample volume was by the sample by the dissection The results were compared with the estimated sample volume by the The software provides an of the tip use on the that the has The for this study was a tip use of at of the tip with milling tip use of were not in the this a a if the tip use on the sample was at and the actual sample volume was at of the estimated sample volume by the of FFPE tissue samples were used in this patient samples were for analysis and in with commercially were as samples were processed using manual and automated AVENIO Millisect System dissection set 1 two FFPE tissue of and of lung tissue, which were of 5- and 10-μm-thick sections and processed in paraffinized and deparaffinized for the downstream extraction procedures using different kits section was with H&E and annotated by an The annotated H&E slide was used as a for the automated tissue dissection by to the of and were dissected for nucleic acid extraction and protein and collected in dissection the manual the H&E slide was with the unstained and the was on the of the glass slide. The tissue area was with a surgical blade, and tissue were collected in an for and and FFPE the AVENIO Tumor FFPE FFPE FFPE FFPE FFPE FFPE high not the AVENIO Tumor in a high not lung and FFPE tissue samples were used to compatibility with the commercially available extraction The from the 10-μm lung tissue sections was used to the of quality samples dissected manually and by automated Millisect The workflow for the AVENIO Tumor (Roche Sequencing for use not for use in diagnostic was used to the to generate sequencing set previously lung FFPE tumor tissue of excisional specimens with tumor content. was in 10-μm sections for both automated Millisect and manual and two paraffinized sections FFPE tumor tissue were processed for extraction by the EGFR (Roche Sequencing using the to the the results on Millisect and manually dissected unstained slide section at 5-μm thickness is recommended for this set was composed of cell lung and FFPE tumor tissue specimens with tumor content from 5% to FFPE tumor tissue were at 10-μm with sections, and collected slides and to at sections were at for 30 and with H&E on an automated using H&E-stained slides were on a slide at slide were annotated by a pathologist for tumor of and digital were created as dissection reference sections from FFPE tumor tissue were workflows slides automated dissection by manual and slide tumor tissue enrichment. In automated dissection by tissue slides were dissected using or large AVENIO Millisect milling tip (Roche Sequencing Solutions, collected with dissection and 1 of was to the dissection buffer tissue and for at a tissue was from the tissue and to at tissue were at nucleic acid extraction and the manual the H&E slide was with the unstained and the was on the of the glass slide. The tissue area was with a surgical blade, and tissue were collected in an the slides, tissue was from the slides and buffer was to these an additional tissue samples collected through different methods were processed for extraction. samples derived from the FFPE tumor tissue were in different were processed through the AVENIO tumor tissue and the results were compared across the of annotated on unstained slides were before and after the automated dissection by Millisect (Supplemental Figure and tissue volumes were dissected by Millisect was collected in the dissection buffer was with 1 of for and the was at manually dissected samples were and at The collected sample material was processed to nucleic acids and and using commercially available extraction kits 1) of or protein from paraffinized and deparaffinized were and assessed for quality In sample were of which were were and were protein from previously lung FFPE tissue sections dissected manually or by Millisect set were using the The is a real-time PCR for the detection of mutations and of the epidermal growth factor receptor in from FFPE lung 10-μm-thick sections FFPE tumor tissue content were used for extraction. 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The downstream analysis of using the high manually and Millisect dissected tissue EGFR and mutations were in both tissue samples dissected by the two different However, were in of tissue samples when dissected by Millisect and four of when dissected manually EGFR on Millisect and Cancer Tumor of tissue number of in a Molecular testing has the of J. Campbell P.J. Genome sequencing during a patient's journey through cancer.N Engl J Med. 2019; 381: 2145-2156Crossref PubMed Scopus (24) Google Scholar there are different used for therapy and tumor profiling, FFPE tissue is the most commonly used tumor content for most is and tumor enrichment is recommended if the R.R. Patel K.P. Routbort M.J. Aldape K. Lu X. Manekia J. Abraham R. Reddy N.G. Barkoh B.A. Veliyathu J. Medeiros L.J. Luthra R. Clinical massively parallel next generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours.Br J Cancer. 2014; 111: 2014-2023Crossref PubMed Scopus (66) Google Scholar in such as massively parallel sequencing and a need for automation of the tissue dissection process for tumor sample enrichment. downstream assays, is critical that input material from the FFPE tissue sections are of high quality and yield and in a The AVENIO Millisect System was to an accurate automated dissection on and FFPE tumor tissue and the dissected material was to be compatible with different commercially available kits for nucleic acid and protein extraction from multiple with very and quality and quality was automated and manually dissected tissue, the yield was higher in the Millisect dissected tissue across nucleic acid extraction The protein yield was these two methods of dissection. quality as well as allelic frequencies were assessed and the manual method to ensure that high-quality input resulted in downstream especially in low tumor content specimens. The results in low tumor content specimens higher allelic frequencies in Millisect dissected samples compared with manual the results were on a small sample Hence, of manual dissection by Millisect in the clinical can be with adequate validation. In addition, previously lung FFPE tissue samples were processed for the samples of Millisect and manual dissection were concordant for The not detect an in sample dissected The be by an in the manual method in of the The of this study is a small sample size of clinical tumor specimens for which representation of the tumor content the and of In addition, the sample set not include specimens with tumor cell or of which from dissection by Millisect to the tumor before nucleic acid and protein isolation these this study that the AVENIO Millisect System may laboratories the preanalytic step of tumor enrichment, and tissue requirements with high for downstream nucleic acid or for input during the and performed and results and the results and the with Figure AVENIO Millisect with Figure pattern with various shapes for dissection performance and are of of which are for dissection through the AVENIO Millisect System The small milling tip was used in with the tool for 1 through with the tool for A and with the tool for shape and with the color picker tool for shape H. The milling tip was used in with the tools for to with the tool for and with the tool for shape and with the color picker tool for shape J. The large milling tip was used in with the tool for through with the tool for and with the tool for shape and with the color picker tool for shape L. with Figure of glass slides before and after dissection by AVENIO Millisect System and and eosin slide annotated for tissue dissection using to be to unstained tissue slides for dissection. slide is annotated using the H&E slide. milling after the Millisect dissection with

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