| Abstract|| |
Background: The programmed cell death protein – 1 (PD-1) – programmed cell death ligand – 1 (PD-L1) axis is emerging as a promising target for immunotherapy in triple-negative breast cancers (TNBC). Aims: We analyzed the expression of PD-L1 in TNBC cases, with special emphasis on lymphocyte-predominant tumors along with correlation of the same with clinicopathological features and outcome. Settings and Design: Tissue microarrays (TMA) were prepared from resection specimens of TNBC cases diagnosed from 2004 to 2008. Subjects and Methods: Immunohistochemical staining was performed on the TMA using the ventana PD-L1 antibody (Clone SP 263). Statistical Analysis: Chi-square test was used for correlation of PD-L1 positivity in tumor and immune cells with clinicopathological features. Univariate and multivariate survival analyses were carried out using the Kaplan Meir and Cox Regression methods, respectively. Results: Overall, PD-L1 staining was seen in 35.9% (66 out of 184) tumors. PD-L1 positivity of tumor cells was seen in 14.7% (27 out of 184 cases), whereas stromal immune cell expression was observed in 21.2% (39 out of 184) cases. Lymphocyte-predominant tumors showed statistically significant expression of PD-L1 in both tumor (P < 0.0001) and immune cells (P 0.036). On univariate analysis, PD-L1 in immune cells was associated with good overall survival (P 0.05) as well as disease-free survival (P 0.013). On multivariate analysis, the same was associated with a significantly decreased risk for recurrence (P 0.018). Conclusion: PD-L1 expression in stromal immune cells proved to be a significant prognostic factor for TNBC. This data can serve as a baseline to plan clinical trials with anti-PD-L1 drugs for TNBC in the Indian setting.
Keywords: India, outcome, Programmed cell death ligand – 1 (PD-L1), triple-negative breast carcinoma
|How to cite this article:|
Gajaria PK, Gupta MR, Patil A, Desai SB, Shet TM. Programmed cell death ligand – 1 expression in triple negative breast carcinoma and its prognostic significance in Indian population. Indian J Pathol Microbiol 2021;64:664-70
|How to cite this URL:|
Gajaria PK, Gupta MR, Patil A, Desai SB, Shet TM. Programmed cell death ligand – 1 expression in triple negative breast carcinoma and its prognostic significance in Indian population. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Dec 1];64:664-70. Available from: https://www.ijpmonline.org/text.asp?2021/64/4/664/328518
| Introduction|| |
Tumor pathogenesis elucidates the fact that mutations cause the cell proliferative pathways to go hay-wire and offer these deleterious cells replication and survival benefits. Immunoediting is one such mechanism wherein tumor cells express inhibitory molecules resulting in either attenuation of host antitumor response or allow them to escape from host immune defenses. Programmed cell death protein – 1, PD-1 (CD 279), an immune checkpoint inhibitor is normally expressed on the surface of activated T-cells, B-cells, natural killer T-cells, dendritic cells, and monocytes. It binds to the ligands programmed cell death ligand-1 (PD-L1) and PD-L2. This linkage causes T-cell apoptosis, decreases T-cell proliferation, leads to downregulation of T-cell activity, decreases cytokine production, and induces tolerance to antigens.,,
Triple negative breast cancers (TNBC) are aggressive tumors with limited options for targeted therapy. These have shown early recurrence, are known to metastasize, and have a shorter overall survival (OS), indicating that these carry a poorer prognosis than other types of breast cancer., Hence, there is a need to identify potential biomarkers that can be exploited for newer targeted therapeutic agents in TNBC.
PD-L1 appears to be a propitious target in this regard, and anti-PD-L1 therapies are now the option in the narrow therapeutic landscape of TNBC. Following the results of IMpassion 130, FDA has approved atezolizumab in nonresectable locally advanced and metastatic TNBC. This offers hope for patients. However, there are several lacunae and issues surrounding the testing for PD-L1 in a laboratory.
