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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 2  |  Page : 709-715

Prognostic value of programmed death ligand-1 expression in breast carcinomas


1 Department of Clinical Oncology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Oncology, Nasser Institute Hospitals, Cairo, Egypt

Date of Submission03-Oct-2021
Date of Decision08-Dec-2021
Date of Acceptance12-Dec-2021
Date of Web Publication27-Jul-2022

Correspondence Address:
Suzy F Gohar
Department of Clinical Oncology, Faculty of Medicine, Menoufia University, Shebin Elkom City, Menoufia Governorate 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_192_21

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  Abstract 


Objectives
To assess the prognostic value of programmed death ligand-1 (PDL-1) expression in patients with nonmetastatic breast carcinoma patients both in tumor cells and tumor-infiltrating lymphocytes (TILs).
Background
PDL-1 has emerged as important immune biomarkers in breast cancer. It can be targeted with immunotherapy, which represents a significant advancement in the management of this disease.
Patients and methods
Paraffin blocks of patients with breast cancer will be tested for the expression of PDL-1 both on TILs and malignant epithelial cells and correlated with patients and disease features.
Results
This retrospective study included 60 patients. PDL-1 expression was positive in 33 (55%) patients on TILs and was positive in tumor cells in 27 (45%) patients of the studied patients. There was no significant relation between PDL-1 expression on TILs and patients and disease features. There was significant relation between positive PDL-1 expression on epithelial cells and triple-negative breast cancer, estrogen receptor negative, progesterone receptor negative status (P = 0.006, 0.006, and 0.006). PDL-1 intensity in intratumoral lymphocytes is significantly related to tumor stage (P = 0.006). PDL-1 H score in malignant epithelial cells was significantly related to disease stage (P = 0.021). Tumor size, HER-2neu status, and PDL-1 H score on malignant epithelial cells were independent risk factors for disease progression.
Conclusion
PDL-1 expression is common in both malignant tumor cells and TILs. A higher PDL-1 H score on malignant epithelial cells was associated with longer time to progression.

Keywords: breast cancer, immune biomarker, programmed death ligand-1, triple-negative neoplasm, tumor-infiltrating lymphocytes


How to cite this article:
Alagizy HA, El Bary NM, Mohamed AS, Mohamoud MT, Gohar SF. Prognostic value of programmed death ligand-1 expression in breast carcinomas. Menoufia Med J 2022;35:709-15

How to cite this URL:
Alagizy HA, El Bary NM, Mohamed AS, Mohamoud MT, Gohar SF. Prognostic value of programmed death ligand-1 expression in breast carcinomas. Menoufia Med J [serial online] 2022 [cited 2024 Mar 29];35:709-15. Available from: http://www.mmj.eg.net/text.asp?2022/35/2/709/352145




  Introduction Top


The immune system plays an important role in cancer. It contributes to either tumor elimination or progression and impacts cancer therapy response[1]. Triple-negative breast carcinomas (TNBC) and human epidermal growth factor-2 positive (HER-2) breast carcinomas contain active tumor immune microenvironments, which can be targeted with immunotherapy[2].

Programmed cell death-1 (PD-1) is an inhibitory surface receptor primarily expressed by cytotoxic effector T-cells, but also expressed by B-cells, natural killer cells, activated monocytes, and dendritic cells. The ligands of PD-1 are programmed death ligand-1 (PDL-1) and PDL-2, which can be expressed by tumor cells, and by other cells in the tumor microenvironment, such as tumor-infiltrating lymphocytes (TILs), macrophages, and fibroblasts[3].

PD-1 and PDL-1 are key physiologic suppressors of the cytotoxic immune response. Results from preclinical studies revealed that inhibition of PDL-1 and PD-1 axis in the tumor microenvironment may promote tumor regression. Various tested agents targeting PD-1 or PDL-1 have demonstrated robust response rates in a variety of tumor types[4].

As antibodies targeting PD-1 or PDL-1 are the most promising immunotherapeutic treatment strategies in breast cancer the role of PDL-1 as a prognostic marker and as a predictive marker for the effectiveness of PD-1/PDL-1 immune checkpoint inhibition is a matter of debate[3].

As the study of PDL-1 expression in tumor cells and/or inflammatory cells in breast cancer remains an active area of research, the aim of the current study was to detect the expression of PDL-1 in patients diagnosed with nonmetastatic breast carcinoma in tumor cells and TILs and detect its relationship with disease features and patient's outcome.


