Expression of Programmed Cell Death-1 (PD-1)and Its Ligand (PD-L1) in Breast Cancers and ItsAssociation with ClinicopathologicalParameters: A Study from North IndiaAnoushika Mehan1 Michael Leonard Anthony1 Pranoy Paul1 Anjum Syed2 Nilotpal Chowdhury3

Shalinee Rao3 Nuzhat Hussain4 Bina Ravi5

1Department of Pathology and Laboratory Medicine, AIIMS,Rishikesh, Uttarakhand, India

2Department of Radiodiagnosis and Integrated Breast Care Centre(IBCC), AIIMS, Rishikesh, Uttarakhand, India

3Department of Pathology and Laboratory Medicine and IntegratedBreast Care Centre (IBCC), AIIMS, Rishikesh, Uttarakhand, India

4Department of Pathology, Ram Manohar Lohia Institute of MedicalSciences, Lucknow, Uttar Pradesh, India

J Lab Physicians

Address for correspondence Shalinee Rao, MD, Department ofPathology and Laboratory Medicine and Integrated Breast Care Centre(IBCC), AIIMS, Rishikesh, 249203, Uttarakhand, India(e-mail: shalineerao@gmail.com).

5Department of Surgery and Integrated Breast Care Centre (IBCC),AIIMS, Rishikesh, Uttarakhand, India

Keywords

► programmed celldeath 1 ligand 1

► programmed celldeath 1 receptor

► breast carcinoma

Abstract Introduction Cancer immunotherapy targeting the programmed cell death ligand 1(PD-L1) and programmed cell death-1 (PD-1) axis has revolutionized cancer therapy.PD-L1 also serves as a predictivemarker for such therapy. To assess the potential of suchtherapy in any cancer, the positivity of PD-1 and PD-L1 in such cancers needs to beassessed. However, such studies for breast cancer are lacking in South Asia. We aimedto estimate the positivity of PD-L1 and PD-1 receptors in breast cancer and its variousclinicopathological groups in our patient population.Materials and Methods We studied the immunoexpression of PD-1 and PD-L1 in 103histologically proven invasive carcinoma breast cases fromOctober 2018 to April 2019.The percent positivity of PD-1 and PD-L1 with 95% confidence intervals (CI) wasestimated for all the cases as well as groups defined by stage, grade,molecular subtype,hormone receptor status, Ki-67, and age.Results PD-1 positivity was seen in 72 (69.9%) cases (95% CI: 60.1–78.6). PD-L1immunoexpression was seen in 61 (59.2%) cases (95% CI: 49.1–68.8) in immune cellsand in 39 (37.9%) cases (95% CI: 28.5–50.0) in tumor cells. No significant association wasfound between PD-1, PD-L1 and age, overall clinical stage, grade, size, estrogen receptor,progesterone receptor, humanepidermalgrowth factor receptor2, andKi-67.Moderate-to-high PD-1 and PD-L1 immunopositivity was seen in all subtypes of breast cancer.Conclusion PD-1 and PD-L1 is expressed in all subgroups of breast carcinoma.Patients in all such groups are amenable to immunotherapy, provided they are foundsuitable otherwise.

DOI https://doi.org/10.1055/s-0041-1736522.ISSN 0974-2727.

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Original Article

Published online: 2021-11-10

Introduction

During cancer progression, immune responses play a pivotalrole. Inhibition of immune responses favors cancer progression.Tumor clearance is enhanced through host immune responsesby inhibiting programmed cell death 1 (PD-1) and its ligand(programmed cell death ligand 1: PD-L1) receptors.1,2 PD-L1expression has been studied in a variety of cancers1,3,4 withevidence of correlations to various clinicopathological features.Immune checkpoint inhibitors have now been U.S. Food andDrug Administration (FDA) approved in many of these cancers.

Recently, atezolizumab (PD-L1 inhibitor) and Abraxanechemotherapy as a combination therapy has been approvedfor the treatment of patients with PD-L1-positive, unresect-able locally advanced or metastatic triple-negative breastcancer.5 It is highly likely that patients having other breastcancer subtypes may also benefit from such immune check-point inhibitors. Prior to applying individualized treatmentprotocol guidelines, we need to study the prevalence ofexpression of immune checkpoint markers in a populationof breast cancer patients to provide guidance about theirpotential utility. We also need to establish the level ofexpression in breast cancer and its different clinicopatholog-ical subgroups to have an idea of whether immunotherapywill be particularly useful in different groups. Therefore, wehave conducted this study to estimate the expression of PD-1and PD-L1 in breast cancer and its different clinicopatholog-ical subgroups. To thebest of our knowledge, such a study hasnot been performed in South Asia and will additionallyprovide local guidance for testing in a South Asian context.