We studied the expression of PD-L1 by immunohistochemistry (IHC) on TNBCs in a cancer referral center and correlated the same with clinicopathological features and patient outcomes in the Indian population, with emphasis on lymphocyte predominant (LyP) tumors.
| Subjects and Methods|| |
The study was conducted after approval from the institutional ethics committee. Tissue microarrays (TMA) consisting of TNBC cases from the year 2004 to 2008 were utilized for the study.. TMA blocks were made for another Institutional Review Board (IRB)–approved study on folate receptors in TNBC, and the same were used for the present study. Each case was represented by two cores (core size – 0.2 mm) on the TMA. Sections were cut at 4-micron thickness, stained with Hematoxylin and Eosin (H and E) for PD-L1 by IHC. Cases with washed-off cores and nonrepresentative material were excluded.
Relevant clinical details of the patients were obtained from the electronic medical records. Other details such as tumor morphology, grade, presence of lymphovascular emboli (LVE), and nodal metastatic status were noted from the histopathology report of either modified radical mastectomy (MRM) or breast conservation surgery (BCS) specimens. The tumor was considered as negative for estrogen receptor (ER), progesterone receptor (PR) when <1% tumor cells showed nuclear staining. The criteria for HER2/neu negativity was a score 0/+1 staining. These IHC findings were documented from the core biopsy reports and were used only for sample selection, core biopsies were not reviewed.
Morphologically, tumors showing dense stromal lymphocytic infiltrate, covering ≥50% tumor area were classified as lymphocyte-predominant (LyP) breast cancers. As the study was done on TMA, scoring of tumor infiltrating lymphocytes (TIL) was not done.
Sections were stained using the Ventana PD-L1 antibody (Clone SP 263) on the Ventana Benchmark XT fully automated platform using the standard recommended protocol. Placenta and lung tissue were used as controls.
Criteria used for the positivity of tumor cells was ≥ 5% cytoplasmic and membrane staining at any intensity. For immune cells, ≥1% cytoplasmic or membrane staining at any intensity was considered positive, based on some studies.,,
All analyses were performed using the Statistical Package for the Social Sciences (SPSS, Version 25) software. Chi-square test was used for correlation of clinicopathologic factors with PD-L1 positivity in tumor and immune cells. Univariate and multivariate survival analyses were carried out using the Kaplan Meir and Cox Regression methods, respectively. OS was calculated from diagnosis to the date of last follow-up or date of death. Disease-free survival (DFS) was calculated from the date of diagnosis to the date of first local/distant recurrence.
| Results|| |
A total of 184 TNBC cases were evaluable on the TMA. The median age of patients was 50 years (Range 21–90 years). About a half (90 out of 184) of the patients were premenopausal at the time of diagnosis. Staging workup at the time of presentation, categorized 33.7% patients (60 out of 178) as locally advanced breast cancer (LABC), whereas 66.3% (118 out of 178) had operable breast cancer (OBC). About 56 of the LABC patients had received neoadjuvant chemotherapy (NACT), and the residual tumor was included in the TMA.
In the study group, 43.5% of the patients (80 out of 184) underwent MRM and 56.5% (104 out of 184) had a BCS. Twelve patients were lost to follow-up and 37.8% (65 out of 172) had local/distant recurrence, of whom 32.6% (56 out of 172) died due to disease. The remainder 62.2% (107 out of 172) patients were alive with no disease.
The majority of the tumors (68.9%) were T2 (2 to 5 cm), whereas 18% and 13.1% tumors were ≤2 cm and >5 cm, respectively. On histopathologic examination, except seven, all tumors were Grade III (according to Nottingham modification of Bloom-Richardson System), 25.7% (47 out of 183) tumors showed the presence of LVE, and 47.5% (85 out of 179) of the tumors had metastasized to the axillary lymph nodes. Using the definition of ≥50% stroma showing TILs, 16.3% (30 out of 184) tumors could be labeled LyP and none had received NACT.
