|Ahead of print
Determining PD-L1 expression in head and neck squamous cell carcinoma using immunohistochemistry
Prabha S Mishra1, Avnit Sidhu1, Gunjan Dwivedi2, Deepak S Mulajker3, Shivali Awasthi4
1 Department of Pathology, Command Hospital, Pune, Maharashtra, India
2 Department of Otorhinolaryngology, Command Hospital, Pune, Maharashtra, India
3 Department of Internal Medicine and Oncology, Command Hospital, Pune, Maharashtra, India
4 Department of Pathology, Armed Forces Medical College, Pune, Maharashtra, India
|Date of Submission||18-Oct-2019|
|Date of Decision||01-Jan-2020|
|Date of Acceptance||28-Mar-2020|
|Date of Web Publication||27-Jan-2021|
Department of Pathology, Command Hospital, Pune, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Advanced head and neck squamous cell carcinoma (HNSCC) has limited treatment options. Programmed death-ligand1 (PD-L1) expressed by tumor cells interacts with PD-1 receptor on T lymphocytes leading to immune evasive response and survival advantage. Therapy with immune check-point inhibitors target PD-1/PD-L1 blockade inducing tumor regression. Immunohistochemistry (IHC) for PD-L1 expression enables patient selection for immunotherapy and may be considered a potential predictor of clinical response.
Methods: A retrospective analysis of IHC for PD-L1 expression using manual laboratory developed technique (LDT) with antibody clone 22C3 (Dako) in 93 cases of HNSCC. PD-L1 expression was correlated with age, gender, tumor site, grade and stage.
Results: PD-L1 IHC was performed in 93 cases and immunopositivity was noted in 59 (63.4%) cases. High expression with combined proportion score (CPS) =50 was seen in 15 (16.1%) cases and low expression with CPS =1 expression was seen in 44 (47.3%) cases. An almost-perfect interobserver agreement was noted by two pathologists for PD-L1 IHC expression (Cohen's kappa coefficient = 0.910). No statistically significant correlation was noted between PD-L1 score and patient demographics, tumor site, grade or stage.
Conclusion: Detection of PD-L1 status by IHC enables identification of HNSCC patients eligible for future targeted immunotherapy.
Keywords: Head and neck squamous cell carcinoma, immunohistochemistry, LDT, PD-L1Key Message Immunohistochemical expression of PD-L1 in head and neck squamous cell carcinoma is used as predictive biomarker for initiating immunotherapy. Validated laboratory-developed techniques need to be developed and assessed for PD-L1 testing.
|How to cite this URL:|
Mishra PS, Sidhu A, Dwivedi G, Mulajker DS, Awasthi S. Determining PD-L1 expression in head and neck squamous cell carcinoma using immunohistochemistry. Indian J Cancer [Epub ahead of print] [cited 2021 May 5]. Available from: https://www.indianjcancer.com/preprintarticle.asp?id=308063
| » Introduction|| |
Squamous cell carcinoma is the commonest malignancy of head and neck region and includes tumors arising from distinct locations in the upper aerodigestive tract, including nasal cavity, oral cavity, pharynx, and larynx.The current management protocols are multimodal. Stage I/II disease is treated by surgery or radiotherapy, whereas locoregionally advanced disease (stage III/IV) is treated by combining surgery, radiotherapy, and chemotherapy., Despite appropriate treatment, a large number of patients present with either recurrence or metastasis. Unfortunately, these patients have limited treatment options. However, in recent times anti-programmed death ligand-1 (PD-1/PD-L1) therapy appears to be promising in advanced and metastatic head and neck squamous cell carcinoma (HNSCC).,
PD-L1 is a cell surface glycoprotein that serves as the physiological ligand for PD-1. It is expressed by the immune effector cells, as well as various tumor cells. PD-1 is a transmembrane receptor of the immunoglobulin superfamily that is expressed on activated T lymphocytes and even natural killer cells and B lymphocytes upon induction. PD-1/PD-L1 interactions are critical in mediating mechanisms of immune tolerance and protecting against autoimmunity. In cancer, tumor cells can over express PD-L1 and this provides survival advantage to the tumor as it can evade host immune system by diminishing anti-tumor activity through anergy or apoptosis of antigen specific T-cells.,
Introduction of immune checkpoint inhibitors has revolutionized cancer treatment by conquering the tumor immune evasion mechanism and this therapy has proved highly effective in advanced nonsmall cell lung cancer, malignant melanoma, and hematolymphoid neoplasm like Hodgkin lymphoma., Although expression of PD-L1 in various malignancies has been reported, data regarding HNSCC especially from Indian subcontinent is limited. Considering the emergence of various licensed immune inhibitory drugs and validated automated diagnostic assays, we attempted to evaluate the technical performance of laboratory developed technique (LDT) of PD-L1 immunohistochemistry (IHC) in HNSCC. An attempt was also made to correlate the expression of PD-L1 with clinicopathological parameters.
