Tumor Infiltrating Lymphocytes 12 as a Prognostic and ...U NCORRECTED PROO F 1 23 Tumor Infiltrating Lymphocytes 12 4 as a Prognostic and Predictive 5 Biomarker in Breast Cancer 6

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2 123 Tumor Infiltrating Lymphocytes4 as a Prognostic and Predictive5 Biomarker in Breast Cancer

6 Murali Janakiram, Hina Khan, Susan Fineberg,7 Xingxing Zang and Joseph A. Sparano

9 Abstract10

11 Tumor infiltrating lymphocytes (TILs) have been recognized in various12 cancers and may reflect a host immune response to malignant cells. TILs13 are a heterogeneous population of various types of mononuclear cells,14 including CD8 or CD4 + T cells and their subsets, B cells, myeloid15 derived suppressor cells (MDSC), macrophages, and other cells. Immuno-16 suppressive factors in the tumor microenvironment (TME) that inhibit17 recruitment and function of TILs include immunosuppressive cells,18 cytokines secreted by tumor or mesenchymal cells, and co-inhibitory19 ligands expressed by tumor cells. Despite this complex interplay of20 immune cells and the TME, higher TIL density is associated with21 favorable prognosis in certain breast cancer subtypes, including HER222 overexpressing cancers, and “triple negative” cancers that do not express23 the estrogen and progesterone receptors or overexpress HER2. TILs24 infiltrating the tumor stroma (sTILs) are associated with higher rates of25 complete pathologic response to neoadjuvant chemotherapy, decreased26 recurrence and improved survival in early stage triple negative and27 HER2-positive breast cancer treated with adjuvant systemic therapy. An28 international working group has published guidelines on reporting TILs in29 pathology specimens. In this chapter we review the composition of TILs,30 mechanisms of immune evasion, recommendations for TILs measurement,

Murali Janakiram and Hina Khan are contributed equallyto this work.

M. Janakiram � H. Khan � X. Zang � J.A. Sparano (&)Departments of Medicine, Montefiore MedicalCenter, Albert Einstein College of Medicine, 1695Eastchester Road, Bronx, NY 10461, USAe-mail: [email protected]

M. Janakiram � H. Khan � X. Zang � J.A. SparanoDepartments of Oncology, Montefiore MedicalCenter, Albert Einstein College of Medicine, 1695Eastchester Road, Bronx, NY 10461, USA

S. FinebergDepartments of Pathology, Montefiore MedicalCenter, Albert Einstein College of Medicine, 1695Eastchester Road, Bronx, NY 10461, USA

M. Janakiram � X. ZangDepartments of Microbiology and Immunology,Montefiore Medical Center, Albert Einstein Collegeof Medicine, 1695 Eastchester Road, Bronx, NY10461, USA

Layout: T4_Grey Book ID: 331091_1_En Book ISBN: 978-3-319-41759-2

Chapter No.: 12 Date: 28-8-2016 Time: 10:10 am Page: 1/20

© Springer International Publishing Switzerland 2016S. Badve and Y. Gökmen-Polar (eds.), Molecular Pathologyof Breast Cancer, DOI 10.1007/978-3-319-41761-5_12

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34343434 and data supporting use of TILs as a prognostic and predictive biomarker35 in breast cancer.

37 Keywords38

39 Tumor infiltrating lymphocytes � CD8 � CD4 � MDSC

4041 12.1 Introduction

42 It has been recognized for decades that some43 primary breast cancers are associated with infil-44 tration by lymphocytes, often referred to as “tu-45 mor infiltrating lymphocytes” , or “TILs”. This46 phenomenon was first described in medullary47 carcinoma of the breast, an uncommon subtype of48 poorly differentiated invasive carcinoma charac-49 terized by dense lymphocytic infiltration, cir-50 cumscription, syncytial growth and absence of51 estrogen receptor (ER), progesterone receptor52 (PR) expression and HER2 overexpression, and a53 relatively favorable prognosis (Moore and Foote54 1949; Richardson 1956; Bloom et al. 1970).55 Recent reports have noted that lymphocytic infil-56 tration to be more prevalent in HER2 overex-57 pressing and “triple negative” invasive ductal58 carcinomas, and distinguished between lympho-59 cytic infiltration of the tumor (iTILs) and stroma60 (sTILs) (Loi et al. 2013). In addition, there is a61 consistent body of evidence indicating a strong62 correlation between the presence of TILs, espe-63 cially sTILs, in the primary tumor and a signifi-64 cantly reduced risk of breast cancer recurrence and65 mortality in both HER2 overexpressing (Dieci66 et al. 2015) and triple negative breast cancer67 (TNBC) (Loi et al. 2013; Dieci et al. 2015; Adams68 et al. 2014). An association between TILs in69 residual tumor after neoadjuvant chemotherapy70 and prognosis has also been reported (Dieci et al.71 2014), and higher TIL density in diagnostic72 pre-treatment core biopsies is also predictive of73 pathologic complete response to neoadjuvant74 chemotherapy (Denkert et al. 2010, 2015; West75 et al. 2011; Ono et al. 2012; Mao et al. 2014). This76 strong association between TILs and clinical out-77 comes has led to an expert group providing78 guidelines for evaluating and scoring TILs in

