Cytopathology in focus: Non-small cell lung carcinoma

Cytology samples and immunotherapy predictive testing

Kaitlin E. Sundling, MD, PhD

January 2020—Requests for predictive biomarkers in oncology patients are becoming increasingly common in the cytology laboratory. At the time of rapid on-site evaluation, cytologists are now keenly aware of the need to collect adequate material not just for a diagnosis of malignancy but also for diagnostic and predictive molecular and immunohistochemical testing. This article provides an overview of current practices and some of the recent literature regarding predictive testing for immunotherapy in cytologic preparations in non-small cell lung carcinoma.

Biological rationale of immunotherapy. Tumor cells may evade the immune system through inhibition of the immune synapse between T cells and antigen presenting cells.1 The inhibitory molecules programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are referred to as immune checkpoint molecules. Immunotherapy using PD-1, PD-L1/2 (programmed cell death ligand 1/2), and CTLA-4 targeted antibodies are immune checkpoint inhibitors. These therapies may restore the ability of the cytotoxic T cells to recognize and attack tumor cells.1 In the appropriate context, high levels of PD-L1 expression on tumor cells suggest that the patient may be more likely to respond to inhibition of PD-1.2

Notably, immune checkpoint inhibitors have a unique set of side effects including immune-related adverse events as well as others seemingly unrelated to the immune system.3 Rarely, these adverse events may prove fatal. Thus, efforts continue to better predict which patients will be most likely to benefit from immunotherapy. While the most evidence has accumulated for non-small cell lung carcinoma, other cancers with the potential to respond to immune checkpoint inhibitors include gastroesophageal adenocarcinoma, cervical carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, and triple-negative breast carcinoma.4

PD-L1 immunohistochemistry. Although specific immune checkpoint inhibitors may be used clinically without testing of the tumor itself, many situations require quantification of the level of PD-L1 expression on tumor cells to allow the patient to receive specific therapies or to participate in clinical trials.2 It is important to know which antibody clone or specific assay is requested, as some assays have been FDA approved/cleared as companion diagnostic tests for a specific drug,4 while others may not be.

PD-L1 is quantified by the percentage of tumor cells with membranous staining. For PD-L1 clone 22C3 in advanced non-small cell lung carcinoma, greater than or equal to 50 percent staining may result in the use of pembrolizumab as first-line therapy, while a greater than or equal to one percent staining is considered positive and pembrolizumab may be considered as second-line therapy.5,6 Interpretive criteria are likely to vary by antibody clone/testing platform,7 and some investigational applications may explore PD-L1 expression in immune cells and/or stroma in addition to tumor cell expression.

Significant intratumoral, intersite, and temporal heterogeneity in PD-L1 expression has been reported. Cytologic preparations, owing to their small size, present a dilemma with respect to sampling. In cases where a small number of cells is present in the cell block (for example, fewer than 100 cells), both false-positives and false-negatives due to intratumoral heterogeneity are a concern. PD-L1 interpretive criteria were developed for histologic sections; in many cases the same criteria may be applied to adequately cellular cytologic preparations. Several studies have shown high concordance between cytologic samples and core biopsy or surgical resection specimens.5,8–11 One study suggests that interobserver reproducibility may be lower in cytologic samples specifically, as compared with histologic samples.12 Reproducibility may improve as pathologists gain more experience in interpreting PD-L1 results in cytologic samples.

An important consideration in predictive immunohistochemical stains is the impact of fixation on quantitative results. Previous studies have shown the potential for both false-negative13 and false-positive14 results with alcohol fixation of cell blocks. To this end, validation may be necessary for predictive immunohistochemical stains if the fixation conditions of cytologic preparations (smears, liquid-based preparations, or cell blocks) differ from those used in the routine validation study.15

Other testing options under investigation. High rates of somatic mutations lead to the production of neoantigens that may be integral to the effectiveness of immune checkpoint inhibitors.16 Thus, molecular testing for the tumor mutation burden, or a global measurement of somatic mutations, may aid in predicting response to immune checkpoint inhibitors. Efforts are underway to standardize tumor mutation burden estimation and reporting.17 Although next-generation sequencing may be successfully performed in cytology samples with adequate tumor cellularity, tumor proportion, and assay validation,18 few studies report tumor mutation burden in cytology samples at this time.

Likewise, mismatch repair defects may result in high rates of mutations and production of neoantigens. Thus, either mismatch repair immunohistochemistry or nucleic acid-based microsatellite instability testing may also help in predicting the potential response to immune checkpoint inhibitors. A CAP template exists for reporting DNA mismatch repair biomarkers for tumors other than endometrium and colon.19 This template is not tailored to cytologic preparations. As with PD-L1 testing, validation may be needed when fixation conditions for cytologic preparations vary from the conditions used in the routine validation.15

In conclusion, cytology samples of non-small cell lung carcinoma represent an opportunity to provide predictive biomarkers with a minimally invasive approach. CAP guidelines on the collection and handling of thoracic small biopsy and cytology specimens are expected soon.20 Guidelines regarding PD-L1 testing are also in progress.21