Materials and Methods

This was an observational study done in the Department ofPathologyandLaboratoryMedicine& IntegratedBreastCancerCenter, of our Institute, after obtaining ethical approval fromthe Institutional Ethics Committee. One hundred and threeconsecutive histopathologically proven needle core biopsiesand mastectomy invasive carcinoma specimens from Octo-ber 2018 till April 2019 with adequate tissue for furtherworkupwere included in the study. Clinical detailswere notedfrom case files and radiology department. Patients on chemo-therapy and radiotherapy before biopsy, recurrent tumors,necrotic cores with no viable tumor cells and incompletelyworked up case were not included in the study.

Hematoxylin and eosin-stained sections of study caseswere observed for lymphocyte load in reference to tumortissue present. Four-micron thick paraffin embedded tissuesections were subjected to immunohistochemistry withfollowing primary antibodies (prediluted) on positivecharged slides alongwith control showing appropriate stain-ing. For PD-1, Clone: EP239; Isotype:Monoclonal, Rabbit IgG,make: Path-in-Situ; and for PD-L1, Clone: SP-263; Isotype:Monoclonal, Rabbit IgG, make: Ventana were used.

Sections were examined under low power (10� ) and higherpower (20� and 40� ) fields to observe immunoreactivity.Expression of PD-1 was studied in lymphocytes, while PD-L1was studied in both tumor cells and immune cells. Immunoex-

pression of PD-1 was considered positive in membrane andcytoplasm of lymphocytes with a cutoff of at least 1% in lym-phocytes. For PD-L1, partial or completemembranous staining ingreater than 1% of tumor cells was considered positive for tumorcells, and cytoplasmic and membranous staining in greater than1%of immunecellswasconsideredaspositivefor immunecells.Acutoffof� 1%was consideredpositive for PD-1andPDL-1 inbothtumor cell membrane and immune cells.

Estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor 2 (Her2neu), and Ki-67wereassessed on immunohistochemistry. For ER and PR, tumorsshowing greater than 1% positivity were considered positive.Scoring for Her2neu was done as per American Society ofClinical Oncology/College of American Pathologists guidelines2018.6 Molecular surrogate subtyping was done according toSt. Gallen Consensus recommendations of 2013.7 Grading oftumorwas done using theNottinghamSystem. Clinical tumor,node, metastasis staging was done based on American JointCommittee on Cancer, 8th edition 20188 recommendations.

The prevalence of PD1 and PDL1 positivity in the overallstudy population and clinicopathological parameters wasassessed as a proportion along with exact 95% confidenceintervals (CIs). The association of the expression with ER, PR,grade,andstagewasstudiedbytheFisher’sexact test,whilethatwith Ki-67, size, and agewas assessed by theMann–Whitney Utest. Statistical analysis was done using R statistical environ-ment version 3.5.0.

Results

One hundred and three consecutive breast carcinoma caseswere studied. PD-1 positivity was seen in 72 (69.9%) cases(95% CI: 60.1–78.6). PD-L1 immunoexpression was seen in 61(59.2%) cases (95% CI: 49.1–68.8) in immune cells and in 39(37.9%) cases (95% CI: 28.5–50.0) in tumor cells. PD-L1 tumorcell positivity showed a statistically significant associationwith nodal status, distant metastasis, and ER status whenusing unadjusted p-values (►Table 1). PD-1 and PD-L1 posi-tivity by immune cell positivity was not significantly associat-ed with any of the variables studied (age, size, node status,distant metastasis, clinical stage, ER, PR, Her2neu, Ki-67%,grade, and molecular subtype). PD-1 and PD-L1 by eitherstaining method showed a moderately high proportion ofpositivity in all groups of breast cancer. When adjustmentfor multiple comparisons was used by the Holm method, novariable retained its statistical significance. Therewas a signif-icant statistical association between PD-1 in immune cells andPD-L1 in immune cells and in tumor cells (p<0.001 by theFisher’s exact test between PD-1 and PD-L1 in immune cells,p¼0.004 between PD-1 and PD-L1 in tumor cells, p¼0.002between PD-L1 in immune cells and PD-L1 in tumorcells).►Fig. 1 gives representative examples of PD-L1 positivi-ty in immune cells and membranous positivity in tumor cells.

Discussion

All subgroups have moderate expression of PD-1 and PD-L1.No statistically significant association after adjustment for

Journal of Laboratory Physicians © 2021. The Indian Association of Laboratory Physicians. All rights reserved.

Expression of PD-1 and Its Ligand (PD-L1) in Breast Cancers Mehan et al.

Table

1Assoc

iation

ofPD

-1,P

D-L1in

tumor

cells

andPD

-L1in

immun

ece

llswithvariou

spa

rametersstud

ied.