  Patients and methods Top


This retrospective study was carried out at the Department of Clinical Oncology, Menoufia University in collaboration with the Pathology Department after ethical approval from the Institutional Review Board (IRB) of Menoufia University (approval number 12/2018 ONCO 32) And after consent.

Patients diagnosed with nonmetastatic invasive duct carcinoma of the breast from January 2016 to December 2016 whose medical records were available together with their paraffin blocks containing enough tissue for further pathological assessment were included in the study. However, patients with incomplete medical records, who were lost to follow up, with lost, inadequate paraffin blocks, or presented with metastatic diseases were excluded.

Formalin-fixed, paraffin-embedded blocks of selected cases were submitted for hematoxylin and eosin staining and then histopathological assessment was done at the Pathology Department for the evaluation of histological tumor type, tumor grade, presence of vascular invasion, and lymph node invasion.

Four-micrometer thick sections from each the tumor block was immune stained using the streptavidin–biotin-amplified system. The primary antibody used was rabbit polyclonal anti-human PDL-1 diluted as 1: 100 (Cat. #YPA1542) (Room 27-2, Building 7, No. 15, Paradise Walk, Jiangbei District, Chongqing, 400020, China). After deparaffinization and rehydration of the tissue, antigen was retrieved using a high pH citrate solution followed by cooling at room temperature. Then slides were incubated with primary antibodies overnight at 4°C. Secondary antibody using UltraVision detection system antipolyvalent HRP/DAB, ready-to-use, NeoMarker was applied, and staining was visualized using a DAB chromogen substrate and Mayer's hematoxylin as a counterstain.

Tumor cells and TILs were assessed for PDL-1 positivity. PDL-1 was assigned as positive if there is brownish cytoplasmic as well as membranous staining in any number of cells. The intensity of staining was graded as 0 (no staining), 1 (mild), 2 (moderate), and 3 (strong). The extent of staining was expressed as a percentage of expression. Then the histoscore (H score) was calculated[5].

Patients' clinical, laboratory, and follow-up data were collected from their medical records. Progression-free survival was defined as the period from the date of start of treatment till disease progression in the form of development of new metastatic lesion or local recurrence.

Statistical analysis

Data were fed to the computer and analyzed using IBM SPSS software package, version 20.0. (IBM Corp., Armonk, New York, USA). The Kolmogorov–Smirnov was used to verify the normality of distribution of variables. Comparisons between groups for categorical variables were assessed using the χ2 test (Monte Carlo correction). Pearson's coefficient was used to correlate between two quantitative variables. Kaplan–Meier survival curve was used for the significant relation with progression-free survival. COX regression was used to detect the most affecting factor for progression-free survival. Significance of the obtained results was judged at the 5% level.


  Results Top


This retrospective study included 60 patients with newly diagnosed nonmetastatic breast cancer. The age of the patients ranged from 35 to 60 years with mean ± SD age of 46.95 ± 7.29 and median of 45 years. Forty-five patients representing 75% of the patients were postmenopausal. Forty-two (70%) patients were of stage II disease and only 18 (30%) patients were of stage III. Only six (10%) patients were triple negative.

Fifty-three (88.3%) patients had grade III disease. Lymph node invasion was positive in 54 (90%) patients. Three (5%) patients had lympho-vascular invasion and two (3.3%) patients had perineural invasion.

PDL-1 expression was positive in 33 (55%) patients in the TILs. Out of the included patients only three (9.1% of positive cases) patients had a strong expression. The mean ± SD of the H score for the positive cases was 8.27 ± 8.24, which ranged from 1.0 to 20.0.

PDL-1 tumor cell expression was positive in 27 (45%) patients of the studied patients. Among those patients only six (22%) patients experienced a strong expression. The H score mean ± SD was 13.59 ± 17.76 (minimum–maximum: 1.0–55.0) [Figure 1].
Figure 1: PDL-1expression in invasive duct carcinoma. (a) A case of poorly differentiated invasive duct carcinoma showing strong membranous PDL-1 expression in epithelial cells and moderate membranous expression in tumor-infiltrating lymphocytes (IHC ×400). (b) Moderate membranocytoplasic PDL-1 expression in most of tumor-infiltrating lymphocytes (IHC ×400). (c) A case of poorly differentiated invasive duct carcinoma showing mild membranous PDL-1 expression in epithelial cells (yellow circle) and strong membranous expression in tumor-infiltrating lymphocytes (red circle) (IHC ×400). (d) Moderately differentiated invasive duct carcinoma lacking expression of PDL-1 (IHC ×200). PDL-1, programmed death ligand-1.