  1. Shoushtari AN, Wolchok J, Hellmann M. Principles of cancer immunotherapy. UpToDate website. www.uptodate.com/contents/principles-of-cancer-immunotherapy. Updated July 1, 2019. Accessed Nov. 3, 2019.
  2. Bernicker EH. What the oncologist needs from the pathologist for immune therapies. Arch Pathol Lab Med. 2019;143(12):1513–1516.
  3. Postow M, Wolchok J. Special considerations and toxicities associated with checkpoint inhibitor immunotherapy. UpToDate website. www.uptodate.com/contents/special-considerations-and-toxicities-associated-with-checkpoint-inhibitor-immunotherapy. Updated Sept. 30, 2019.
  4. List of cleared or approved companion diagnostic devices (in vitro and imaging tools). U.S. Food and Drug Administration website. www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools. Updated Dec. 4, 2019. Accessed Nov. 3, 2019.
  5. Heymann JJ, Bulman WA, Swinarski D, et al. PD-L1 expression in non-small cell lung carcinoma: comparison among cytology, small biopsy, and surgical resection specimens. Cancer Cytopathol. 2017;125(12):896–907.
  6. PD-L1 IHC 22C3 pharmDx interpretation manual—non-small cell lung cancer (NSCLC). Santa Clara, Calif.: Agilent Technologies; 2019.
  7. Udall M, Rizzo M, Kenny J, et al. PD-L1 diagnostic tests: a systematic literature review of scoring algorithms and test-validation metrics. Diagn Pathol. 2018;13(1):12.
  8. Skov BG, Skov T. Paired comparison of PD-L1 expression on cytologic and histologic specimens from malignancies in the lung assessed with PD-L1 IHC 28-8pharmDx and PD-L1 IHC 22C3pharmDx. Appl Immunohistochem Mol Morphol. 2017;25(7):453–459.
  9. Ilie M, Juco J, Huang L, Hofman V, Khambata-Ford S, Hofman P. Use of the 22C3 anti-programmed death-ligand 1 antibody to determine programmed death-ligand 1 expression in cytology samples obtained from non-small cell lung cancer patients. Cancer Cytopathol. 2018;126(4):264–274.
  10. Russell-Goldman E, Kravets S, Dahlberg SE, Sholl LM, Vivero M. Cytologic-histologic correlation of programmed death-ligand 1 immunohistochemistry in lung carcinomas. Cancer Cytopathol. 2018;126(4):253–263.
  11. Wang H, Agulnik J, Kasymjanova G, et al. Cytology cell blocks are suitable for immunohistochemical testing for PD-L1 in lung cancer. Ann Oncol. 2018;29(6):1417–1422.
  12. Tsao MS, Kerr KM, Kockx M, et al. PD-L1 immunohistochemistry comparability study in real-life clinical samples: results of blueprint phase 2 project. J Thorac Oncol. 2018;13(9):1302–1311.
  13. Sauter JL, Grogg KL, Vrana JA, Law ME, Halvorson JL, Henry MR. Young investigator challenge: validation and optimization of immunohistochemistry protocols for use on cellient cell block specimens. Cancer Cytopathol. 2016;124(2):89–100.
  14. Williams SL, Birdsong GG, Cohen C, Siddiqui MT. Immunohistochemical detection of estrogen and progesterone receptor and HER2 expression in breast carcinomas: comparison of cell block and tissue block preparations. Int J Clin Exp Pathol. 2009;2(5):476–480.
  15. Fitzgibbons PL, Bradley LA, Fatheree LA, et al. Principles of analytic validation of immunohistochemical assays: guideline from the College of American Pathologists Pathology and Laboratory Quality Center. Arch Pathol Lab Med. 2014;138(11):1432–1443.
  16. Rizvi NA, Hellmann MD, Snyder A, et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science. 2015;348(6230):124–128.
  17. Stenzinger A, Allen JD, Maas J, et al. Tumor mutational burden standardization initiatives: Recommendations for consistent tumor mutational burden assessment in clinical samples to guide immunotherapy treatment decisions. Genes Chromosomes Cancer. 2019;58(8):578–588.
  18. Roy-Chowdhuri S, Stewart J. Preanalytic variables in cytology: lessons learned from next-generation sequencing—the MD Anderson experience. Arch Pathol Lab Med. 2016;140(11):1191–1199.
  19. Bartley AN, Fitzgibbons PL, Broaddus RR, Shi C. Template for reporting results of DNA mismatch repair testing in patients being considered for checkpoint inhibitor immunotherapy. College of American Pathologists website. https://documents.cap.org/protocols/cp-general-dnamismatchrepair-18biomarker-1001.pdf. Published January 2018. Accessed Nov. 2, 2019.
  20. Collection and handling of thoracic small biopsy and cytology specimens for ancillary studies. College of American Pathologists website. www.cap.org/protocols-and-guidelines/upcoming-cap-guidelines/collection-and-handling-of-thoracic-specimens. Accessed Nov. 3, 2019.
  21. Upcoming CAP guidelines. College of American Pathologists website. www.cap.org/protocols-and-guidelines/upcoming-cap-guidelines. Accessed Nov. 3, 2019.

Dr. Sundling is faculty director of the cytotechnology program, clinical instructor, and cytopathologist at the University of Wisconsin and Wisconsin State Laboratory of Hygiene, Madison. She is a member of the CAP Cytopathology Committee.