Adequacy in cytopathology: focus on cytology specimen use in molecular testing

Varsha Manucha, MD
Martin J. Magers, MD
Sinchita Roy-Chowdhuri, MD, PhD
Derek B. Allison, MD
Efrain A. Ribeiro, MD, PhD

May 2023—In the first article in our series on adequacy in cytology, published in January 2023 (bit.ly/3MDNVzr), we summarized current approaches to defining adequacy for the purpose of primary diagnosis in the majority of specimen types encountered routinely in cytology practice. As we saw, while the essence of adequacy is constant across reporting systems, the technical definitions can vary significantly by specimen type. A major advantage of adopting standardized reporting systems for cytology specimens is a unification of the language that pathologists use to communicate with clinicians and classify a diagnosis accurately. In cases in which a specimen is inadequate, the diagnosis should be interpreted with caution.

While current adequacy standards have excelled at facilitating the practice of cytopathology by providing a unified language for primary diagnosis, they do not account for a more complex understanding of adequacy that is evolving rapidly in clinical practice. Namely, they fail to account for the emerging use of cytology specimens in molecular testing. In many cases, the adequacy standards used for primary diagnosis do not consider the subsequent need for molecular testing from the same specimen. In this article, we will address the hurdles that are still in the way of establishing adequacy standards for cytology specimens with a focus on molecular diagnostic testing.

Several studies have demonstrated that cytology specimens are suitable for molecular testing.1-3 Cytology specimens are now being used for routine molecular testing, specifically for prognostic and therapeutic testing in lung cancer, and guidelines from the CAP/IASLC/AMP for the use of cytology specimens for molecular testing were published in 2018.4 Endobronchial ultrasound-guided fine-needle aspiration has a prominent role in establishing the diagnosis of primary lung cancers, and collecting tissue biopsies can be challenging, leaving only glass slide smears with or without a cell block. Rapid onsite evaluation (ROSE) services have greatly improved diagnostic adequacy of specimens for these cases, which has resulted in fewer re-operations and lower costs. However, as cytology specimens are increasingly used for molecular testing, onsite adequacy assessments will have to evolve to account for the collection of sufficient additional tissue for molecular testing.

According to a recent survey of health care professionals in 102 countries about molecular testing failures in lung cancer cases, the main reasons for testing failures were an insufficient amount of tumor cells (83 percent) and inadequate tissue quality (55 percent).5 As it stands now, these minimum standards for a specimen to be used for molecular testing, such as next-generation sequencing, can vary among molecular laboratories. Moreover, as the importance of molecular testing continues to grow, more patients are needing to be re-operated on solely to obtain additional tissue for molecular testing. As things stand with our diagnostic reporting systems today, there is a gap in the definition of adequacy between adequacy for diagnosis and adequacy for ancillary testing. This survey highlights a growing recognition that while a specimen may be considered adequate for a primary diagnosis, it does not mean that the same specimen will be adequate for the molecular testing required for prognostic and therapeutic decision-making.

Numerous roadblocks will have to be overcome to establish universal adequacy criteria for cytology specimens to be used for molecular testing in the context of ROSE services where a diagnosis of malignancy is suspected onsite. These roadblocks come in the form of preanalytic variables that are affected by the way cytology specimens are collected and transported and how they are subsequently processed and handled in the laboratory. While cytology specimens may simply be glass slides with smears, a growing number of liquid-based cytology specimens require specialized processing to get cells onto a slide in a standardized fashion for primary diagnosis (i.e. ThinPrep or SurePath). Cytology specimens may also generate cell blocks for ancillary testing. However, there are currently no adequacy standards that address how many additional passes should be collected to ensure that a cell block is sufficient for ancillary testing beyond immunohistochemistry. To complicate matters further, different molecular labs may have significantly different requirements for the kind (i.e. smears, cell blocks, centrifuged supernatants) and/or amount of tissue needed to obtain enough nucleic acids for molecular testing.