| » Materials and Methods|| |
The study cohort consisted of 93 consecutive patients diagnosed as HNSCC either on biopsy or surgical resection at an Indian tertiary care hospital between January 2017 and December 2018. Patient demographics and relevant clinical data were obtained from the hospital medical records. Details regarding tumor site, grade, and clinical stage were also recorded. Only treatment naive patients were included. PD-L1 status should ideally be assessed on the initial diagnostic tissue as chemotherapy may lead to confounding results by inducing PD-L1 expression. The representative, formalin-fixed paraffin-embedded blocks were retrieved, hematoxylin and eosin (H&E) stained slides were prepared and independently reassessed for the histological diagnosis by two pathologists. IHC for PD-L1was performed on whole mounted sections and reported independently by two pathologists.
The study was conducted after obtaining due approval of the institutional ethics committee.
IHC and scoring
Manual method of IHC was performed on deparaffinized 3 μm thick sections. Antigen retrieval was done by heating the sections with the retrieval solution (pH 6) provided by the company (Dako) followed by hydrogen peroxide block for 5 minutes. Incubation was done with primary mouse monoclonal PD-L1 antibody clone 22C3 (1:50 dilution; PD-L1 IHC 22C3 pharmDx; Dako SK006) for 60 minutes at 37°C. Further incubation of bound antibodies was done with HRP and mouse linker for 30 minutes. Subsequent addition of 3,3'-diaminobenzidine (DAB) tetrahydrochloride chromogen for 10 minutes followed by DAB enhancer at room temperature resulted in a visual reaction. Sections were finally counterstained with hematoxylin. Each batch was run with the commercially provided positive and negative controls. In addition section of tonsil was run as positive control as well as negative control. Tonsil displays a spectrum of PD-L1 expression, intense staining of crypt epithelium and absence of staining of germinal centers, mantle zone, and surface epithelium.
IHC was evaluated using combined proportion score (CPS) at 200× magnification. CPS is the number of PD-L1 staining tumor cells, macrophages, and lymphocytes divided by total viable tumor cells, multiplied by 100. CPS =1 was scored as positive. CPS <1 or no expression in tumor or immune cells was scored as negative. Tumor cells showing partial or complete membranous staining at any intensity were positive, and any staining for immune cells was considered as positive. Positive cases were further scored by a two-tiered system into low (=1–49) and high expression (=50).
Chi square test was performed by SPSS version 21 software to analyze the statistical correlation between PD-L1 expression and clinicopathological parameters.
| » Results|| |
In the patient cohort of 93; 11 (11.83%) were women and 82 (88.17%) were men; the median age was 60.13 (range 36–80 years). The tissue sample included 65 biopsy and 28 resection specimen. Histomorphologic grading showed 24 (25.8%) cases as well differentiated, 57 (61.29%) cases moderately differentiated, and 12 (12.9%) cases poorly differentiated.
PD-L1 expression and clinicopathological features
PD-L1 immunopositivity was noted in 59 (63.4%). The IHC staining patterns are depicted in [Figure 1]. The association between PD-L1 expression and clinicopathological features is provided in [Table 1]. Men outnumbered women in both the positive and negative categories. Oral cavity was the commonest site of tumor. Lymph node involvement was seen in 24 (40.7%) PD-L1 positive cases and in 9 (26.4%) PD-L1 negative cases. Based on the American Joint Committee on Cancer 8th edition staging, 15 out of 93 cases were stage I, 32 out of 93 stage II, 24 out of 93 stage III and 22 out of 93 stage IV. In the positive category, 15 out of 59 cases were stage III and 17 out of 59 cases were stage IV. There was no correlation between PD-L1 expression with tumor, site, grade, stage or lymph node status.
