F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.110031.1 Research Article Articles Evaluation of PD-L1 expression in vulvar cancer

[version 1; peer review: 1 not approved]

 Sahraoui Ghada Conceptualization Formal Analysis Methodology Resources Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0001-5361-9083 a 1 2 Manai Marwa Conceptualization Data Curation Resources Writing – Review & Editing https://orcid.org/0000-0003-4037-3402 3 Yaïche Rahma Conceptualization Data Curation Writing – Review & Editing https://orcid.org/0000-0002-3635-0825 1 Charfi Lamia Resources Visualization Writing – Review & Editing 1 2 Ghalleb Montassar Conceptualization Data Curation Investigation Writing – Review & Editing 4 Douik Hayet Project Administration Writing – Review & Editing 2 Mrad Karima Project Administration Software Supervision Validation Writing – Review & Editing 1 2 Doghri Raoudha Methodology Resources Writing – Review & Editing 1 2 Pathology Department, Salah Azaiez Institute, Tunis, 1006, Tunisia Precision Medicine, Personalized Medicine and Oncology Investigation Laboratory, LR21SP01, Salah Azaiez Institute, Tunis, 1006, Tunisia Human Genetics Laboratory LR99ES10 University of Medecine of Tunis, University of Tunis El Manar, Tunis, 1068, Tunisia Surgical Oncology Department, Salah Azaiez Institute, Tunis, 1006, Tunisia ghada_sahraoui@yahoo.com

No competing interests were disclosed.

 27 10 2022 2022 11 1225 19 10 2022 Copyright: © 2022 Sahraoui G et al. 2022 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: The PD-1/PD-L1 inhibitory immune checkpoint seems to have a significant prognostic impact in the evolution of certain cancers such as bronchopulmonary cancer, breast cancer, lymphomas etc. Indeed, it has been suggested that PD-L1 expression was associated with a worse prognosis due to its immunosuppressive activity within tumor tissue. Currently, the evaluation of the expression of these biomarkers is of increasing interest in gynecological cancers. Hence, we proposed to study the expression of PDL1 in vulvar cancer. Our aims were to study the expression profile of PD-L1 in vulvar cancer by immunohistochemistry and to correlate its expression with overall and relapse-free survival rates.

Methods: This is a retrospective study conducted at the pathological anatomy and cytology department of the Saleh Azaiez Institute, Tunis, Tunisia involving 55 patients followed for vulvar cancer over a period of 13 years from 2008 to 2021. Clinicopathologic data was collected from medical records and pathology reports.

Results: PD-L1 expression in vulvar squamous cell carcinoma was observed in 44% of cases. This expression was noted in 33% of cases in tumor cells and in 11% of cases in lymphocytes. The median follow-up period was 40 months. During this period, 30% of patients relapsed. For all recurrence cases, 77% were during the first two years. The overall survival rate was 68.4% at two years and 50.3% at five years. The recurrence-free survival rate was 63.8% at five years. Overall survival as well as recurrence-free survival were more reduced in the case of PD-L1 expression. However, this difference was not significant (p=0.07).

Conclusions: These results, although insufficient, emphasize the prognostic value that PDL1 could play in vulvar cancer as described in the literature. Despite the low numbers and the unavailability of data for some cases, our results encourage carrying out this work on larger populations.

 vulvar cancer squamous cell carcinoma PD-L1 immunohistochemistry The author(s) declared that no grants were involved in supporting this work. Introduction

Vulvar cancer accounts for 3–5% of all female genital malignancies. 1 It mainly affects postmenopausal women over the age of 60 years. 2

Squamous cell carcinoma is the most frequent histological type with an incidence reported between two and seven per 100,000 women. Its particularity is that it can arise from pre-cancerous lesions or vulvar intraepithelial neoplasia. 3

Recently, immunotherapy has become one of the therapeutic bases in the management of some cancers by blocking of the PD-1/PD-L1 axis in solid tumors. 4

PD-L1 is a membranous protein of nucleated cells. It acts by binding to its PD-1 receptor located on T lymphocytes, inactivating its action. Until now, it played a key role in the treatment of bronchopulmonary cancers. 5

Our aims in this work were to study the immunohistochemical expression profile of PD-L1 in vulvar cancer and to correlate its expression with survival rates.

