Evaluation of PD-L1 expression in vulvar cancer

Ghada Sahraoui; Marwa Manai; Rahma Yaïche; Lamia Charfi; Montassar Ghalleb; Hayet Douik; Karima Mrad; Raoudha Doghri

doi:10.12688/f1000research.110031.1

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Research Article

Evaluation of PD-L1 expression in vulvar cancer

[version 1; peer review: awaiting peer review]

Ghada Sahraoui ^1,2, Marwa Manai³, Rahma Yaïche¹, [...] Lamia Charfi^1,2, Montassar Ghalleb⁴, Hayet Douik², Karima Mrad^1,2, Raoudha Doghri^1,2

Ghada Sahraoui ^1,2, Marwa Manai³, [...] Rahma Yaïche¹, Lamia Charfi^1,2, Montassar Ghalleb⁴, Hayet Douik², Karima Mrad^1,2, Raoudha Doghri^1,2

PUBLISHED 27 Oct 2022

Author details Author details

¹ Precision Medicine, Personalized Medicine and Oncology Investigation Laboratory, LR21SP01, Salah Azaiez Institute, Tunis, 1006, Tunisia
² Pathology Department, Salah Azaiez Institute, Tunis, 1006, Tunisia
³ Human Genetics Laboratory LR99ES10 University of Medecine of Tunis, University of Tunis El Manar, Tunis, 1068, Tunisia
⁴ Surgical Oncology Department, Salah Azaiez Institute, Tunis, 1006, Tunisia

Ghada Sahraoui
Roles: Conceptualization, Formal Analysis, Methodology, Resources, Writing – Original Draft Preparation, Writing – Review & Editing

Marwa Manai
Roles: Conceptualization, Data Curation, Resources, Writing – Review & Editing

Rahma Yaïche
Roles: Conceptualization, Data Curation, Writing – Review & Editing

Lamia Charfi
Roles: Resources, Visualization, Writing – Review & Editing

Montassar Ghalleb
Roles: Conceptualization, Data Curation, Investigation, Writing – Review & Editing

Hayet Douik
Roles: Project Administration, Writing – Review & Editing

Karima Mrad
Roles: Project Administration, Software, Supervision, Validation, Writing – Review & Editing

Raoudha Doghri
Roles: Methodology, Resources, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS AWAITING PEER REVIEW

This article is included in the Oncology gateway.

Abstract

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.

Keywords

vulvar cancer, squamous cell carcinoma, PD-L1, immunohistochemistry

Corresponding author: Ghada Sahraoui

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

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

How to cite: Sahraoui G, Manai M, Yaïche R et al. Evaluation of PD-L1 expression in vulvar cancer [version 1; peer review: awaiting peer review]. F1000Research 2022, 11:1225 (https://doi.org/10.12688/f1000research.110031.1) First published: 27 Oct 2022, 11:1225 (https://doi.org/10.12688/f1000research.110031.1) Latest published: 27 Oct 2022, 11:1225 (https://doi.org/10.12688/f1000research.110031.1)

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 (H₂O₂) 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 H₂O₂ 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 cells⁶ (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

Figure 1. Immunostaining of 60% of tumor cells with moderate expression intensity (×400).

Figure 2. Immunostaining of 30% of tumor cells with high expression intensity (×400).

Figure 3. Immunostaining of 15% of tumor cells with moderate expression intensity (×400).

Figure 4. Negative immunostaining for tumor cells and lymphocytes (×400).

Figure 5. Immunostaining of 10% of lymphocytes with moderate expression intensity (×400).

Figure 6. 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.

Table 1. 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

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).

Figure 7. Overall survival for women treated for squamous cell carcinoma of the vulva, according to the Kaplan-Meier method.

Figure 8. 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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