79�breast cancer, with the ultimate goal of capturing80�the prognostic information in an accurate and81�reproducible manner that provides sufficient ana-82�lytic validity to permit further investigation and83�eventually clinical application (Salgado et al.84�2015b). Although the infiltrating cells comprising85�the infiltrate have been dubbed “lymphocytes”,86�they are identified morphologically as mononu-87�clear cells, and hence actually consist of a mixed88�population of cells including not only cytotoxic89�and suppressor T lymphocyte and B lymphocyte90�populations, but also natural killer (NK) cells,91�plasma cells, macrophages, dendritic cells, and92�myeloid derived progenitor cells (Fig. 12.1). With93�the emergence of immune checkpoint blockade as94�a new strategy to treat a wide variety of cancers,95�there has also been interest in more precisely96�characterizing the composition of the TIL popu-97�lation with the ultimate goal of developing pre-98�dictive biomarkers that identify tumors more99�susceptible to eradication by immune checkpoint100�blockade or other immunotherapeutic approaches.

101102�12.2 Characterization of TILs103�in Breast Cancer

104�As described above, the International TILs105�Working Group is an expert panel that has pro-106�vided recommendations for evaluation of TILs in107�breast cancer (Salgado et al. 2015b). The rec-108�ommendations of the panel are summarized in109�Table 12.1, and several key recommendations110�are described herein. First, the panel recom-111�mended that all mononuclear cells within the112�border of the primary invasive tumor be identi-113�fied as TILs in whole sections excluding areas114�with necrosis, crush artifact, or hyalinization; the115�panel did not recommend use of tissue

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2 M. Janakiram et al.



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116 microarrays (TMA) or evaluating only “hot117 spots” in either whole sections or TMAs.118 Although pretreatment core biopsies may also be119 used for TIL assessment prior to administration120 of neoadjuvant chemotherapy, there is limited121 information about analytic validity of assessing122 TILs in residual tumor after therapy. Second, the123 panel recommended distinguishing between124 iTILs and sTILs (Fig. 12.2), and to report pri-125 marily sTILs when assessing TIL status. The126 group defined iTILs as those in tumor nests127 having cell-to-cell contact with no intervening128 stroma and directly interacting with carcinoma129 cells. sTILs were defined as TILs dispersed in the130 stroma between the carcinoma cells that are not131 directly in contact with the malignant cells, and132 should be reported as percentage of stromal areas133 occupied by sTILs. The panel pointed out that134 trafficking between tumor and stromal microen-135 vironment is likely a dynamic process captured136 in a static manner by histologic evaluation at a137 single time point, and hence distinguishing138 between iTILs and sTILs may be artifactual.

139�Characterization of sTILs is more practical140�because of the greater abundance of sTILs rela-141�tive to iTILs, greater ease in recognizing and142�enumerating sTILs, and no additional or more143�accurate prognostic information provided above144�and beyond that provided by sTILs enumeration.145�Third, the panel recommended that sTILs be146�characterized and reported in a continuous man-147�ner (ex. deciles of <10 %, 10–20 %, etc.), rather148�than a binary manner (ex. lymphocyte predomi-149�nant breast cancer with at least 50 % sTILs),150�because very densely infiltrated tumors may be151�uncommon (<5–10 %) as there is a linear rela-152�tionship between sTILs and prognosis without a153�prognostically relevant binary threshold.154�The essential characteristics of a prognostic155�and/or predictive biomarker include analytic156�validity, clinical validity, and clinical utility.157�Although there is strong evidence supporting the158�association between sTILs and prognosis in early159�stage breast cancer treated with adjuvant160�chemotherapy and response to neoadjuvant cyto-161�toxic therapy inTNBC, and hence clinical validity,

Fig. 12.1 TILs are composed of a heterogeneous population of cells that may promote or suppress the development ofcancer [Reproduced with permission from Salgado et al. (2015b)]

12 Tumor Infiltrating Lymphocytes as a Prognostic … 3



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162 the clinical utility of this information remains163 uncertain. For example, there is currently insuffi-164 cient level of evidence to spare chemotherapy165 based on TIL assessment, or to select for patients166 most likely to benefit from immune checkpoint167 blockade. In addition, few studies have evaluated168 the intra and inter observer reproducibility, or169 analytic validity, of TILs assessment. For exam-170 ple, in the report by Adams et al., inter observer171 correlation was evaluated in a subset of 99 evalu-172 able cases. Rates of agreement within 10

173�percentage points between two expert breast174�pathologists were 85 % (95 % confidence inter-175�vals [CI] 76–91 %) for sTILs and 97 % (95 % CI176�91–99 %) for iTILs. If categorical cut points from177�the Kaplan-Meier analysis were used, the kappa178�statistic showed moderate agreement between the179�two pathologists (sTILs, 0.40; iTILs, 0.43)180�(Adams et al. 2014). Hence further studies or181�guidelines are required to study methods to182�improvise interobserver agreement in TIL evalu-183�ation in breast cancer.

