|Year : 2020 | Volume
| Issue : 8 | Page : 4086-4091
Effect of neoadjuvant chemotherapy (NAC) on programmed cell death ligand (PD-L1) in patients of carcinoma breast: A prospective study in Indian tertiary care setting
Vipul Srivastava, BR Akshay, Sweety Kumari, Ram Niwas Meena, Rahul Khanna
Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Submission||28-Mar-2020|
|Date of Decision||25-Apr-2020|
|Date of Acceptance||08-Jun-2020|
|Date of Web Publication||25-Aug-2020|
Dr. Ram Niwas Meena
Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh - 221005
Source of Support: None, Conflict of Interest: None
Context: Several studies have reported that PD-L1 has shown therapeutic activity in various tumor types. However, its expression changes in a person on administration of NAC which is reported by very few studies. Aims: To find out the difference in the expression of PD-L1 by tumor cells after the administration of NAC. Settings and Design: This prospective study was conducted on 30 patients who were diagnosed with locally advanced breast carcinoma (LABC) between 2017 and 2019 and those who received NAC followed by surgery. Methods and Material: Breast cancer specimens were collected using core needle biopsy prior to administration of NAC and IHC was performed. Frequency and staining intensity of PD-L1 by tumor cells were analyzed. PD-L1 expression was dichotomized into two groups according to the frequency distributions of the H-scores. Statistical Analysis Used: The differences in expression of PD-L1 along with various parameters were analyzed using Chi-square test and Student's t test. Results: The mean age of the patients in our study was 51.37 ± 11.37 years. The response of NAC according to the RECIST criteria showed that most of patients (83.3%) showed complete response. Of the 30 cases, 11 (36.7%) patients were PD-L1 positive before the administration of NAC. We found a significant change in expression from positive to negative status, i.e., seven patients changed from positive to negative (p = 0.036). Upon comparing the PD-L1 expression before NAC, significant association was observed between the primary tumor (T) and tumor stage with high PD-L1 expression (p = 0.020 and P = 0.034). After NAC, 18 (69.2%) patients who were ER positive and 18 (69.2%) patients who were PR positive showed negative PD-L1 expression while none of them were positive in PD-L1 positive patients (p = 0.018 and P = 0.018). Conclusion: PD-L1 expression in a same person changes upon administration of NAC which may indirectly be used as a predictor of response to NAC.
Keywords: Breast cancer, neoadjuvant chemotherapy, Programmed cell death ligand
|How to cite this article:|
Srivastava V, Akshay B R, Kumari S, Meena RN, Khanna R. Effect of neoadjuvant chemotherapy (NAC) on programmed cell death ligand (PD-L1) in patients of carcinoma breast: A prospective study in Indian tertiary care setting. J Family Med Prim Care 2020;9:4086-91
|How to cite this URL:|
Srivastava V, Akshay B R, Kumari S, Meena RN, Khanna R. Effect of neoadjuvant chemotherapy (NAC) on programmed cell death ligand (PD-L1) in patients of carcinoma breast: A prospective study in Indian tertiary care setting. J Family Med Prim Care [serial online] 2020 [cited 2021 Apr 11];9:4086-91. Available from: https://www.jfmpc.com/text.asp?2020/9/8/4086/293032
| Introduction|| |
Breast cancer is the most common cancer among women, affecting 2.1 million women each year and causing the largest number of women with cancer-related deaths. In 2018, breast cancer deaths were reported at 627,000, i.e., 15% of all women cancer-related deaths. While the breast cancer rates among women in developed countries are higher, their rates are rising globally in almost every region. In the Indian scenario, breast cancer is the most common cancer among women in metropolitan cities. Despite the remarkable advances in therapies for breast cancer, there are still women who die of this disease at the end. In Western population, the incidence of advanced stage disease has decreased due to routine screening protocols. However, we face an increase in the number of locally advanced and metastatic cases in the Indian setting even at a younger age at the time of diagnosis. In such cases, NAC remains the cornerstone of treatment.
Immunotherapy is a newly emerging tool in breast cancer management and can be safely applied as an adjunct or alternative to chemotherapy in the treatment of breast cancer. Cancer cells frequently express tumor antigens that, in principle, can be recognized by the patient's immune system; however, the resultant immune responses are ineffective and often do not parallel clinical tumor regression.
Adaptive and innate immune responses play an important role in tumor immune monitoring and may restrict neoplasm development and growth. Tumors develop in a complex network of epithelial cells, blood vessels, lymph channels, cytokines and chemokines, and invade immune cells known as tumor microenvironments. It has been shown that adaptive immune system cells conduct monitoring and can remove nascent tumors via various immune modulatory pathways.
