Jennifer L. Sauter, MD, Memorial Sloan Kettering Cancer Center

Jordan P. Reynolds, MD, Cleveland Clinic Foundation

On behalf of the American Society of Cytopathology Clinical Practice Committee

Immune checkpoint inhibition, or immunotherapy (IO), has revolutionized the treatment of cancer. Many tumor cells carry programmed death ligand (PD-L1), a transmembrane protein which can bind to programmed death (PD1) receptor on cytotoxic T-cells and allow cancer cells to evade destruction by immune cells.1 This mechanism has been demonstrated in many different tumors, for which several immune checkpoint inhibitor therapies can successfully treat. IO initially emerged as a treatment for melanoma and non small cell lung carcinoma (NSCLC), but its use has rapidly expanded to the treatment of several other tumor types. Several IO drugs including Pembrolizumab, Nivolumab, Ipilumumab, Atezolizumab, Avelumab  and Durvalumab have recently been approved for the treatment of various tumors including cervical carcinoma, gastrointestinal tumors including colorectal, gastroesophageal junction and gastric carcinomas, hepatocellular carcinoma, triple negative breast carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, urothelial carcinoma, renal cell carcinoma and some lymphomas including classic Hodgkin lymphoma and primary mediastinal diffuse large B-cell lymphoma.2-5 6

For some IO drugs, approved use in specific tumor types requires a corresponding companion diagnostic or complementary diagnostic PD-L1 test by immunohistochemistry (IHC). While companion diagnostics are required for use of a specific drug, complementary diagnostics are tests developed for use with a specific drug but their use is not absolutely required prior to treatment. Several different PD-L1 antibody clones have been FDA approved as companion or complementary diagnostics for specific IO drugs, and their interpretation and thresholds established for treatment eligibility vary considerably. For instance, the percentage of tumor cells expressing PD-L1 by IHC is scored in some tumor types and for others, PD-L1 expression in stromal and/or tumor infiltrating inflammatory cells is also included. To highlight the complexity of PD-L1 testing, Table 1 lists the various FDA approved PD-L1 antibodies for NSCLC.

Due to this rapidly evolving landscape, cytopathologists are now facing requests to perform PD-L1 testing on cytology specimens. However, PD-L1 testing by immunochemistry in cytology has several limitations, most importantly lack of validation studies on cytology specimens, but also lack of architecture in cytology specimens limiting PD-L1 interpretation in some cases and prohibiting PD-L1 testing for some tumor types, pre-analytical variables including differences in fixation, issues related to tumor heterogeneity of PD-L1 expression, tumor quantity in small specimens and potential inter-observer variability in scoring. This bulletin discusses these issues and highlights the emerging body of literature regarding the successes and challenges of performing PD-L1 testing in cytologic specimens.

An important issue regarding the use of cytology specimens for PD-L1 testing by immunochemistry is that cytology specimens were not included in the original validation studies that led to the FDA approvals of companion and complementary diagnostic tests.  In the development of these tests, specific cut-offs for percentage of positive tumor cells, and in some settings, stromal and/or immune cells were generated to determine patient eligibility for IO to be used as first or second-line therapy. These initial validation studies utilized traditional formalin-fixed paraffin embedded (FFPE) tissue. There are important differences between cytology material and FFPE that must be considered when immunochemical testing is applied to cytology specimens, and these differences are particularly important in the context of predictive marker testing where the results are used to determine patient eligibility for treatment.

In the context of PD-L1 testing, there is unfamiliarity and concern over the quality and amount of material present in cytology specimens. The lack of architecture inherent to cytology specimens can be problematic for PD-L1 interpretation. A variety of non-neoplastic cells often express PD-L1. For example, macrophages can show positive staining for PD-L1, and sometimes it can be difficult to discern tumor cells from macrophages in lung fine needle aspiration or pleural effusion specimens. In these cases, TTF-1 IHC may be useful in distinguishing tumor cells from non-neoplastic cells and may aid in the interpretation of PD-L1. In the situation of PD-L1 testing with the SP142 antibody for triple negative breast carcinoma, cytology specimens are not suitable since the reporting requires interpretation of PD-L1 expression within the immune infiltrate. Unfortunately, it is not possible to accurately assess PD-L1 expression within the immune infiltrate in a cytology specimen given the lack of architecture present in this material.

            Pre-analytical variables, particularly alcohol fixation commonly used for cytology specimens in many laboratories, may alter the performance of immunochemistry on cytology material. A common issue reported is decreased staining and even false negative staining with some antibodies.7  Prior to clinical use, antibodies for immunocytochemistry should be validated on cytology material if that material is processed differently from the material on which the antibodies were initially validated, which, in most laboratories, is FFPE tissue.8 In the context of predictive marker testing, proper validation of antibodies is particularly important. In fact, current guidelines for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 IHC in breast cancer state that testing should only be performed on formalin-fixed material.9  Official recommendations regarding appropriate specimen types and addressing pre-analytical variables for PD-L1 predictive biomarker testing have yet to be generated, but collaborations among various societies will likely result in evidence-based guidelines for PD-L1 testing in the near future.