Table 12.1 TIL working group recommendations for evaluating TILs in breast cancer

Specimen source

∙ Full sections are preferred over biopsies whenever possible. Cores can be used in the pre-therapeutic neoadjuvantsetting; currently no validated methodology has been developed to score TILs after neoadjuvant treatment

∙ One Section (4–5 μm, magnification Å * 200–400) per patient is currently considered to be sufficient

Methodology for characterizing TILs

∙ TILs should be evaluated within the borders of the invasive tumor∙ A full assessment of average TILs in the tumor area by the pathologist should be used. Do not focus on hotspots∙ Exclude TILs outside of the tumor border and around DCIS and normal lobules. Exclude TILs in tumor zones withcrush artifacts, necrosis, regressive hyalinization as well as in the previous core biopsy site

∙ All mononuclear cells (including lymphocytes and plasma cells) should be scored, but polymorphonuclearleukocytes are excluded

∙ TILs should be reported for the stromal compartment (=% stromal TILs). The denominator used to determine the %stromal TILs is the area of stromal tissue (i.e. area occupied by

mononuclear inflammatory cells over total intratumoral stromal area), not the number of stromal cells (i.e.fraction oftotal stromal nuclei that represent mononuclear inflammatory cell nuclei)∙ The percentage of stromal TILs is a semiquantitative parameter for this assessment, for example, 80 % stromal TILsmeans that 80 % of the stromal area shows a dense mononuclear infiltrate. For assessment of percentage values, thedissociated growth pattern of lymphocytes needs to be taken into account. Lymphocytes typically do not form solidcellular aggregates; therefore, the designation ‘100 % stromal TILs’ would still allow some empty tissue spacebetween the individual lymphocytes

Reporting results

∙ Pathologist should report their scores in as much detail as the pathologist feels comfortable with∙ The working group’s consensus is that TILs may provide more biological relevant information when scored as acontinuous variable, and thus should TILs should be assessed as a continuous parameter (as deciles)

∙ Lymphocyte predominant breast cancer can be used as a descriptive term for tumors that contain ‘more lymphocytesthan tumor cells’. However, the thresholds vary between 50 % and 60 % stromal lymphocytes

Clinical implications of results

∙ No formal recommendation for a clinically relevant TIL threshold(s) can be given at this stage. The consensus wasthat a valid methodology is currently more important than issues of thresholds for clinical use, which will bedetermined once a solid methodology is in place

Adapted from Salgado et al. (2015b)




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184185 12.3 TILs Are an Immunologic186 Response to Tumor187 Neoantigens

188 Tumors occurring in subjects harboring189 germ-line BRCA1 mutations are associated with190 more TILs than sporadic breast cancers (Lakhani191 et al. 1998). BRCA-mutation associated cancers192 have a higher mutational burden than sporadic193 cancers due to impaired homologous recombi-194 nation and consequently less error free DNA195 repair. This results in a greater neoantigen burden196 and hence induces an immune response. Vari-197 ability in the somatic mutational burden of cancer198 has been described, with tumors associated with199 exposure to tobacco and sunlight (e.g., lung

200�cancer and melanoma, respectively) associated201�with the highest mutational burden, and breast202�cancer having a mutational burden in the inter-203�mediate range when considered in the context of204�all human cancers that have been characterized205�thus far (Alexandrov et al. 2013). Non-BRCA-206�associated TNBCs are also frequently character-207�ized by defective DNA repair mechanisms due to208�germ-line defects in other DNA repair pathways209�(e.g., PALB2, RAD51) and BRCA1 promoter210�hypermethylation. Assays that identify tumors211�harboring “genomic scars” as a consequence of212�these deficiencies are likewise characterized by213�greater TIL density, providing additional evi-214�dence supporting the link between mutational215�burden and immune response (Telli et al. 2015).216�On the other hand, tumors harboring defective