|Figure 1: (a) Photomicrograph depicting PD-L1 immunohistochemistry (a) Negative with combined proportion score (CPS) <1 at ×200. (b) Positive immunostaining of both tumor cells and immune cells with combined proportion score; 1 at ×100. (c) Positive immunostaining of only immune cells and CPS; 50 at ×100. (d) Membranous staining of tumor cells ×400|
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|Table 1: Association of clinicopathological features of head and neck squamous cell carcinoma with programmed death-ligand 1 (PD.L1) expression|
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A low CPS =1 was observed in 44 (47.3%) cases and high CPS of =50 was observed in 15 (16.1%) cases. Significant correlation (P = 0.010) was observed between PD-L1 score and lymph node status. No statistically significant correlation was noted between low and high PD-L1 score with patient demographics, tumor site, grade or stage as tabulated in [Table 2].
|Table 2: Clinicopathological features of head and neck squamous cell carcinoma with low and high PD-L1 expression|
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Discrepancy of scoring was found in 4 cases between the two reporting pathologists, as there was lack of concordance at cut-off score of 1. After consensual decision 3 cases were scored positive and 1 case was scored negative. An almost-perfect interobserver agreement was noted for PD-L1 expression as assessed by Cohen's kappa coefficient with a value of 0.910.
| » Discussion|| |
PD-1 immunotherapy has gained immense recognition and acceptance subject to realizing its scope in management of various metastatic and recurrent carcinomas, sarcomas, and hematolymphoid neoplasms. [8, 9, 10]Pembrolizumab is recommended by UsS Food and Drug Administration (FDA) as single agent or in combination with platinum-containing chemotherapy for metastatic or with unresectable or recurrent HNSCC expressing PD-L1 with CPS =1.
The most reliable biomarker for patient selection prior to institution of anti-PD-1 immunotherapy in HNSCC is PD-L1 (22C3) IHC performed on only the automated platform Dako link 48. IHC being a simple, standardized and reliable diagnostic technique is employed widely in histopathology laboratories and we exploited the manual LDT of IHC for PD-L1 using approved kits instead of the FDA approved automated platform. The available literature cites that despite testing on automated platform, PD-L1 IHC testing and interpretation is marred with complexity and variation owing to the antibody clone used, cut-off scores, tumor site, and tumor type.We feel that to enhance the clinical utility of PD-L1 immunotherapy in resource constrained laboratories, LDT need to be developed and encouraged.
Meta-analysis of various PD-L1 IHC assays by Torlakovic et al. has suggested that a validated LDT may be used for the same purpose as approved by FDA when the companion assay cannot be performed instead of replacing the companion assay with another. They reported specificity and sensitivity of 100% for 22C3 LDT.
Our data is reflective of results from greater number of biopsies in comparison to resection specimens because stage III and IV cases accounted for one third of the patient cohort. In such cases biopsy would be the only sample available for histopathology diagnosis. This recapitulates the true clinical scenario. The immunopositivity observed in 63.4% cases was not statistically significant and nor was there any significant association between PD-L1 expression and the clinicopathological parameters. Similar results have been reported by Muller et al  and Schneider et al.Literature review is provided as per [Table 3]. Different studies have evaluated different PD-L1 clones and used variable cut-off scores.
|Table 3: Comparative studies of head and neck squamous cell carcinoma (HNSCC)|
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The inter-observer concordance rate of our study is comparable to that investigated by Downes et al. reflecting reliable reporting of IHC. Disagreement among pathologists in our study was noted in only 4 cases for rendering low CPS score of 1 which we feel possibly would have been better addressed with an automated platform. It is reiterated that appropriate patient selection is essential to harness the desirable outcomes of immunotherapy. Undoubtedly, the histopathologist plays a pivotal role in guiding and optimizing treatment decisions.
The main limitation of our study is that it represents the retrospective data of a single center and patient follow-up has not been carried out to assess overall survival (OS), disease-free survival (DFS) or progression-free survival (PFS ). Meta-analysis done by Yang et al. concluded that PD-L1 expression was predictive of only better PFS in cases of advanced HNSCC. They did not report any association of PD-L1 expression with OS or DFS in HNSCC.
In conclusion, despite certain limitations, our study holds extreme relevance as it is the first from India to comprehensively investigate and stratify a cohort of HNSCC based on PD-L1 immunohistochemical expression using LDT. PD-L1 expression in HNSCC cases was independent of age, gender, tumor site, tumor grade or stage. PD-L1 immunohistochemistry is a reliable and promising laboratory technique with wider impact in guiding therapeutic decisions. Validated LDT assays need to be developed ensuring quality, economy, and clinical utility. Future multi-center studies will be needed to validate the findings on a larger cohort.
We sincerely acknowledge the contributions of our laboratory technicians for their invaluable support during the conduct of this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]