PD-L1 staining was seen in 35.9% (66 out of 184) tumors of which tumor cells were stained in 14.7% (27 out of 184) cases, and PD-L1 expression in stromal immune cells was observed in 21.2% (39 out of 184) cases. In four tumors, PD-L1 stained both the tumor as well as immune cells. As regards PD-L1 staining in tumor cells, eight showed staining in 5% cells (weak), seven tumors had 10% to 30% tumor cell staining (moderate), and 12 had strong staining with >30% of tumor cells staining [Figure 1]. Amongst the 30 LyP tumors, tumor cell as well as immune cell positivity was seen in 40% (12 cases), each.
|Figure 1: (a) Shows the lymphocyte-predominant type of tumor (H and E, 200×) (b) PD-L1 staining highlighting the macrophages around the tumor (200×) (c) PD-L1 staining of the lymphocytes around the tumor (200×) (d) Weak staining intensity of the tumor cells by PD-L1 (200×). (e) Moderate staining intensity of the tumor cells by PD-L1 (200×). (f) Strong staining intensity of the tumor cells by PD-L1 (200×)|
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Association of PD-L1 with clinicopathological factors
The correlation of PD-L1 positivity in tumor and immune cells with the various clinicopathological features is shown in [Table 1]. It was observed that tumor cell positivity for PD-L1 was significantly associated with younger age (≤ 50 years) at diagnosis (P 0.003), premenopausal status (P 0.036), OBC at presentation (P 0.002), and with LyP tumors (P < 0.0001).
|Table 1: Association of clinicopathologic factors with PD-L1 positivity in tumor and immune cells (ORIGINAL)|
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Stromal immune cell positivity for PD-L1 showed a significant association with tumor size being between 2 and 5 cm (P 0.044), absence of LVE in the tumor (P < 0.0001), but not with the above factors. As immune cells are more in LyP type tumors, an obvious correlation was found with PD-L1 (P 0.006).
Univariate analysis revealed that patients with locally advanced disease (P < 0.0001), tumor size > 5 cm (P 0.019), and axillary nodal metastasis at surgery (P 0.004) were associated with significantly worse survival outcomes in our TNBC cohort. PD-L1 in immune cells in our study cohort was associated with good OS (P 0.05) as well as DFS (P 0.013) [Table 2] and [Graph 1].
Multivariate analysis showed that locally advanced disease at the time of diagnosis was associated with increased risk of mortality and recurrence (P < 0.0001), whereas positivity for PD-L1 in the immune cells was associated with significantly decreased risk for recurrence (P 0.018). This was also associated with better outcomes in terms of patient survival, though this association was not statistically significant (P 0.060) [Table 3].
| Discussion|| |
The burden of TNBC is on the rise in India, and immunotherapy is a ray of hope for the successful treatment of these patients. PD-L1 is one such marker, that can be targeted by immunotherapeutic agents and has shown promising results in the treatment of Non-small-cell lung carcinoma (NSCLC), melanomas, and urothelial carcinomas.,, Monoclonal antibodies targeting this protein prevent its binding with PD-1 on T-cells, thereby preventing suppression of T-cell function and enhancing host immune response against tumor cells. To stratify patients who would benefit from these immunotherapeutic agents targeting PD-L1, it is important to identify subsets of tumors expressing the same.
We hereby report the first study from India describing the incidence of LyP tumors in TNBC along with the expression and prognostic significance of PD-L1 in TNBC.
Host antitumor immune response holds importance for the outcome in TNBCs, as observed by Loi S. In our cohort, only about a fifth of patients showed LyP phenotype, which was lower as compared to most studies reported in the literature.,, This variability could be due to differences in the genetic make-up of the hosts, presence of genetic aberrations in cancer cells, environmental factors, and retrospective TMA-based nature of our study. Adams S et al. proved that there was a significant reduction of recurrence or death for every 10% increase in tumor stromal lymphocytes. In our study too, LyP tumors showed a trend towards better OS of TNBC patients, though this association was not statistically significant (P 0.083).
In our cohort of TNBCs, 14.7% of tumor cells and 21.2% of immune cells showed positive staining for PD-L1, which was comparable to most studies reported in the literature as shown in [Table 4].
|Table 4: Comparison of various studies on PD-L1 expression in TNBC (ORIGINAL)|
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All studies have used different antibody clones, and our study used the clone SP 263 with comparative results. This, along with differences in the criteria used to define positivity, types of samples tested (TMA versus whole sections, prechemotherapy versus postchemotherapy samples, primary tumor versus metastatic site) and composition of cohorts can be a cause for the varied prognostic significance with PD-L1 across studies. Bonito MD et al. had also used the Ventana clone SP 263 in TNBC, before our study. In order to establish standardization with respect to PD-L1 scoring in TNBC, they suggested a “tumor score” for evaluation.