Fig. 12.2 Various levels of TIL infiltration in differentbreast cancer samples are shown. Top left sTILs < 1 %(20x)—The stroma is clearly visible in pink and is devoidof TILs, Top right sTILs—50 % (20x)—The stroma isvisible and has a considerable infiltration of TILs, Bottom

left sTILs—90 % (20x)—Intervening stroma is not seenand is nearly replaced by lymphocytes, Bottom rightIntratumoral TILs (40x)—The lymphocytes are in directcontact with the tumor cells with no intervening stroma




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217 DNA repair mechanisms, whether due to218 germ-line or somatic alterations, are more sen-219 sitive to DNA damaging agents such as plat-220 inums, alkylating agents, and anthracyclines.221 Thus, it is unclear at this time as to whether the222 more favorable prognosis associated with TILs223 represents an effective immune response, greater224 sensitivity of tumors with high TILs to cytotoxic225 therapy, or both. Higher mutational burden has226 been shown to correlate with clinical response to227 anti-PD1 directed therapy in non-small cell lung228 cancer (Rizvi et al. 2015), suggesting that TILs229 may serve as a surrogate predictive biomarker for230 response to immune checkpoint blockade in a231 variety of cancers.

232233 12.4 Composition of TILs234 and Subpopulations

235 The diagnosis of clinical cancer represents236 escape from cancer immunoediting, an237 immunologic process that reduces cancer burden238 through elimination or equilibrium (Dunn et al.239 2004). Although the escape from or failure of this240 process results in the clinical detection of cancer,241 the association between TILs and prognosis242 suggests that host immunity is still relevant after243 cancer is diagnosed. TILs are composed of a244 heterogeneous population of cells having both245 immunostimulatory and immunosuppressive246 effects, and the balance of these effects contribute247 to tumor tolerance (Quezada et al. 2011).248 The subpopulations of cells are shown in249 Fig. 12.1. Some cells in the TIL population250 suppress tumor progression, including CD8+ T251 cells, helper CD4+ T (Th1) cells, natural killer252 (NK) cells, whereas others promote tumor pro-253 gression, including Th2 cells, myeloid derived254 stem cells, and T regulatory (Tregs) cells.255 Subpopulations of macrophage and dendritic256 cells can suppress (M1, DC1) or promote tumor257 (M2, DC2) tumors, while other cell populations258 may be either tumor suppressive or promoting,259 including B cells and Th17 cells (Salgado et al.260 2015b). Although neutrophils are not considered261 in characterizing TILs, they may likewise have

262�either tumor suppressing or promoting subpop-263�ulations (Sagiv et al. 2015).264�TIL subpopulations may be evaluated using a265�variety of methodologies, including immunohis-266�tochemistry, RNA in situ hybridization, and flow267�cytometry. Gene expression profiling has identi-268�fied tumor associated immune signatures that269�reflect the composition of the subpopulations. To270�date, classification of subpopulations has largely271�been described using immunohistochemistry272�including a panel of antibodies directed at CD4,273�CD8, CD25, and FOXP3+. Using this method-274�ology, the major subtypes of immunosuppressive275�cells constituting TILs include T regs (CD4276�+ CD25+ FOXP3+) and myeloid derived sup-277�pressor cells (MDSC) (Jiang and Shapiro 2014).278�Tregs produce RANKL which binds to RANK279�on human breast cancer cell lines and promotes280�lung metastases in mouse models (Tan et al.281�2011). Depletion of Tregs with a vaccine and low282�dose cyclophosphamide results in increased283�cytolytic activity of T cells in Her-2 Neu trans-284�genic mice (Weiss et al. 2012). MDSC suppress285�T cell proliferation through production of reac-286�tive oxygen species, disrupt binding of antigen287�specific peptides to CD8 T cells by inactivating288�tyrosinases in the TCR-CD8 (T cell289�receptor-CD8) complexes, and inhibit antigen290�presentation by tumor cells via nitration of tumor291�MHC class I expression (Jiang and Shapiro292�2014). Hence these cell populations through293�pleiotropic effects can prevent or suppress an294�effective immune response.

295296�12.5 The Tumor Microenvironment297�and TIL Function

298�TILs comprise only one component of the tumor299�microenvironment (TME), which also includes300�mesenchymal cells, and extracellular matrix/301�stroma. The extracellular matrix functions as a302�scaffold for tissue architecture, and also provides303�biochemical and biomechanical signals that304�influence cell growth, survival, migration and305�differentiation, as well as vascular development306�and immune function, and hence modulates the