 Methods Material

Our study was retrospective, and it was conducted at the Pathological Anatomy and Cytology Department of the Salah Azaiez Institute of Tunis in Tunisia. It involved 55 patients followed for vulvar cancer who underwent vulvectomy in Cancer Surgery Department from January 2008 to December 2021. Clinicopathologic data were collected from medical records and pathology reports, which were archived in the Salah Azaiez Institute (Cancer Surgery and Pathology departments).

Immunohistochemical analysis was performed using an automaton (BOND-MAX, Leica Biosystems, Melbourne Pty Ltd). Immunohistochemical steps of the protocol were performed with EnVision FLEX visualization system (Dako) for 60 min, on a Lenovo Ideapad 3-15IIL05, Type 81 WE (Windows 10 Professional Version 21H1) connected to the automaton. Immunohistochemistry samples were imaged using an Olympus BX51 microscope with camera (Olympus U-TV0.63XC SN 1H50935 T7 TOKYO, JAPAN) with a 40× oil objective.

The main steps of immunohistochemistry were as follows:

Step 1: Preparing the slides

The Tissue MicroArray (TMA) blocks were cut into 4 μm paraffin sections by a microtome and adhered to glass slides then incubated at 37°C overnight to remove excess paraffin and promote their attachment to the slides, then placed in a steam room.

Step 2: Deparaffinization and rehydration

We started with deparaffinization of the slides by immersion in three baths of toluene for 5 min in each to eliminate the rest of the paraffin, then rehydration in alcohol for 10 min in three baths of decreasing concentration (100°, 95° and 85°). We rinsed with distilled water and then put them on a support.

Step 3: Antigenic unmasking

The antigenic unmasking was carried out initially by heating in a water bath at 97°C for 40 min at pH 6 or pH 9.

Step 4: Cooling the slides

The slides were allowed to cool and then rinsed with Tris Saline Buffer (TBS) for 5 min to remove traces of the unmasking solution.

Step 5: Blocking antigenic sites

To block non-specific sites, we incubated the slides in hydrogen peroxide (H 2O 2) for 5 min then rinsed with TBS.

Step 6: Adding the antibody

The antibody used was PD-L1, monoclonal Mouse (clone 22C3, Isotype: IgG1 kappa, Ref M3653, Batchcode 11146263). The concentration required was 1/50 and the manufacturing company was Dako (North America INC, 6392 Via Real, Carpinteria CA, 93013, USA). It was incubated in a humid tank for 1 hr to avoid the risk of tissue dehydration. It was then rinsed with TBS.

Step 7: Revealing the antigen-antibody complex

The peroxidase activity was initiated by the addition of its DAB substrate in the presence of H 2O 2 for 10 min, the latter was oxidized by the peroxidase from which an insoluble brown precipitate was observed.

Step 8: Microscopic observation

A microscopic preparation was then ready for analysis by the pathologist.

The PD-L1 expression study was performed on 4-μm tissue sections cut from formalin-fixed, paraffin-embedded lymph node biopsies. Immunostaining by PD-L1 was considered positive in case of membrane labeling associated or not with cytoplasmic labeling. We considered the cut-off ≥5% of immunostained tumor cells and inflammatory immune cells 6 ( Figures 1– 6). Recurrence was defined as tumor relapse in the first five years after treatment at any site: local recurrence, regional recurrence, and/or distant metastasis. All underlying data are provided. 7

Immunostaining of 60% of tumor cells with moderate expression intensity (×400). Immunostaining of 30% of tumor cells with high expression intensity (×400). Immunostaining of 15% of tumor cells with moderate expression intensity (×400). Negative immunostaining for tumor cells and lymphocytes (×400). Immunostaining of 10% of lymphocytes with moderate expression intensity (×400). Immunostaining of 30% of tumor cells with moderate expression intensity and 60% of lymphocytes with high expression intensity (×400). Statistical analysis

Statistical analysis was performed using Microsoft Excel 2019 for Microsoft 365 MSO (Version 2208 Build 16.0.15601.20148) (RRID:SCR_016137) and SPSS software (version 20) (RRID:SCR_019096). Continuous data were summarized using descriptive statistics. Kaplan–Meier curves were plotted.