There is an emerging body of literature regarding the use of cytology specimens for PD-L1 testing. Data from several studies show a high concordance in PD-L1 reporting between paired cell block and histologic material from the same site, although there is some variation in concordance among the various studies. These data are outlined in Table 2. There are several potential reasons for discordant PD-L1 results in a matched cytology and histology specimen from the same tumor: (1) PD-L1 is notorious for demonstrating tumor heterogeneity in its expression; (2) pre-analytical factors including differences in fixation; (3) fragmentation of tissue in cytology material; (4) inter- and intra-observer variability and (5) difficulty in distinguishing tumor from non-neoplastic cells that also may express PD-L1. Discrepant cases in these studies may be attributed to any of the above factors. However, the finding of some cases to be positive on cytology but interpreted as negative on corresponding surgical specimen suggest that in some cases, a cytology result may lead to patient treatment whereas the PD-L1 result in the surgical specimen may not. Additional studies utilizing cytology specimens for PD-L1 IHC that include clinical follow up and incorporate patient response to anti-P(D)-L1 therapy are needed to further explore the potential impact of false positive or false negative cases.

Since IO is often considered as treatment in advanced stage patients, cytology specimens may be the only material available for a substantial number of these patients. Therefore, PD-L1 testing in cytology material is clinically relevant despite the challenges and limitations outlined in this review. The College of American Pathologists, International Association for the Study of Lung Cancer and the American Molecular Pathology Society released guidelines in 2018 that supported the use of cytology material for molecular testing for the selection of patients for clinical trials. Without validation of cytology material for PD-L1 testing, many patients may not have the opportunity to receive potentially effective treatment with IO unless they undergo a surgical procedure to procure FFPE tissue. Therefore, additional data are needed to support the use of cytology specimens for PD-L1, and universally accepted validation guidelines for PD-L1 biomarker testing on cytology material may benefit of these patients.

References

1. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12: 252-264.

2. Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab for the Treatment of Non–Small-Cell Lung Cancer. New England Journal of Medicine. 2015;372: 2018-2028.

3. Herbst RS, Baas P, Kim DW, et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet. 2016;387: 1540-1550.

4. Schmid P, Adams S, Rugo HS, et al. Atezolizumab and Nab-Paclitaxel in Advanced Triple-Negative Breast Cancer. New England Journal of Medicine. 2018;379: 2108-2121.

5. Sonpavde G. PD-1 and PD-L1 Inhibitors as Salvage Therapy for Urothelial Carcinoma. New England Journal of Medicine. 2017;376: 1073-1074.

6. Fuchs CS, Doi T, Jang RW, et al. Safety and Efficacy of Pembrolizumab Monotherapy in Patients With Previously Treated Advanced Gastric and Gastroesophageal Junction Cancer: Phase 2 Clinical KEYNOTE-059 Trial. JAMA Oncol. 2018;4: e180013.

7. 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: 89-100.

8. 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: 1432-1443.

9. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med. 2010;134: e48-72.

10. Udall M, Rizzo M, Kenny J, et al. PD-L1 diagnostic tests: a systematic literature review of scoring algorithms and test-validation metrics. Diagnostic Pathology. 2018;13: 12-12.

11. 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: 896-907.

12. Stoy SP, Rosen L, Mueller J, Murgu S. Programmed death-ligand 1 testing of lung cancer cytology specimens obtained with bronchoscopy. Cancer Cytopathol. 2018;126: 122-128.

13. 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: 453-459.

14. 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: 264-274.

15. Torous VF, Rangachari D, Gallant BP, Shea M, Costa DB, VanderLaan PA. PD-L1 testing using the clone 22C3 pharmDx kit for selection of patients with non-small cell lung cancer to receive immune checkpoint inhibitor therapy: are cytology cell blocks a viable option? J Am Soc Cytopathol. 2018;7: 133-141.

16. Noll B, Wang WL, Gong Y, et al. Programmed death ligand 1 testing in non-small cell lung carcinoma cytology cell block and aspirate smear preparations. Cancer Cytopathol. 2018;126: 342-352.

17. 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: 253-263.

18. 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: 1417-1422.

19. Hernandez A, Brandler TC, Zhou F, Moreira AL, Schatz-Siemers N, Simsir A. Assessment of Programmed Death-Ligand 1 (PD-L1) Immunohistochemical Expression on Cytology Specimens in Non-Small Cell Lung Carcinoma. Am J Clin Pathol. 2019;151: 403-415.