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308308 hallmarks of cancer (Pickup et al. 2014). Thus,309 crosstalk between TIL subpopulations and other310 components of the TME, mediated in part by311 chemokines and cytokines, results in a complex312 interplay that can influence the balance between313 tumor promotion and suppression, as described314 below.315 Cytokines and chemokines: The stromal cells316 and other immune cells secrete various cytokines317 that profoundly influence different subpopula-318 tions of immune effector cells. For example,319 TGF-beta induces transcriptional repression of320 genes in CD8+ T cells resulting in impaired321 cytolytic activity (Thomas and Massague 2005).322 Products of altered steroid metabolism can323 accumulate and inhibit CCR7 thus preventing324 dendritic cell maturation and translocation into325 the lymphoid organs (Villablanca et al. 2010).326 Tumors secrete soluble ligands such as MHC327 class I polypeptide-related sequence (MIC) that328 deplete T cell receptors and attenuate response of329 specific effector T cells in response to tumor330 antigens (Groh et al. 2002). Thus cytokines331 secreted in the TME can inhibit presentation of332 antigens to T cells and can also inhibit responses333 of T cells to the tumor. Other cytokines, most334 notably interferon-gamma, play an important role335 in enhancing cell immunity.336 Co-inhibitory and co-stimulatory receptors337 and ligands: T cell activation depends on338 recognition of antigens on host antigen present-339 ing cells (APCs) and the presence of a simulta-340 neous co-stimulatory or co-inhibitory signal341 delivered through the CD28 –B7 family of342 receptor-ligand interaction between T cells and343 APC or malignant cells. Certain B7 ligands,344 including PD-L1, B7-H3, B7x and HHLA2345 inhibit T cell responses when expressed on APCs346 or tumor cells. Expression of these ligands in347 breast cancer and other cancers has been asso-348 ciated with unfavorable clinical features349 (Janakiram et al. 2012; 2014). For example,350 PD-L1 expression on human pancreatic and351 ovarian cancers is inversely correlated with CD8352 + TILs (Ohaegbulam et al. 2015). Several studies353 also show an inverse correlation of another354 co-inhibitory ligand B7x and TILs in various355 cancers. Tumor cell B7x expression is inversely

356�correlated with the intensity of TILs in renal cell357�carcinoma (Zhang et al. 2013), with the number358�of CD3+ and CD8+ TILs in uterine endometri-359�oid carcinoma (Miyatake et al. 2007) and with360�the densities of CD3+ TILs in tumor nest and361�CD8+ TILs in tumor stroma in esophageal car-362�cinoma (Chen et al. 2011). These results suggest363�that tumor-expressed B7x may be important in364�limiting TILs infiltration. The expression of365�coinhibitory receptor PD-1 on TILs has also been366�shown to be associated with decreased overall367�survival in breast cancer (Sun et al. 2014; Muenst368�et al. 2013). In summary, TILs recruitment and369�function is influenced by a complex interplay of370�neoantigens and co-inhibitory ligands on the371�tumor, coinhibitory receptors on TILs, subpop-372�ulation of cells in the infiltrate and the tumor373�microenvironment.

374375�12.6 Clinical Validity of TILs376�as a Prognostic Biomarker377�in Patients with Breast Cancer378�Treated with Adjuvant379�Chemotherapy

380�The results of studies evaluating the association381�between TILs and prognosis in operable breast382�cancer are summarized in Table 12.2.383�Relationship between TILs and Breast Cancer384�Subtype: Loi et al. (2013) first described the385�variability of TILs by breast cancer subtype, and386�the strong association between TILs and prog-387�nosis in TNBC. TILs were evaluated indepen-388�dently by two expert pathologists in primary389�tumor specimens from 2009 patients with axil-390�lary node-positive breast cancer enrolled on the391�BIG 02-98 adjuvant phase III trial comparing392�anthracycline chemotherapy given without a393�taxane (doxorubicin followed by cyclophos-394�phamide, methotrexate, and fluorouracil395�(CMF) or doxorubicin plus cyclophosphamide396�followed by CMF), concurrently or sequentially397�with a taxane (doxorubicin plus docetaxel fol-398�lowed by CMF or doxorubicin followed by399�docetaxel followed by CMF). iTILs and sTILs400�were found to be significantly higher in TNBC401�and HER2-positive breast cancer compared with




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Table

12.2

Clin

ical

stud

iesof

TILsas

abiom

arkerin

adjuvant

therapyof

breastcancer

Trial—

author

and

reference

No.

ofsamples

analyzed

Method

— H&E/IHC

TILs

analyzed:

i-TILs,

s-TILsor

t-TILs

Lym

phocytepredom

inant

breastcancer

(LPB

C)

AdjuvantTherapy

used

Survival

parameter

inHighTILssubgroup

TNBC

Loi

etal.

(2013)

256

H&E

s-TILsand

i-TILs

HighTILs(>50

%)wereseen

in10.6

%patients

Anthracyclin

ebasedcombinatio

nregimenswith

taxanes

∙Every

10%

increase

ini-TILsands-TILswereassociated

with

17%

(p=0.1)

and15

%(p

=0.025)

reducedrisk

ofrelapse,

and27

%(p

=0.03)and17

%reducedrisk

ofdeath(p

=0.023).

