Maximizing derivable information from cytologic specimens for pathologic and molecular diagnostics

doi:10.1016/j.jasc.2015.01.007

Journal of the American Society of Cytopathology

Volume 4, Issue 3, May–June 2015, Pages 141-147

https://doi.org/10.1016/j.jasc.2015.01.007 Get rights and content

Introduction

The advent of precision medicine will increase the demand for molecular testing on patient tumor specimens. Cytology specimens have been shown to be ideal substrates for molecular testing, but their often paucicellular nature can lead to conflicts in prioritizing sample management. A microfluidic platform was investigated to determine whether cytologic and molecular data could be procured from the same cells, obviating the need for partitioning a sample by multiplexing it instead.

Materials and methods

Cytology samples were created from a tissue source, stained with a supravital dye, and enriched using immunomagnetic beads. These cells and the attached immunomagnetic beads were then run through a microfluidic channel, temporarily immobilized for cytologic examination, and then recovered. The cytologic characteristics of these cells was compared with cells from the same source prepared by conventional cytologic preparatory means. DNA was extracted from the cells recovered from the microfluidic channel and the nature of their integrity was assessed.

Results

Cytologic features between cells run in a microfluidic channel and prepared by conventional means were similar. The DNA recovered from the cells run through the microfluidic channel was of high molecular weight.

Conclusions

Microfluidics enables multiplex testing of cytologic specimens, allowing for cytology-based diagnostic examination and recovery of high-quality DNA. This approach will be of particular benefit for cytology specimens that are paucicellular and will need molecular testing.

Introduction

The medical community is in a period of transformation. Rapid advances in biotechnology have put genomics in the forefront of oncologic care. Molecular alterations are now used to guide the treatment of patients with a growing number of malignancies.¹ Clinical demand for molecular testing of patient-derived tissue samples has correspondingly increased. In response, the field of pathology has been accommodating without drastically reconstructing traditional modalities of diagnosis and specimen processing. The conventional approach of fixing tissue in formalin and embedding them in paraffin wax results in a template appropriate for morphologic evaluation but with problems associated with molecular analysis. Problems with low recovery of nucleic acids, fragmentation of nucleic acids, allele dropouts, and artifactually induced alterations at the molecular level are perils associated with today’s established fixation paradigm.2, 3, 4, 5, 6 Recently, the value of cytologic specimens as substrates for combined pathologic and molecular diagnostics has been demonstrated to have clinical value.7, 8 Cytologic specimens, although different than traditional biopsies in that architecture is lost in the former, have proven to yield concordant diagnoses when compared with the latter.⁹ The currently undervalued importance of these specimens relative to biopsy specimens is because they are not processed by formalin fixation. This has proven to result in improved yields of nucleic acids at both the qualitative and quantitative levels.¹⁰ In fact, molecular analysis from a variety of cytology formats (fluids, washes, aspirations, and cell blocks) have been documented to be reliable sources for molecular analysis.11, 12, 13, 14, 15 Looking to further increase the value of cytology specimens, we have developed a microfluidic platform from which the cells in a cytologic specimen can be viewed for diagnostic purposes and later recovered for molecular analysis, all in the context of a formalin-free environment. Herein we demonstrate the utility of applying microfluidics to the cytologic evaluation of cells and the recovery of high–molecular weight DNA from those same cells.

Section snippets

Materials and methods

Cytologic specimens were created by dissociating viable cells from recently excised, unfixed surgical resection specimens. Briefly, thin strips of nontransformed human colonic mucosa were excised from excess regions of segmental colonic resection specimens extirpated for medically indicated purposes. Use of excess human tissue was approved by the University at Buffalo Institutional Review Board. Colonic tissue was selected for testing based on the availability at this institution, the columnar

Results

Two cell types from colonic epithelium could be readily identified in the specimens prepared to create cytology-like specimens, absorptive cells, and goblets cells.²² In both the cytosmears and cytospin specimens, the absorptive cells were columnar in shape and either in small cohesive groups or single and partially detached (Fig. 3). The nuclei in these cells was located either in the middle of the cells or toward, but not at, the basal pole. The nuclei were oval to oblong and with smooth

Discussion

The field of oncology treatment is in a period of rapid transition. The current, evolving paradigm of precision medicine emphasizes administration of the right therapeutic agent to the right patient, with identification of the “right” patient based on stratifying patients into responders and nonresponders. Responders can be identified via molecular tests that link targeted therapeutic agents to specific molecular alterations.²³ As our knowledge of the dynamics and complexity of malignancy at

Conclusions

We have demonstrated the ability to process a specimen as a cytology sample on a microfluidic device. The microscopic features of cells evaluated in the microfluidic device tested are comparable to those prepared by current methods. The advantage of a microfluidic device for the evaluation of cytologic specimens with malignant cells is in its potential to capture cells for bright-field diagnosis and recover the same cells for molecular analysis. This will be optimal when there are low numbers

Funding sources

Authors Furlani and Oh would like to acknowledge financial support from the U.S. National Science Foundation, through award number CBET-1337860.

Conflict of interest disclosures

The authors made no disclosures.