There is a complex interaction between the T-cells and antigen-presenting-cells (APC) which involves T-cell receptor along with multiple co-stimulatory receptors. This can either activate or inhibit T-cell function. Programmed cell death-1 (PD-1) is an example of such co-stimulatory receptor that belongs to CD28/CTLA-4 family. It conveys an inhibitory signal to T-cell, thus impeding immune response; a protective mechanism designed for self-antigens. Tumor cells mimic this interaction and escape from our immune system which otherwise would have been destroyed, this phenomenon is known as “tumor evasion”.
PD-1 binds on the surface of cancer cells to programmed cell death ligand-1 (PD-L1), which suppresses the T lymphocyte antitumor functions. Thus, the expression of PD-1 suggests depleted lymphocyte activity, and a high level of PD-1 + tumor lymphocyte infiltration correlates with worse breast cancer survival. PD-L1 expression has been shown in various cancers such as lung, melanoma, ovary, colon, and breast cancers.,
Several studies indicated that chemotherapy had an immunogenic function. They improve the immunogenicity of tumor cells, or they can modulate the immune cells and thus exert antitumor response. Doxorubicin can induce the immunogenic death of cancer cells by the release of high-mobility group box-1 (HMGB-1) protein from dying cancer cells. Paclitaxel enhances lymphocyte anti-tumor function by activating various cytokines. In view of the clinical applications of the PD-1/PD-L1 axis and after reviewing previous studies, we aimed to find out the difference in the expression of PD-L1 by tumor cells after the administration of NAC which may have significant implications for the treatment of breast cancer.
| Methods|| |
The cohort used in this study consists of 30 patients with locally advanced breast carcinoma (LABC) diagnosed between 2017 and 2019 and received NAC followed by surgery at the Department of General Surgery, Institute of Medical Sciences in Banaras Hindu University. Breast cancer specimens were collected using core needle biopsy prior to the administration of NAC and IHC was performed. Then, three cycles of NAC was given and modified radical mastectomy was performed and this sample was used for IHC this time. Most of our patients (76.7%) received CAF regimen (cyclophosphamide, Adriamycin, and 5-fluorouracil) followed by Taxane-based chemotherapy in 23.3% of individuals. The ethical approval for the study was obtained from the Institutional Ethics Committee and the protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki. A proper written informed consent was obtained from all the patients. Approval from institute ethical committee was taken. It was done on 24/10/2017.
Estimation of PD-L1 using Immunohistochemistry (IHC)
Tissue microarray (TMA) was built using the most representative areas from each single case. Immunohistochemical staining was done after the preparation of slides from formalin-fixed, paraffin embedded tissues (after antigen retrieval from TRIS-EDTA buffer followed by peroxidase block and power block). Slides were incubated overnight at 4°C with a prediluted primary rabbit-anti-human PD-L1 polyclonal antibody (Abcam, Cambridge, UK). The standard DAB-technique (Optiview DAB IHC Detection Kit, Ventana Medical Systems, Tuscon, AZ, USA) was employed for immunostaining. The results were interpreted using a light microscope (Olympus BX 51, Tokyo, Japan) [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d.
|Figure 1: (a) Photograph of PD-L1 expression in breast cancer tissue specimen showing diffuse positivity of PD-L1 seen after IHC staining on 10X field. (b) Photograph of PD-L1 expression in breast cancer tissue specimen showing membranous and cytoplasmic positivity of PD-L1 seen on tumor cells after IHC staining through a 40X field. (c) Photograph of PD-L1 expression in breast cancer tissue specimen without any staining seen after IHC staining (PD-L1 negative) through a 10X field. (d) Photograph of PD-L1 expression in breast cancer tissue specimen without any staining seen after IHC staining (PD-L1 negative) through a 20X field|
Click here to view
The frequency and staining intensity of PD-L1 by tumor cells were analyzed, and PD-L1 expression was quantified using the modified Histo-score (H-score) with a range of possible scores from 0 to 300. PD-L1 expression was dichotomized into two groups according to the frequency distributions of the H-scores, using a cut-off score of ≥100 (H-score 0–99 = negative/low expression and 100–300 = high/positive expression).