PD-L1 positivity showed a significant inverse association with age and menopausal status in our study, and this was reported in some of the other studies as well., Neither age nor menopausal status was found to be significant by Guo L et al. and Li M et al.,
PD-L1 on tumor cells was not prognostically significant in our study, but immune cell positivity correlated significantly with a good prognosis, both in terms of longer DFS and OS, as has been reported by most authors.,,,
In one study from Australia, PD-L1 expression was very common in TNBC, reported in the tumor cell membrane (64%), cytoplasm (80%), and stromal (93%) cellular compartments. In their study, cytoplasmic expression of PD-L1 in tumor cells was associated with a lower risk of breast cancer–specific death (HR 0.45, P 0.035), whereas stromal PD-L1 expression was associated with a lower rate of deaths from all causes (HR 0.305, P 0.0042). Membranous expression of PD-L1 was not associated with the outcome. Botti G et al. also observed tumor cell PD-L1 to be of significance for better DFS.
All the authors had unique study designs and have observed varied factors to be of prognostic significance. Sun WY et al. compared three antibody clones, utilized different criteria for each clone to define positivity, and reported that clone 28-8 was associated with shorter DFS and OS. Mori H et al. clubbed the variables of PD-L1 expression with TILs and in their multivariate analysis, PD-L1-positive/TILs-high subgroup had the longest DFS and OS. With the ≥25% cut-off for PD-L1 in tumor cells, Cerbelli B et al., observed that a significant number of patients achieved pathologic complete response with neoadjuvant chemotherapy.
The beneficial impact of PD-L1 in immune cells is due to the favorable nature of the immune microenvironment with such cells. The expression of this biomarker on both tumor as well as immune cells was significantly associated with the LyP subtype in our study, again highlighting the importance of reporting TILs in TNBC.
A phase I trial, testing anti-PD-L1 drug atezolizumab or MPDL3280A in metastatic TNBC patients, showed an overall objective response rate of 19%. Another recent milestone study in this regard is the IMpassion130, comparing atezolizumab plus nab-paclitaxel with placebo plus nab-paclitaxel, as a first-line therapy in locally advanced/metastatic TNBCs. For PD-L1 positivity, they considered a cut-off of ≥ 1% positivity in the immune cells (using the clone, SP 142), and accordingly in patients with PD-L1 positive tumors, the response rate was 58.9% with atezolizumab–nabpaclitaxel as compared to only 42.6% in patients treated with placebo–nab-paclitaxel.
After the data from Impassion 130 recommending the use of clone SP 142 in TNBC, it can be argued that the clone, SP 263 is no longer valid for testing in TNBC. However, other trials such as GeparNuevo study have used the clone SP 263 and compared the utility of durvalumab (in the neoadjuvant setting along with anthracycline-taxane–based regimen) versus placebo, in their cohort of early TNBC patients. In their trial, the overall pathologic complete response rate was increased in PD-L1-positive tumors in all the therapy groups. This clinical trial highlights the importance of tumor cell PD-L1 expression. Thus, the clone SP 263 may be lower down in the list for choice of PD-L1 testing in TNBC but is not totally out of the same. Testing for PD-L1 in TNBC with newer companion diagnostics will open new avenues for treatment of this poor prognostic group of patients.
We identified a subset of PD-L1 positive TNBCs that could serve as a prognostically and therapeutically beneficial group. However, some limitations of our study could be due to NACT given to some patients that could have had an impact on the number of immune cells as well as tumor cells and also on the IHC expression. Also, archival blocks can show loss of antigenicity over the years, but this phenomenon is biomarker specific, and more studies are needed to test this with regards to PD-L1 expression.
| Conclusion|| |
About a third of TNBC in this retrospective study cohort were PD-L1 positive, and PD-L1 expression in immune cells was associated with better behavior. This data can serve as a baseline for planning immune therapy–based treatment and trials for TNBC in India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Tanuja M Shet
Department of Histopathology, 8th Floor, Annexe Building, Tata Memorial Centre, Dr E Borges Road, Parel, Mumbai - 400 012, Maharashtra
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]