 Ethical considerations

We respected ethical considerations in our work. We received ethical approval for this study from the ethics committee of Salah Azaiez Institute, Tunis. The ethics committee approval number is ISA/2021/12 dated 12 th January 2021.

 Results Clinical data

This study included 55 patients followed for vulvar cancer. All clinicopathological data are summarized in Table 1. The average age of our patients was 66 years with extremes of 45 and 91 years. The most affected age group was 70–79 years, which represented 31% of cases.

Immunostaining by PD-L1.
Case number Age Immunostaining of tumor cells Immunostaining of lymphocytes Percentage of PD-L1 (%) Intensity of PD-L1
1 48 + - 60 Moderate
2 71 + - 30 Strong
3 53 + - 15 Moderate
4 65 Negative
5 75 + - 80 Moderate
6 70 Negative
7 66 Negative
8 72 Negative
9 59 Negative
10 57 Negative
11 68 Negative
12 82 Negative
13 80 Negative
14 45 + - 30 Moderate
15 87 Negative
16 55 Negative
17 79 Negative
18 66 Negative
19 72 - + 1 Moderate
20 59 Negative
21 60 + - 10 Moderate
22 78 Negative
23 58 - + 1 Moderate
24 91 Negative
25 57 Negative
26 52 Negative
27 73 - + 5 Moderate
28 78 + - 1 Weak
29 60 + - 90 Strong
30 63 + - 90 Strong
31 72 Negative
32 83 Negative
33 52 Negative
34 75 - + 10 Moderate
35 61 Negative
36 79 Negative
37 69 + - 1 Weak
38 80 + - 1 Weak
39 74 Negative
40 47 Negative
41 51 + - 10 Moderate
42 49 + - 40 Moderate
43 72 + - 1 Moderate
44 57 Negative
45 59 Negative
46 65 Negative
47 62 Negative
48 73 Negative
49 48 - + 1 Moderate
50 68 Negative
51 69 + - 50 Moderate
52 72 + - 20 Strong
53 74 - + 10 Strong
54 59 + - 1 Moderate
55 65 + - 1 Moderate

+: Presence of immunostaining by PD-L1.

-: Absence of immunostaining by PD-L1.

The disease was incidentally discovered in 31 (56%) cases. It was revealed by a vulvar pruritus in 23 (42%) cases. Pelvic pain associated with burning on urination was the chief complaint in one (2%) case. These symptoms evolved over a period ranging from one month to 15 years. Further paraclinical explorations such as chest standard radiography, abdomino-pelvic ultrasound, cervico-vaginal smear, mammography and abdomino-pelvic scanner did not reveal secondary localizations.

 Pathological data

Macroscopic characteristics

We had 54 surgical sections following vulvectomy and a single biopsy. The tumor was strictly lateral in 31 (56%) cases with the labia majora being the most common site, noted in 18 (58%) cases. The tumor was medial (clitoris, vulvar range and paraurethral) in seven (13%) cases and both medial and lateral in 17 (31%) cases. It was unifocal in 52 (95%) cases and multifocal in three (5%) cases. Macroscopic tumor size varied between 5 and 170 mm an average tumor size of 15 mm. In our study series, 15 (27) patients had a tumor larger than 30 mm. The tumor was ulcerated in 19 (34%) cases. The mean depth of invasion was 6 mm (range 1–30 mm).

Microscopic characteristics

All our cases corresponded to squamous cell carcinoma, well differentiated in 50 (91%) of cases. Vulvar intraepithelial neoplasia (VIN) was noted in 34 (62%) cases, lichen sclerosis in 27 (49%) cases and lichen planus in three (5%) cases.

Surgical margins were free of invasion in 34 (62%) cases.

Inguinal dissection was performed in 51 (93%) cases and showed lymph node metastases in 17 (31%) cases.