Statistical analysis was performed using statistical package for the social sciences (SPSS), Version 23.0. IBM Corp., NY). Simple descriptive statistics were used (mean ± standard deviation) for quantitative variables, and frequency with percentage distribution for categorized variables. All the clinical and related parameters studied during observation period were compared using Chi-square test for parametric variables. Statistical analysis was performed using the paired and unpaired Student's t test, the Wilcoxon test, and the Mann–Whitney U test, with the comparison of absolute and relative values for cells obtained before and after NAC. The value of P < 0.05 was considered as the significant difference for comparison.
| Results|| |
The mean age of the patients in our study was 51.37 ± 11.37 years (ranging from 30 to 71 years). Majority (33.3%) of the patients were aged 41–50 years followed by >60 years (26.7%) and 51–60 years (23.3%), respectively. There were 29 women and only 1 male patient. 13 (43.3%) of our patients had T2-stage primary tumor followed by T4 stage in 12 (40%) patients as per TNM staging of AJCC 8th 2017. In addition, majority of them (63.3%) were in the cN1 stage. Triple negative breast cancer (TNBC) patients were seven in number (23.3%). The demographic and clinicopathologic characteristics of the patients are shown in [Table 1].
The response of NAC according to RECIST criteria showed that most of patients (83.3%) showed complete response followed by those with partial response (13.3%) after NAC; one patient (3.3%) was stable and did not show any decrease or increase in size.
Of the 30 cases, there were 11 (36.7%) patients who were PD-L1 positive before the administration of NAC. After giving three cycles of NAC only four (13.3%) patients remained PD-L1 positive while the rest of them, i.e., seven patients out of 11 changed from positive to negative with a significant P value of 0.036 [Figure 2].
|Figure 2: Comparison of change in PD-L1* on administration of NAC *Here positive and negative is based on PD-L1 expression in IHC which was calculated using Histo Score which ranged from 0-300. Negative implies values between 0-99 while positive implies values from 100-300|
Click here to view
Upon comparing the clinicopathological parameters with PD-L1 expression before NAC, all of the PD-L1 positive patients (n = 8, 72.7%) were in the T4 category followed by T2 category in two (18.2%) patients. Most of the PD-L1 negative patients (n = 11, 57.9%) were in T2 category which showed significant association (p = 0.020). Also among PD-L1 positive patients, nine (81.8%) patients were in stage IIIA & IIIB while in negative patients, 11 (57.9%) patients were in Stage IIA & IIB which showed a significant association (p = 0.034). Sixteen (84.2%) ER positive patients and 16 (84.2%) PR positive patients who were negative for PD-L1 expression showed significant association (p < 0.001). Her2-neu status and histological grade of tumor were also compared with PD-L1 expression but did not show any significant association [Table 2]. After NAC, among negative PD-L1 patients, 18 (69.2%) were ER positive and 18 (69.2%) PR positive, while none of them were positive in PD-L1 positive patients. Other parameters such as primary tumor (T), tumor stage, histological grade, and Her2-neu status were also compared but they did not show any significant association [Table 3].
|Table 2: Comparison of clinico-pathological parameters with expression of PD-L1 before NAC|
Click here to view
|Table 3: Comparison of clinico-pathological parameters with expression of PD-L1 after NAC|
Click here to view
| Discussion|| |
Locally advanced breast cancer (LABC) is a heterogeneous group of patients with uncertain prognosis and a 5-year survival rate of 50%–80%. Among developing countries, the prevalence of LABC is higher than in Western countries, possibly due to the late-stage disease when diagnosed. NAC is one of the standard therapy options for the management of LABC. The objectives of preoperative systemic therapy in LABC include early eradication of distant subclinical micrometastases and downstage of the primary tumor to enable operability. This method has the added benefit of making an in vivo measurement of chemotherapy responsiveness to tumors.
It was clearly evident in this analysis that most of our participants were 41–50 years of age. This is slightly below the age of 52 in Western countries. This is further confirmed by a study conducted by Surakasula et al., who also reported breast cancer in India as opposed to their Western counterparts in a younger age group. Only seven (23.3%) patients in our sample had triple negative breast cancer, the result was close to different studies, i.e., the prevalence of triple negative breast cancer to be in the range of 10–30%.
PD-L1 has been shown to be directly involved in the protection of cancer cells from destruction by activated T lymphocytes by inhibiting anti-cancer immune response. The interaction of PD-1/PD-L1 between the tumor cells and T-lymphocytes results in the impairment of both cytokine development and T-lymphocyte apoptosis. Patients with breast cancer already have immune defects such as a lower absolute number of peripheral blood lymphocytes and an increased number of functionally immunosuppressive CD4 + CD25 + T-reg lymphocytes in both the peripheral blood and tumor microenvironments.