∙Fo

rhigh

versus

low

TILs(>50

%)

–DFS

-HR:0.30,95

%CI:0.11

–0.81

–OS-HR:0.29,95

%CI:0.091–

0.92

Adams

etal.

(2014)

481

H&E

s-TILsand

i-TILs

HighTILs(≥50

%)wereseen

in4.4%

patients

AC,A

Cfollo

wed

bytaxane

orCMF

∙With

every10

%increase

ins-TILs,a14

%reductionof

risk

ofrecurrence

ordeath(p

=0.02),18

%reductionof

risk

ofdistantrecurrence

(p=0.04),and19

%reduction

ofrisk

ofdeath(p

=0.01)was

seen.sTILswerean

independentprognostic

markerof

DFS

,DRFI,andOS

∙Fo

rpresence

vsabsenceof

sTILs

–HR:0.69,95

%CI:0.49

–0.98

∙Fo

rpresence

vsabsenceof

iTILs

–HR:0.69;95

%CI:0.45

–1.06

Dieci

etal.

(2015)

199

H&E

s-TILsand

i-TILs

High-TILs(>50

%)wereseen

in5%

patients

FEC

orFA

Ccomparedto

nochem

otherapy

∙Each10

%increase

ini-TILsor

s-TILswas

associated

with

14and13

%reductionin

risk

relapsein

thehigh

and

low-TILsgroups

∙Fo

rhigh

versus

low

TILs(>50

%)

–DFS

-HR:0.43,95

%CI:0.20

–0.94

–10

yearsDFS

rate

was

85and53

%

Loi

etal.

(2014b)

134

H&E

s-TILsand

i-TILs

TILsused

ascontinuous

variable,HighTILsdefined

as≥50

%forstatistical

analyses

Docetaxel

orvinorelbine×3cycles,

follo

wed

bythreecycles

ofFE

C∙Fo

reach

10%

increase

insTILstherewas

13%

decrease

inrisk

ofdistantrecurrencein

TNBC;and

improvem

entin

DFS

.Nodifference

inOSwas

observed

∙Fo

rhigh

versus

low

TILs(≥50

%)

–DFS

-HR:0.77,95

%CI:0.61

–0.98

(con

tinued)




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Table

12.2

(con

tinued)

Trial—

author

and

reference

No.

ofsamples

analyzed

Method

— H&E/IHC

TILs

analyzed:

i-TILs,

s-TILsor

t-TILs

Lym

phocytepredom

inant

breastcancer

(LPB

C)

AdjuvantTherapy

used

Survival

parameter

inHighTILssubgroup

HER2

+Loi

etal.

(2014b

)209

H&E

s-TILsand

i-TILs

TILsused

ascontinuous

variable,HighTILsdefined

as≥50

%forstatistical

analyses

Docetaxel

orvinorelbine×3cycles,

follo

wed

bythreecycles

ofFE

C,

with

orwith

outn

inedosesof

weekly

trastuzumab

∙Fo

reach

10%

increase

inlymphocytic

infiltration,

there

was

an18

%decrease

inrelativ

erisk

ofdistantrecurrence

inpatientsrandom

ized

tothetrastuzumab

arm.Higher

levelsof

TIL

areassociated

with

increasedtrastuzumab

benefitin

HER2+

disease

∙Fo

rHighversus

low

TILs(≥50

%)

–DFS

-HR:0.96,95

%CI0.82

–1.3

Perez

etal.

(2014)

945

H&E

s-TILs

HighTILs(≥60

%)wereseen

in9.9%

patients

AC

follo

wed

byTaxol

with

orwith

outTrastuzum

ab∙sTILsof

≥60

%was

associated

with

RFS

inchem

otherapy

only

arm,butnotin

trastuzumab

arm.The

10year

RFS

was

90.9

%forhigh

S-TILsgroupin

the

chem

otherapy

arm

∙10

year

RFS

was

notsignificant

different(80%)in

either

groups

inthetrastuzumab

arm

∙Fo

rHighversus

low

TILs(≥60

%)

–10

year

DFS

HR:0.23;95

%CI:0.073–

0.73

Dieci

etal.

(2015)

112

H&E

s-TILsand

i-TILs

High-TILs(>50

%)wereseen

in24

%patients

FEC

orFA

Ccomparedto

nochem

otherapy

∙10

year

DFS

rate

was

higher

inthehigh-TIL

group

∙Fo

rhigh

versus

low

TILs(>50

%)

–DFS

-HR:0.48;95

%CI:0.21

–1.07

Liu

etal.