Immunohistochemical data

PD-L1 immunostaining was as follows:

tumor cells = 18 cases (33%) with variable intensity;

lymphocytes = six cases (11%) with moderate intensity in all cases.

Follow-up and survival

The median follow-up period was 39 months (ranging from 1 to 15 years). During follow-up, 25 (45%) patients had recurrence, which was local in seven (28%) cases, regional in three (12%) cases, loco-regional in 4% and distant in one (4%) case.

The OS rates were 58% at five years with a median period of eight years ( Figure 7) and the RFS rate was 60% at five years with a median period of 96 months ( Figure 8). Overall survival as well as recurrence-free survival were more reduced in case of PD-L1 expression. However, this difference was not significant ( p = 0.8).

Overall survival for women treated for squamous cell carcinoma of the vulva, according to the Kaplan-Meier method. Recurrence-free survival for women treated for squamous cell carcinoma of the vulva, according to the Kaplan-Meier method. Discussion

The PD-1/PD-L1 inhibitory immune checkpoint seems to have a significant prognostic impact in the evolution of certain cancers (bronchopulmonary cancer, breast cancer, lymphomas, etc.). 8 – 10 Indeed, it has been suggested that the expression of PD-L1 by tumor cells was associated with a worse prognosis due to its immunosuppressive activity within the tumor tissue. 9 Some in vitro studies have demonstrated that the binding of PD-L1 to its PD-1 receptor negatively regulates the synthesis of IL-2 and IFN-γ. Therefore, suppression of the secretion of these cytokines leads to apoptosis of cytotoxic T cells. 10 – 14

According to some studies, the expression of PD-L1 in intra-tumoral lymphocytes may also play a role in immune suppression, even when it is not expressed by tumor cells. 15 – 19 However, the expression of PD-L1 by tumor cells and/or the microenvironment varies according to tumor type and the prognostic implications remain controversial. 20 – 25 In addition, the expression of PD-L1 by tumors suggests a possible prognostic impact and the possibility of immunotherapy via PD-1 blockade. 26

Numerous studies on different types of tumors (particularly lymphomas) have demonstrated that blocking PD-L1 improves the response of T lymphocytes and promotes antitumor activity. This suggests that these molecules represent potential biomarkers in certain tumors. 27 – 29

Several research studies have attempted to determine the prognostic factors of vulvar cancer in order to optimize therapeutic management. The expression of PDL1 in this type of neoplasia has not been previously studied in the literature, hence the interest of our work.

Indeed, we observed PDL1 expression in vulvar squamous cell carcinoma in 44% of cases. This expression was noted in tumor cells in 33% of cases and in lymphocytes in 11% of cases.

In accordance with data from the literature, in the cases of our series expressing PDL1, there was a decrease in the rates of overall survival and survival without relapse (without being significant). This reflects a poorer prognosis for PDL1 positive tumors. These results are encouraging and encourage us to carry out this research work on a larger population.

Highlights of our study

It is true that the PDL1/PD1 checkpoint in cancers is a topical subject and there are many works that focus on it; nevertheless, our series concerns a cancer that is not yet treated, which is the cancer of the vulva. Our study could thus enrich the various works that have been devoted to the study of this regulatory checkpoint in gynecological cancers.

 Limits of our work

The main limitation of our study was the relatively small size of the study population, as well as missing data for a number of tumors. In addition, we could enrich our work with a more complete statistical study where we study the level of expression of PDL1 in relation to the various histoprognostic factors of vulvar tumors in order to better detect the prognostic role of PDL1 in this type of tumor.

In addition, our study was centralized on a single technique which was immunohistochemistry. A cytogenetic and molecular study, in addition to immunohistochemistry, could on the one hand catch up with the false negatives of the first technique, and, on the other hand, better characterize the ultrastructural profile of these tumors.

 Conclusions

In summary, in accordance with data from the literature, in the cases of our series expressing PD-L1, we found histological factors of poor prognosis in the cases of PD-L1 plus at the level of the tumor cells. These results are encouraging and encourage us to carry out this research work on a larger population.