We found the PD-L1 expression in breast cancer specimens to be 36.7% before NAC administration, which is consistent with the study by Ghebeh et al. who reported the expression of PD-L1 in 34% of breast cancers. Schalper et al. reported the expression of PD-L1 mRNA in 58% of their specimens of breast cancer. Since giving NAC, there was a significant reduction in the expression PD-L1, which had now decreased to 13.3%. Nonetheless, the higher expression of PD-L1 in NAC patients could be due simply to the fact that the patients who receive it typically have LABC. Most of the cases were negative at baseline and remained negative after chemotherapy in a study done by Pelekanou et al. They also reported that PD-L1 expression decreased in most of the initially positive PD-L1 tumors following chemotherapy that is in line with our study.
Cytotoxic drugs are widely used to supplement the activation of immune checkpoints, which in some cases can provide immunogenic benefits. Contrary to our study, Peng et. al reported that anthracycline and taxane upregulated PD-L1 and Gal-9 expressions, independent of the effect of IFN-γ, in vitro and also that cytotoxic drugs could induce PD-L1 upregulation through the NFκB-dependent pathway in an ovarian cancer model.
Upon comparing PD-L1 with various clinicopathological parameters it was further found that PD-L1 expression was associated significantly with poor prognostic factors like higher tumor grade (72.7% of PD-L1 positive cases were in T4 category), tumor stage (81.8% of PD-L1 positive cases had stage III tumor), and hormone receptor status (81.8% of PD-L1 positive cases were both ER and PR negative). Histological grade and Her-2-neu status did not show any significant association. This in accordance with various other previous studies.,,
Once expressed by antigen-presenting cells, PD-L1 generates an inhibitory signal to T-lymphocytes by inducing T-lymphocyte apoptosis. Therefore, by inhibiting the immune system, it is likely that PD-L1 can establish a favorable environment for the tumor to develop. Another likelihood of lower survival may be due to the association of tumors with higher growth rates (large tumor size and high histological grade) or poor differentiation (small histological grade and negative estrogen and progesterone receptors), which is known to reduce the survival of patients with cancer.
We agree that our research has some limitations such as the fact that the use of tissue microarrays (TMAs) may not accurately represent PD-L1 protein expression due to the intratumoral heterogeneity of expression. Also, there are questions about the efficacy of immunohistochemical staining for PD-L1, due to the lack of a standardized procedure for staining and analysis, as well as the variety of antibodies. The relatively small number of patients in each group makes it difficult to draw a conclusion. While the majority of our patients (76.7%) received CAF regimen (cyclophosphamide, adriamycin and 5-fluorouracil), some were given Taxane-based chemotherapy (23.3%), limiting any interpretation of treatment-specific results.
| Conclusion|| |
Manipulating the immune system to directly identify and kill tumor cells can be an effective alternative approach to cancer treatment in the days ahead. Blocking the PD-1/PD-L1 pathway has become a recent therapeutic strategy promising in oncology. Taking these findings into account, anti-PD-L1 antibodies combined with chemotherapy agents may be suitable candidates for chemoimmunotherapy. The area of PD-L1 assessment is rapidly evolving and several corresponding and complementary diagnostic applications in various other malignancies are approved by FDA. Also, we conclude that change in PD-L1 expression may serve as an indirect indicator to assess the response of NAC in a patient.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394-424.
Mohan S, Asthana S, Labani S, Popli G. Cancer trends in India: A review of population-based cancer registries (2005–2014). Indian J Public Health 2018;62:221-3.
] [Full text]
Dermime S, Gilham DE, Shaw DM, Davidson EJ, Meziane el K, Armstrong A, et al
. Vaccine and antibody-directed T cell tumour immunotherapy. Biochim Biophys Acta 2004;1704:11-35.
Disis ML. Immune regulation of cancer. J ClinOncol 2010;28:4531-8.
Bour-Jordan H, Esensten JH, Martinez-Llordella M, Penaranda C, Stumpf M, Bluestone JA. Intrinsic and extrinsic control of peripheral T-cell tolerance by costimulatory molecules of the CD28/B7 family. Immunol Rev 2011;241:180-205.
Muenst S, Soysal SD, Gao F, Obermann EC, Oertli D, Gillanders WE. The presence of programmed death 1 (PD-1)-positive tumor-infiltrating lymphocytes is associated with poor prognosis in human breast cancer. Breast Cancer Res Treat 2013;139:667-76.
McLaughlin J, Han G, Schalper KA, Carvajal-Hausdorf D, Pelekanou V, Rehman J, et al
. Quantitative assessment of the heterogeneity of PD-L1 expression in non-small-cell lung cancer. JAMA Oncol 2016;2:46-54.