(2014)

498

IHC—

FOXP3

cells

s-TILsand

i-TILs

Medianused

toclassify

high

andlowTILs.FO

XP3

TILs(≥2

IHC)wereprevalentin

42.4

%samples

CMF,

ACor

FAC

∙HighFO

XP3

+TILswereassociated

with

improved

survival

intheHER2+/ER—

subgroup,with

co-existent

CD8+

T-cellinfiltrates.In

CD8+

group,

thepresence

ofhigh

levelsof

FOXP3

+TILswas

independentof

standard

clinical

prognostic

factors

∙Fo

rhigh

versus

low

TILs(m

edian)

–OS-HR:0.48,95

%CI:0.23

–0.98

Loi

etal.

(2013)

297

H&E

s-TILsand

i-TILs

HighTILs(>50

%)wereseen

in11.1

%patients

Anthracyclin

ebasedcombinatio

nregimenswith

taxanes

∙Each10

%increase

insTILswas

significantly

associated

with

benefitwith

anthracycline-only

regimen.In

the

LPB

C(>50

%TILs)

phenotype,

a5-year

DFS

of78.6

%was

seen

intheanthracyclinearm

versus

57.9

%DFS

intheanthracycline-docetaxelarm.

∙Fo

rhigh

versus

low

TILs(>50

%)

–5year

DFS

inLPB

C–HR:0.45;95

%CI:0.12

–1.71

–5year

DFS

innon-LPB

C-H

R:2.05;95

%CI:1.41

–

2.97

(con

tinued)




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407�407�407�407�407�407�ER-positive, HER2-negative breast cancer. In408�addition, there was no significant prognostic409�association in the entire population (n = 2009) or410�ER-positive, HER2-negative population411�(n = 1079).412�Relationship between TILs and Prognosis in413�Triple Negative Breast Cancer: Although no414�association was found between TIL and prog-415�nosis in the overall population and in416�ER-positive, HER2-negative disease in analysis417�of the BIG 02-98 specimens reported by Loi418�et al. (Loi et al. 2013), each 10 % increase in419�iTILs and sTILs in the TNBC population was420�associated with 17 and 15 % reduced risk of421�relapse (adjusted p = 0.1 and p = 0 0.025),422�respectively, and 27 and 17 % reduced risk of423�death irrespective of chemotherapy type used424�(adjusted p = 0.035 and p = 0.023), respectively.425�This report therefore provided the first evidence426�indicating the strong associated between TILs427�and prognosis in TNBC. Adams et al. (2014)428�reported a confirmatory analysis that focused on429�481 patients with stages I-III TNBC enrolled on430�two large adjuvant phase III trials (ECOG 2197431�and ECOG 1199) in which all patients received432�anthracycline-cyclophosphamide-containing,433�usually in combination with a taxane. In contrast434�to the analysis by Loi et al., TILs were read435�independently rather than in tandem by two436�expert pathologists. Similar to the report by Loi437�et al., however, among the 481 tissue samples438�analyzed; for every 10 % increase in sTIL there439�was a 14 % reduction in risk of recurrence or440�death, 18 % reduction in risk of distant recur-441�rence and a 19 % reduction in risk of death was442�seen. These two independent reports therefore443�demonstrated that sTILs were a strong and444�independent prognostic marker for disease-free445�survival (DFS) and overall survival (OS) in446�patients with stages I-III TNBC treated with447�adjuvant anthracycline-containing chemotherapy.448�The relationship between TILs and prognosis449�was also evaluated retrospectively using breast450�cancer specimens obtained from two randomized451�trials comparing adjuvant chemotherapy with no452�chemotherapy in 817 patients with node-positive453�and node-negative breast cancer (Dieci et al.454�2014). In the TNBC subgroup, both iTILs andTa

ble

12.2

(con

tinued)

Trial—

author

and

reference

No.

ofsamples

analyzed

Method

— H&E/IHC

TILs

analyzed:

i-TILs,

s-TILsor

t-TILs

Lym

phocytepredom

inant

breastcancer

(LPB

C)

AdjuvantTherapy

used

Survival

parameter

inHighTILssubgroup

ER+/

PR+

Dieci

etal.

(2015)

463

H&E

s-TILsand

i-TILs

High-TILs(>50

%)wereseen

in15

%patients

FEC

orFA

Ccomparedto

nochem

otherapy

∙Noprognostic

effect

was

seen

intheER+/HER2—

subgroup

Liu

etal.