 Consent

Written informed consent for publication of the patients’ details and their images was obtained from the patients.

 Data availability Underlying data

Zenodo: Underlying data for ‘Evaluation of PD-L1 expression in vulvar cancer’, https://doi.org/10.5281/zenodo.7195826. 7

This project contains the following underlying data:

Supplementary figures 1–6: The different figures provided are raw and unedited, uploaded as they were taken with our camera. They demonstrate various degree of intensity of PD-L1 expression in both tumor cells and lymphocytes.

Supplementary figures 7,8: These figures correspond to the Kaplan-Meier 5-year overall and relapse-free survival curves.

Data file 1: Table (1) raw clinicopathological data.xlsx. These data include age of anonymous patients at which cancer was diagnosed, symptomatology, the type of sample taken, tumor location, focality and size, the presence or absence of ulceration, hisological tumor type, immunostaining profile of tumor cells and lymphocytes, the percentage of PD-L1 expression and its variable intensity, margins status, inguinal dissection, lymph node metastasis status and recurrence at follow-up.

Data file 2: TMA protocol.xls This file contains the TMA protocol table explaining all the procedures.

Data file 3: Readme clinicopathological PD-L1 vulvar cancer_Data.txt

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)

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Histopathology. 2016;69(2):177–186. 27196116 10.1111/his.12996 Ferreira CR Manohar V Zhao S : Genetic Subtypes of Systemic Anaplastic Large Cell Lymphoma Show Distinct Differences in PD-L1 Expression and Regulatory and Cytotoxic T Cells in the Tumor Microenvironment. Appl. Immunohistochem. Mol. Morphol. 2020;28(1):10–16. 31809310 10.1097/PAI.0000000000000798 Sahraoui G : Evaluation of PD-L1 expression in vulvar cancer.[Dataset]. Zenodo. 2022. 10.5281/zenodo.7195826 Hind ZEA : Profil épidemio-clinique du cancer de la vulve. Thèse pour l’obtention du doctorat en médecine. Université Cadi Ayyad. 2012. Canavan TP Cohen D : Vulvar cancer. Am. Fam. Physician. 2002;66(7):1269–1274. 12387439 Tan A Bieber AK Stein JA : Diagnosis and management of vulvar cancer: A review. J. Am. Acad. Dermatol. 2019;81(6):1387–1396. 10.1016/j.jaad.2019.07.055 Weinberg D Gomez-Martinez RA : Vulvar Cancer. Obstet. Gynecol. Clin. N. 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Blood. 2015;126(19):2193–2201. 26239088 10.1182/blood-2015-02-629600 10.5256/f1000research.121601.r227074 Reviewer response for version 1 Chang Tao 1 Referee https://orcid.org/0000-0002-1450-1936 Department of Gynaecology, Women’s Hospital School of Medicine Zhejiang University, Hangzhou, China

Competing interests: No competing interests were disclosed.

 11 5 2024 Copyright: © 2024 Chang T 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

1. The authors cited relatively insufficient literature on PDL1 expression in vulvar squamous cell carcinoma.

2.  Immunohistochemistry methodology description is more like an experimental report than a scientific paper, and some sentences are confusing, such as 'The PD-L1 expression study was performed on 4-μm tissue sections cut from formalin-fixed, paraffin-embedded lymph node biopsies'. 

3. Why was ≥5% chosen as the cutoff value for PDL1 expression? Since it was Dako's PDL1, why doesn't it use TPS and CPS as the scoring criteria?

4. The research goal of this article is the correlation between PDL1 expression and prognosis of VSCC. The clinical data only has age but no information such as stage, grade, lymph node metastasis, etc., which is unreasonable.

5. The KM graph is puzzling in single line form, why is there no comparison between PDL1 positive and negative?

6. There are flaws in statistical methods and immunohistochemistry pictures.

In summary, the results of the article do not support the conclusion stated.

Is the work clearly and accurately presented and does it cite the current literature?

No

If applicable, is the statistical analysis and its interpretation appropriate?

No

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Gynecologic Oncology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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