Kluger HM, Zito CR, Barr ML, Baine MK, Chiang VL, Sznol M, et al
. Characterization of PD-L1 expression and associated T-cell Infiltrates in metastatic melanoma samples from variable anatomic sites. Clin Cancer Res 2015;21:3052-60.
Chen G, Emens LA. Chemoimmunotherapy: Reengineering tumor immunity. Cancer Immunol Immunother 2013;62:203-16.
Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al
. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 2007;13:1050-9.
Javeed A, Ashraf M, Riaz A, Ghafoor A, Afzal S, Mukhtar MM. Paclitaxel and immune system. Eur J Pharm Sci 2009;38:283-90.
Mathew J, Asgeirsson KS, Cheung KL, Chan S, Dahda A, Robertson JF. Neoadjuvant chemotherapy for locally advanced breast cancer: A review of the literature and future directions. Eur J Surg Oncol 2009;35:113-22.
Franceschini G, Terribile D, Magno S, Fabbri C, D'Alba PF, Chiesa F, et al
. Update in the treatment of locally advanced breast cancer: A multidisciplinary approach. Eur Rev Med Pharmacol Sci 2007;11:283-9.
Petit T, Wilt M, Velten M, Millon R, Rodier JF, Borel C, et al
. Comparative value of tumour grade, hormonal receptors, Ki-67, HER-2 and topoisomearase II alpha status as predictive markers in breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy. Eur J Cancer 2004;40:205-11.
Surakasula A, Nagarjunapu G, Raghavaiah K. A comparative study of pre- and post-menopausal breast cancer: Risk factors, presentation, characteristics and management. J Res Pharm Pract 2014;3:12-8.
] [Full text]
Tan EY, Yan M, Campo L, Han C, Takano E, Turley H, et al
. The key hypoxia regulated gene CAIX is upregulated in basal-like breast tumours and is associated with resistance to chemotherapy. Br J Cancer 2009;100:405-11.
Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci USA 2002;99:12293-7.
Caras I, Grigorescu A, Stavaru C, Radu DL, Mogos I, Szegli G, et al
. Evidence for immune defects in breast and lung cancer patients. Cancer ImmunolImmunother 2004;53:1146-52.
Ghebeh H, Mohammed S, Al-Omair A, et al
. The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors. Neoplasia 2006;8:190-8.
Schalper KA, Velcheti V, Carvajal D, et al
. In situ
tumor PD-L1 mRNA expression is associated with increased TILs and better outcome in breast carcinomas. Clin Cancer Res 2014;20:2773-82.
Pelekanou V, Carvajal-Hausdorf DE, Altan M, Wasserman B, Carvajal-Hausdorf C, Wimberly H, et al
. Effect of neoadjuvant chemotherapy on tumor-infiltrating lymphocytes and PD-L1 expression in breast cancer and its clinical significance. Breast Cancer Res 2017;19:91.
Peng J, Hamanishi J, Matsumura N, Abiko K, Murat K, Baba T, et al
. Chemotherapy induces programmed cell death-ligand 1 overexpression via the nuclear factor-κB to foster an immunosuppressive tumor microenvironment in ovarian cancer. Cancer Res 2015;75:5034-45.
Wimberly H, Brown JR, Schalper K, Haack H, Silver MR, Nixon C, et al
. PD-L1 expression correlates with tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy in breast cancer. Cancer Immunol Res 2015;3:326-32.
Bae SB, Cho HD, Oh MH, Lee JH, Jang SH, Hong SA, et al
. Expression of programmed death receptor ligand 1 with high tumor-infiltrating lymphocytes is associated with better prognosis in breast cancer. J Breast Cancer 2016;19:242-51.
Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al
. Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. Nat Med 2002;8:793-800.
Chang J, Clark GM, Allred DC, Mohsin S, Chamness G, Elledge RM. Survival of patients with metastatic breast carcinoma: Importance of prognostic markers of the primary tumor. Cancer 2003;97:545-53.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
|This article has been cited by|
||PD-L1 Expression after Neoadjuvant Chemotherapy in Triple-Negative Breast Cancers Is Associated with Aggressive Residual Disease, Suggesting a Potential for Immunotherapy
| ||Beatriz Grandal,Manon Mangiardi-Veltin,Enora Laas,Marick Lať,Didier Meseure,Guillaume Bataillon,Elsy El-Alam,Lauren Darrigues,Elise Dumas,Eric Daoud,Anne Vincent-Salomon,Laure-Sophie Talagrand,Jean-Yves Pierga,Fabien Reyal,Anne-Sophie Hamy |
| ||Cancers. 2021; 13(4): 746 |
|[Pubmed] | [DOI]|