Parameter

Allca

ses

PD-1

PD-L1in

tumor

cells

PD-L1in

immun

ecells

Positive

Neg

ative

p-Value

Positive

Neg

ative

p-Value

Positive

Neg

ative

p-Value

Agein

years

51(25–

82)

52.5

(25,

78)

48(30,

82)

0.1

45(25,

81)

52.5

(30,

82)

0.1

52(25,

81)

50.5

(30,

82)

0.5

Size

incm

3.4(0.8–1

7)3.3(0.8,17

)4.4(1.9,10

.6)

0.2

3.5(0.8,16

.5)

3.2(1.0,17

)1

3.4(0.8,17

)3.2(1.0,10

.6)

0.5

Lymph

node

Positive

6745

220.5

2047

0.03

3532

0.06

Neg

ative

3627

919

1726

10

Distant

metastases

Presen

t14

86

0.3

113

0.02

95

0.8

Absent

8964

2538

5152

37

Estroge

nrece

ptora

Positive

6341

220.2

1944

0.04

3726

0.8

Neg

ative

3931

820

1924

15

Proge

steron

erece

ptora

Positive

5134

170.5

1635

0.2

2823

0.4

Neg

ative

5138

1323

2833

18

Her2n

eua

Positive

3925

140.2

1524

0.7

2217

0.4

Neg

ative

5442

1222

3235

19

Equivocal

95

42

74

5

Ki-6

7in

%25

%(2–9

5%)

25(2,95

)27

.5(3,95

)0.9

30(5,90

)25

(2,95

)0.3

30(3,90

)20

(2,95

)0.1

Molecu

larsu

btyp

eaTriple

nega

tive

1813

50.3

117

0.1

108

0.3

Luminal

A13

103

49

58

Luminal

B51

3219

1536

3318

Her2n

euen

rich

ed20

173

911

137

Grade

a1

63

30.2

24

0.9

33

0.8

226

1610

1115

1511

367

5017

2542

4225

Stag

eI

54

10.6

23

0.06

41

0.6

II38

299

1622

2414

III46

3115

2026

2422

IV14

86

113

95

Abb

reviations

:ER,estroge

nrece

ptor;H

er2n

eu,h

uman

epidermalgrow

thfactor

rece

ptor

2;IHC,immun

ohistoc

hemistry;PD

-1,p

rogrammed

cellde

ath-1;PD-L1,

prog

rammed

cellde

athlig

and1;

PR,p

roge

steron

erece

ptor.

Note:

Age

,size,

andKi-6

7arepresen

tedas

med

ianwithrang

ein

parenthe

ses.Fo

rag

e,size,a

ndKi-6

7inde

x,theMan

n–Whitney

Utest

was

used

,and

fortheothe

rva

riab

les,theFish

er’sex

acttest

was

used

toco

mpu

tethestatistica

lsignificanc

e.aIn

four

cases,

grad

eco

uldbe

assessed

;in

onecase

IHCforER

,PR

,Her2n

eu,an

dmolec

ular

subtyp

ingco

uldno

tbe

done

.

Journal of Laboratory Physicians © 2021. The Indian Association of Laboratory Physicians. All rights reserved.

Expression of PD-1 and Its Ligand (PD-L1) in Breast Cancers Mehan et al.

multiple comparisons of either PD-1 or PD-L1 expression (byboth tumor cell and immune cell positivity) was found withanyspecific typeorgroupof breast cancer. Thisunderlines thatpatients of all subgroups may be important candidates forimmunotherapy. Others have found that PD-L1 shows in-creased expression in triple-negative breast cancers.3 Wefeel that this difference may be explained based on eitherimmunohistochemistry antibody differences or tumor loca-tion. At the same time, the positivity rate in our study fortriple-negative breast cancers is comparable to similar otherstudies.While the efficacy of immune checkpoint inhibitors inbreast cancers has mainly been evaluated just on triple-nega-tive breast cancers, our results reflect that the potentiallyimportant benefits for immunotherapy may be trialed in allbreast cancer groups and not just the triple-negative patients.

PD-L1 expression in tumor cells, but not immune cells,does seem to have a mild association with lymph nodepositivity, distant metastasis, and ER status. The statisticalsignificance (by unadjusted p-values) reached in these threecases was associated with a mild-to-moderate effect size,supporting claims that PD-L1 may be associated with prog-nosis. Some studies have found a positive significant associ-ation of PD-L1 with ER, PR, and Her2 status.9–11 Thestatistical significance is however lost when adjusted formultiple comparisons due to the high number of compar-isons done due to the exploratory nature of this study. Todraw a robust conclusion about association with prognosticfactors, a follow-up-focused confirmatory study is required.Nonetheless, there is a relatively high proportion of PD-L1cases in all subgroups defined by nodal status as well as ER

status to suggest that these factors should not negativelyinfluence a decision to treat such groups by immunotherapyprovided other eligibility criteria are met.