Recently, a consensus was reached among the CAP and other organizations (American College of Chest Physicians, Association for Molecular Pathology, American Society of Cytopathology, American Thoracic Society, Pulmonary Pathology Society, Papanicolaou Society of Cytopathology, Society of Interventional Radiology, and Society of Thoracic Radiology) on the appropriate specimen use for ancillary testing in the context of thoracic oncology.6 The goal of this project was similar to the goals of well-known reporting systems used in cytology: to establish guidelines and a language for use by pathologists and clinicians with regard to the appropriate way to obtain and triage specimens in cases in which molecular testing will be required for clinical decision-making. The group focused on providing evidence-based recommendations for the various preanalytic variables affecting the use of cytology specimens for molecular testing, many of which are still being elucidated. These included recommendations for proceduralists (i.e. needle sizes, number of passes for core needle biopsies and cell blocks, fluid volumes for pleural effusions where molecular testing will be indicated) and pathologists (type of fixative, types of collection media, ischemic time, and testing algorithms in practice). There are not strong recommendations for all such factors. However, the article sheds light on specific areas that should be the focus of further studies to help standardize the way cytology specimens are triaged and processed for molecular testing. As is the case with the standardized reporting systems used in cytology, this consensus provides a foundation for effective communication between clinicians and pathologists regarding whether cytology specimens are being handled appropriately, thus reducing the need to re-operate solely to obtain additional tissue for molecular testing.

In summary, just as cytology practice has been aided by the development of adequacy standards in reporting systems for routine diagnosis, the field must continue to evolve by incorporating a broader definition of adequacy into routine practice. To enable the increasing use of cytology specimens for molecular testing, efforts must be made to re-educate providers who perform onsite evaluations to be aware that a malignant diagnosis is likely to trigger the need for molecular testing from the same specimen. Therefore, the cytology lab must understand the molecular testing laboratory’s preferred specimen preparation. Further research is needed to determine the optimal ways to handle cytology specimens in the laboratory to ensure that the tissue is processed in a way that facilitates its incorporation into existing workflows within molecular diagnostic laboratories. As many of these pipelines require automated manual labor for tasks such as nucleic acid extraction, it will be critical to consider these physical limitations when optimizing cytology specimen processing, starting at the step of onsite evaluation. This work will require a close relationship between radiologists, oncologists, cytopathologists, and molecular pathologists within institutions to fully integrate cytology specimens into routine molecular testing at a larger scale.

  1. Baum JE, Zhang P, Hoda RS, et al. Accuracy of next-generation sequencing for the identification of clinically relevant variants in cytology smears in lung adenocarcinoma. Cancer Cytopathol. 2017;125(6):398–406.
  2. Rekhtman N, Roy-Chowdhuri S. Cytology specimens: a goldmine for molecular testing. Arch Pathol Lab Med. 2016;140(11):1189–1190.
  3. VanderLaan PA, Roy-Chowdhuri S. Current and future trends in non-small cell lung cancer biomarker testing: the American experience. Cancer Cytopathol. 2020;128(9):629–636.
  4. Lindeman NI, Cagle PT, Aisner DL, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn. 2018;20(2):129–159.
  5. Smeltzer MP, Wynes MW, Lantuejoul S, et al. The International Association for the Study of Lung Cancer global survey on molecular testing in lung cancer. J Thorac Oncol. 2020;15(9):1434­–1448.
  6. Roy-Chowdhuri S, Dacic S, Ghofrani M, et al. Collection and handling of thoracic small biopsy and cytology specimens for ancillary studies: guideline from the College of American Pathologists in collaboration with the American College of Chest Physicians, Association for Molecular Pathology, American Society of Cytopathology, American Thoracic Society, Pulmonary Pathology Society, Papanicolaou Society of Cytopathology, Society of Interventional Radiology, and Society of Thoracic Radiology. Arch Pathol Lab Med. 2020;144(8):933–958.

Dr. Manucha is clinical professor, Department of Pathology, University of Mississippi Medical Center; Dr. Ribeiro is a resident physician, Department of Pathology, Johns Hopkins University School of Medicine; Dr. Magers is a staff pathologist, Trinity Health IHA Pathology and Laboratory Management; Dr. Roy-Chowdhuri is associate professor, Department of Pathology, Division of Pathology/Laboratory Medicine, University of Texas MD Anderson Cancer Center; and Dr. Allison is assistant professor, Department of Pathology and Laboratory Medicine, University of Kentucky College of Medicine. Drs. Manucha, Magers, and Allison are members of the CAP Cytopathology Committee. Drs. Ribeiro and Roy-Chowdhuri are former members of the committee.