Acknowledgments

The authors wish to thank John Nyquist, MS, CMI, for the illustration of Fig. 2.

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Cited by (3)

Ultrarapid EGFR Mutation Screening Followed by Comprehensive Next-Generation Sequencing: A Feasible, Informative Approach for Lung Carcinoma Cytology Specimens With a High Success Rate
2020, JTO Clinical and Research Reports
Citation Excerpt :
Cytology specimens are often the sole source of tumor tissue for molecular profiling in patients with NSCLC, in view of the increased emphasis on minimally invasive procedures to obtain diagnostic material. Although studies have demonstrated the suitability of these samples with limited tissue for molecular testing,21–26 including comprehensive NGS,27 success rates vary widely across institutions and are lower when a sample is subjected to serial single-gene testing instead of an upfront multiplexed assay.21,28–30 Improvement in success rates relies on a careful choice of technology and optimization of tissue handling and testing protocols.
For patients with advanced NSCLC, cytologic samples may be the only diagnostic specimen available for molecular profiling. Although both rapid and comprehensive assessment are essential in this setting, an integrated multitest approach remains an important strategy in many laboratories, despite the risks and challenges when working with scant samples. In this study, we describe our experience and high success rate in using a multitest approach, focusing on the clinical validation and incorporation of ultrarapid EGFR testing using the Idylla system followed by comprehensive next-generation sequencing (NGS).
Cytology samples received for routine molecular testing were included in this study. The performance characteristics of the EGFR Idylla assay were assessed; tissue suitability parameters and interpretation criteria to supplement automated mutation calling were established. The assay performance was monitored for 1 year, comparing the results with those of concurrent NGS testing by MSK-IMPACT (primarily) or MSK-AmpliSeq and MSK-Fusion solid panel in a subset of cases.
Overall, 301 samples were studied; 83 samples were included in validation (60.2% [50 of 83] were positive for EGFR mutations). Concordance with the reference method was 96.4% (80 of 83) of the samples with excellent reproducibility. The limit of detection was variable depending on the total tissue input and the specific mutation tested. Unextracted tissue inputs that maintained total EGFR cycle of quantification at less than 23 allowed all mutations to be detected if present at greater than 5% variant allele frequency. Mutations could be detected at 1% variant allele frequency with total EGFR cycle of quantification of 18. During the clinical implementation phase, 218 NSCLC samples were tested by Idylla (24.3% [53 of 218] were EGFR mutation positive). Concurrent NGS testing was requested on 165 samples and successfully performed on 96.4% (159 of 165) of the samples. The Idylla automated results were concordant with those obtained by NGS in 96.2% (153 of 159) of cases and improved to 98.7% (157 of 159) after incorporation of manual review criteria to supplement automated calling, resulting in a diagnostic sensitivity of 95.6% (95% confidence interval: 84.9%–99.5%). In general, 9% (14 of 159) of the cases tested by NGS had EGFR mutations not covered by the Idylla assay, primarily insertions in exon 19 and 20 and minor mutations co-occurring with canonical sensitizing mutations.
Comprehensive molecular testing is feasible and has a high success rate in NSCLC cytology samples when using a multitest approach. Testing with the Idylla system enables rapid and accurate determination of the EGFR status without compromising subsequent NGS testing.
Optimizing workflows and processing of cytologic samples for comprehensive analysis by next-generation sequencing: Memorial Sloan Kettering Cancer Center experience
2016, Archives of Pathology and Laboratory Medicine
Core needle biopsy wash optimization: Enabling specimen integrity for both cytological and histological evaluation
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View full text

Original ArticleMaximizing derivable information from cytologic specimens for pathologic and molecular diagnostics

Introduction

Materials and methods

Results

Conclusions

Introduction

Section snippets

Materials and methods

Results

Discussion

Conclusions

Funding sources

Conflict of interest disclosures

Acknowledgments

Am J Pathol

J Mol Diagnostics

Mod Pathol

J Am Soc Cytopathol

A new frontier in personalized cancer therapy: mapping molecular changes

Future Oncol

Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples

Clinical Chem

Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation

Nucleic Acids Res

Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing

BMC Med Genomics

Next-generation sequencing improves the diagnosis of thyroid FNA specimens with indeterminate cytology

Histopathology

Next-generation sequencing of lung cancer EGFR exons 18-21 allows effective molecular diagnosis of small routine samples (cytology and biopsy)

PLoS One

Multiplatform comparison of molecular oncology tests performed on cytology specimens and formalin-fixed, paraffin-embedded tissue

Cancer Cytopathol

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues

BMC Res Notes

Molecular profiling of tumor cells in cerebrospinal fluid and matched primary tumors from metastatic breast cancer patients with leptomeningeal carcinomatosis

Cancer Res

Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer

J Natl Cancer Inst

High- throughput molecular analysis from leftover of fine needle aspiration cytology of mammographically detected breast cancer

Transl Oncol

A new fabrication process for SU-8 thick photoresist structures by simultaneously removing edge-bead and air bubbles

J Micromechan Microeng

Original Article
Maximizing derivable information from cytologic specimens for pathologic and molecular diagnostics