Evaluation of PD-L1 immunoexpression is challenging dueto lack of specific antibodies for use and their validation informalin fixed paraffin embedded tissues in addition todifferent cutoff used and evaluation methods in differenttumors depending upon the immune checkpoint inhibitorapproved. To date, no standardized assays for companiondiagnostics have been approved in immunohistochemistryfor breast cancer, except SP-142 assay approved in 2019 fortriple-negative breast cancer for the drug atezolizumab, butthere are other clones that may be potentially useful includ-ing SP-263, 22C3, and 28–8 belonging to different companiesand having different staining characteristics.9,12

Similar differences appear in defining the area to be exam-ined for PD-L1 positivity.While tumoral immune cell positivi-ty bySP-142 clone is avalidmethod as a companiondiagnosticfor atezolizumab in triple-negative breast cancer, there are nostandardized guidelines for the assessment of PD-L1 in othersubtypes of breast cancer. Issues have also been raised aboutthe lower sensitivity of the SP-142 assay compared with theother assays, and the difficulty to accurately validate theimmune cell positivity the same assay.13 Hence, we assessedPD-L1separatelybyboth tumorcell positivityand immunecellpositivity. The assay we used (SP-263) is also more sensitivecompared with the SP-142 assay.13 While we found in thisstudy that all patients whowere positive for tumor cells werealsopositive for cells,with a strong associationbetween tumorcell positivity and tumoral immune cell positivity for PD-L1,there were some cases of immune cell positivity while beingtumor cell negative. Therefore, in subsequent trials, for im-mune checkpoint inhibitors, both tumor cell and immune cellpositivity need to be tested for optimal identification ofsuitable patients for therapy.

We faced some difficultieswith PD-L1 assessment. Immu-noexpression of PD-L1 is heterogeneous and thus evaluationof PD-L1 expression by counting positive cells in hotspotsmay be appropriate.14,15 Difficulty was encountered in eval-uating PD-L1 immunoexpression in cases with PD-L1 posi-tive tumor cells in a background of necrosis and in caseswithPD-L1 positivity in tumor cells along with PD-L1 positiveimmune cells in background.

This is one of the first studies done in an Indian setting,and thusmay be useful for setting guidelines for treatment inthis subcontinent. Only a few studies havebeen done on PD-1and PD-L1 in primary and metastatic breast cancer else-where in the world.9,16 FDA approval of targeted therapy inbreast cancer has openednewavenue for cancermanagementin breast by immunotherapy. The relatively high prevalence ofPD-1 and PD-L1 in breast cancer cases supports routinescreening of these biomarkers as this may significantlyimprove overall survival and outcome in these patients.

The most significant limitation of this study is that cloneSP-263 was used instead of clone SP-142 that has beenapproved in triple-negative breast cancer. However, theusefulness of SP-142 clone is not given for other types ofbreast cancer, especially since the SP-142 clone itself has

Fig. 1 Programmed cell death ligand 1 (PD-L1)–positive immune cells inthe tumoral stroma and infiltrating the tumor at low (A), moderate (B), andhigh (C) intensity (magnification�200 forA,B,C). Also, tumors cells havingmembranous PD-L1 positivity at low (D), moderate (E), and high (F)intensity (magnification �400, �200, and �200 for D, E, F).

Journal of Laboratory Physicians © 2021. The Indian Association of Laboratory Physicians. All rights reserved.

Expression of PD-1 and Its Ligand (PD-L1) in Breast Cancers Mehan et al.

been found in lung cancers to have poor reproducibilitycompared with the other PD-L1 clones as well as havinglesser sensitivity for detecting tumor cell positivity.17 Theclone SP-263 has a better reproducibility with the otherclones and thus is likely to be a better indicator of “true” or“consensus” PD-L1 positivity. Whatever the method used toassess the PD-L1 status in this and other observationalstudies, the differences in PD-L1 positivity among the usedPD-L1 clones need to be kept in mind and more than oneclone needs to be studied according to specific reproduciblecriteria for reliable treatment. We conclude that PD-1 andPD-L1 show a moderately high degree of positivity in breastcancer across all subgroups in our setting in Northern India,and thus selected patients from all clinicopathological sub-groups should be eligible for immunotherapy.

Conflict of InterestNone.

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Journal of Laboratory Physicians © 2021. The Indian Association of Laboratory Physicians. All rights reserved.

Expression of PD-1 and Its Ligand (PD-L1) in Breast Cancers Mehan et al.