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There was no significant relation between PDL-1 expression on TILs and patients' outcomes and disease features. On the other hand, there was significant relation between positive PDL-1 expression and TNBC, estrogen receptor (ER)-negative, progesterone receptor (PR)-negative status P values were 0.006, 0.006, and 0.006, respectively. However, there was no significant relation between PDL-1 expression in the epithelium and other risk factors [Table 1].
Table 1: Relation between programmed death ligand-1 expression in malignant epithelial cells and tumor-infiltrating lymphocytes and different parameters (n=60)

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There was significant relation between PDL-1 intensity in intratumoral lymphocytes and tumor stage (P = 0.006); higher intensity was associated with stage III disease [Table 2].
Table 2: Relation between programmed death ligand-1 intensity in tumor-infiltrating lymphocytes and malignant epithelial cells and different parameters (n=60)

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Regarding PDL-1 H score in malignant epithelial cells, it was significantly related to disease stage (P = 0.021). A higher H score is associated with stage II disease [Table 3].
Table 3: Relation between programmed death ligand-1 H score in tumor-infiltrating lymphocytes and malignant epithelial cells and different parameters (n=60)

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Correlation between PDL-1 expression in intratumoral lymphocytes and malignant epithelial cells is shown in [Figure 2].
Figure 2: Correlation between PDL-1 expression in tumor-infiltrating lymphocytes and malignant epithelial cells. PDL-1, programmed death ligand-1.

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Univariate analysis for factors affecting progression-free survival were age, tumor size, HER-2neu expression, and PDL-1 expression on malignant epithelial cells and PDL-1 H score in malignant epithelial cells. Multivariate analysis revealed that tumor size, HER-2neu status, and PDL-1 expression on malignant epithelial cells were independent risk factors for disease progression [Table 4].
Table 4: Univariate and multivariate COX regression analysis for the parameters affecting progression-free survival (n=21 vs. 39) for different parameters

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  Discussion Top


PDL-1 gene is located on chromosome 9p24.1 and encodes a 33 kD protein. It has an extracellular immunoglobulin V-like and C-like domains and a hydrophobic transmembrane region, followed by a 30-amino acid cytoplasmic tail. PDL-1 gene has a long 3′-UTR region and multiple acting elements, which are important in posttranscriptional regulation[6].

PDL-1 expression is noticed at low levels in multiple hematopoietic and nonhematopoietic cells, such as dendritic cells, macrophages, B cells, T cells, mast cells, and certain types of epithelial and endothelial cells, including muscles and nerves. Furthermore, PDL-1 is found in many types of tumor cells, where its surface expression level correlates with T-cell function suppression[7].

The binding of PDL-1 to its receptor PD-1 prevents autoimmunity by reducing T-cell expansion and decreasing cytokine production. However, in case of tumor cells, the same immunosuppressive effect permits the tumor to evade immune destruction. Some types of breast cancer (e.g., TNBC) are immunogenic and may overexpress PDL-1. These types have been shown to be more sensitive to immune therapy drugs such as immune checkpoint inhibitors[8].

The aim of the current study was to detect PDL-1 expression in both tumor cells and inflammatory cells in patients diagnosed with nonmetastatic breast carcinoma and detect its relationship with disease features and patient's outcome.

PDL-1 expression was positive in TILs in 55% of the patients and in malignant epithelial cells in 45% of the included patients. This incidence is much greater that that reported by Wang and Liu[9], who observed that PDL-1 expression was detected in both tumor cells and TILs at 8.5 and 25.1% of the patients, respectively.

This difference can be explained by the difference in patient features as their study included triple-negative patients only and they considered membranous cell staining in more than 1% as positive.

We also found that positive PDL-1 expression on tumor cells was associated by negative PDL-1 expression on TILs indicating the inverse relationship between them. These findings are logic as tumor cell expression of PDL-1 is associated with good immunity against tumor. Conversely, PDL-1 expression in TILs is associated with immune evasion by tumor cells.

In the same context, Solinas et al.[10], Beckers et al.[11], Sánchez-Cousido et al.[12], Assaf et al.[13], and Quintana et al.[14] reported that PDL-1 expression on tumor cells correlated with TILs. As positive PDL-1 expression in malignant cells was associated with high TILs in the tumor tissue confirming its immune stimulant effects. Also, Wang and Liu[9] found that PDL-1 expression in TILs was associated with an abundance of TILs.

There was no significant relation between PDL-1 expression on TILs and any of the patients' outcomes or disease features. However, Wang and Liu[9] found that PDL-1 expression in TILs was associated with higher tumor grade.

On the other hand, PDL-1 expression in malignant epithelial cells is significantly related to TNBC and lack of ER and PR expression. We noticed that all patients with TNBC had positive PDL-1 expression in malignant epithelial cells. Although PDL-1 expression in malignant cells was frequently noted in HER-2 neu-positive patients in the current study, this difference was not statistically significant. This is suggesting that triple TNBC and HER-2-positive breast carcinomas contain active tumor immune microenvironments, which can be harnessed or targeted with immunotherapy; this may have significant impact on patient's management.

Kitano et al.[15] reported that the PDL-1 expression rate in malignant cells was highest in TNBC tumors, followed by hormone receptor negative with HER-2-positive disease. Similarly, Noske et al.[16] found that the expression of PDL-1 in tumor cells was significantly associated with negative ER and PR expression, the TNBC subtype.

Furthermore, Beckers et al.[11] found that PDL-1 expression was very common in TNBC, seen in 94.4% of patient samples. Also, Solinas et al.[10] concluded that PDL-1 expression was correlated with TNBC. And Shash et al.[17] found that PDL-1was positive in malignant epithelial cells in 41.5% of breast cancer patients with locally advanced TNBC and HER-2neu overexpression.

All these findings suggest the possible role of immune system in the pathogenesis of these types of breast cancers, that is, TNBC and hormone receptor-negative tumors, and may point to the possible role of immunotherapy in those patients with limited therapeutic options.

Against our results Uhercik et al.[18] did not find significant differences in PDL-1 expression according to ER status.

In our study, a strong PDL-1 expression intensity in TILs was found in only 9.1% of positive cases. This percent is lesser than that reported by Abdeljaoued et al.[19], who found a high PDL-1 expression in 64.5% of their patients and we can explain this by the difference in patients features because they included male breast cancer patients only which is known by its aggressive nature.

We also found a strong PDL-1 expression intensity in TILs was significantly related to more advanced stage III disease, and this can be explained by its immune evasion effect, while PDL-1 expression intensity in tumor cells was not significantly related to any of the disease features.

Also, Kiriacos et al.[20] in their study conducted on young TNBC patients reported that PDL-1 overexpression was directly associated with lymph node metastasis and stage of disease.

A higher H score of PDL-1 on malignant epithelial cells was significantly related to less advanced stage II disease indicating an immunostimulant effect against tumor cells.

Multivariate analysis revealed that negative HER-2 expression, smaller tumor size, and PDL-1 H in epithelial cells score were independent prognostic factors for progression-free survival in our patients. Close to our findings, Shash et al.[17] suggested that there was a relation between PDL-1 expression on tumor cells at initial presentation and event-free survival and in Uhercik et al.[18] higher PDL-1 transcript levels were also associated with better overall and disease-free survival. Similarly, in the Parvathareddy et al.[21] study, PDL-1 overexpression in tumor cells was an independent prognostic marker in TNBCs from Middle Eastern ethnicity.

Both Yue et al.[22] and AiErken et al.[23] found in their studies conducted on a group of TNBC patients that PDL-1 high expression in tumor cells was associated with disease-free survival and overall survival in univariate analysis. And in the multivariate analysis, patients with PDL-1 expression showed significantly more favorable prognosis in disease-free survival and improve the overall survival compared with the PDL-1-negative group.

However, Beckers et al.[11] found that PDL-1 expression was associated with better outcomes but was not an independent prognostic factor in triple-negative patients. Sánchez-Cousido et al.[12] also reported that PDL expression was associated with better overall survival but has not reached a statistically significant level.

Like Noske et al.[16] who did not find any relation between PDL-1 expression in TILs, we did not find any relation between PDL-1 expression, intensity, or H score in TIL and progression-free survival. Against our results Wang and Liu[9] found that PDL-1-positive expression in TILs was an independent prognostic factor for overall survival. Abdeljaoued et al.[19] found that a higher expression of PDL-1 in TIL was an independent prognostic factor for overall survival in male breast cancer patients.

There are some limitations for the current study like the small number of the studied cases and this can be explained by the absence of tissue or block bank; so unfortunately many patients were excluded due to problems regarding lost blocks or exhausted blocks. Similarly, other patients were excluded due to incomplete files and missed data.


  Conclusion Top


PDL-1expression was frequent in breast cancer patients. Cross-talk between tumor cells and TILs is evidenced by an inverse correlation between PDL-1 expression in both. PDL-1 H score in epithelial cells were independent prognostic factors for progression-free survival.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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