(2014)

2761

IHC—

FOXP3

cells

s-TILsand

i-TILs

Medianused

toclassify

high

andlowTILs.FO

XP3

TILs(≥2

IHC)wereprevalentin

27.2

%samples

CMF,

ACor

FAC

∙HighFO

XP3

+TILswereassociated

with

poor

survivalin

ER+breastcancersthat

lacked

CD8+

T-cellinfiltrates

∙Fo

rHighversus

low

TILs[m

edian]

–OSHR:1.30,95

%CI:1.02

–1.66

TILs

Tum

orinfiltratinglymphocytes;sTILs

Stromal

tumor

infiltratinglymphocytes;i-TILsIntratum

oral

tumor

infiltratinglymphocytes;tTILs

Totaltumor

infiltratinglymphocytes;H

&EHem

atoxylin

andeosin;

IHCIm

munohistochem

istry;

TNBCTriplenegativ

ebreastcancer;O

SOverallsurvival;H

RHazardratio

;CIConfidenceinterval;D

FSDisease

free

survival;R

FSRecurrencefree

survival;

DRFIDisease

recurrence

free

interval;FOXP3Fo

rkhead

boxP3

;HER2Hum

anepidermal

grow

thfactor

receptor

2;CMFMethotrexate,

cyclophosphamide,

5-fluorouracil;

AC

Doxorubicin

and

Cyclophospham

ide;

FEC5-Fluorouracil,

EpirubicinandCyclophospham

ide;

FACDoxorubicin,cyclophosphamide,

5-fluorouracil




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455 sTILs were significantly associated with DFS,456 with each 10 % increase associated with 14 and457 13 % reduction in risk of relapse (HR: 0.86,458 95 % CI: 0.78–0.94 and HR: 0.87, 95 % CI:459 0.80–0.94), respectively. TILs were prognostic in460 both the chemotherapy treated and untreated461 population, with no statistical interaction462 observed. The FinHer study was another phase463 III multicenter adjuvant trial that included 1010464 patients with high-risk node-negative or465 node-positive breast cancer; in this cohort each466 10 % increase in TILs was significantly associ-467 ated with a 13 % decrease in risk of distant468 recurrence (HR: 0.77, 95 % CI: 0.61–0.98,469 p = 0.02) in 134 TIL evaluable primary TNBC470 cases (Loi et al. 2014b). Thus, several reports471 have confirmed the prognostic role of increased472 TIL in patients with operable TNBC treated with473 or without adjuvant chemotherapy.474 HER2-Positive Breast Cancer: The associa-475 tion between TILs and prognosis was also476 demonstrated in HER2-positive operable breast477 cancer. As previously described, the FinHer478 study was a multicenter phase III trial that479 included not only patients with TNBC, but also480 patients with HER2 overexpressing breast cancer481 who received adjuvant chemotherapy alone or in482 combination with trastuzumab. In 209 patients483 with HER2-positive breast cancer treated with484 adjuvant trastuzumab in the FinHER study, a485 10 % increase in sTILs was associated with an486 increase in distant disease free survival (HR =487 0.77; 95 % CI: 0.61–0.98). The association488 between TILs and prognosis was also analyzed in489 the N9831 study, which compared adjuvant490 chemotherapy alone or in combination with491 trastuzumab in HER2 overexpressing operable492 breast cancer (Perez et al. 2014). Lymphocyte493 predominant breast cancers (LPBC) with high494 sTILs (>60 %), which accounted for 9.9 %495 (n = 94) of the population, was independently496 associated with improved recurrence-free sur-497 vival (RFS) in patients treated with chemother-498 apy alone, but not in the chemotherapy plus499 trastuzumab group, and did not predict benefit500 from trastuzumab. In patients treated with501 chemotherapy alone, the 10 year RFS rates were502 90.9% and 64.5 % for LPBC and non-LPBC

503�groups, respectively (HR: 0.23; 95 % CI: 0.073–504�0.73). Subgroup analysis from the BIG-02-98505�adjuvant phase III trial of lymph node positive506�breast cancer patients also showed a notable507�benefit of increasing TILs (10 % increments) in508�the HER2-positive cohort treated with509�anthracycline-only chemotherapy without trastu-510�zumab, although this was not seen in anthracy-511�cline and docetaxel arm. It is unclear currently512�why such an interaction should be present with513�the type of chemotherapy regimen, although a514�higher dose of anthracycline could be responsible515�for the immune mediated response. Based on all516�these studies, a higher TILs infiltration is pre-517�dictive of outcome in HER2+ breast cancer518�especially in the anthracycline only treated519�subgroup.520�ER-Positive, HER2-Negative Breast Cancer:521�Data on the prognostic effect of TIL in the hor-522�mone receptor positive breast cancer groups is523�limited. Recent preliminary data from two524�ongoing randomized adjuvant trials has shown525�that there is no prognostic impact of TIL in the526�ER+/HER2– subgroup (Maria Vittoria Dieci et al.527�2014).

528529�12.7 Clinical Validity of TILs530�as a Prognostic and Predictive531�Biomarker in Patients532�with Breast Cancer Treated533�with Neoadjuvant534�Chemotherapy

535�Neoadjuvant chemotherapy of localized breast536�cancer leads to clinical responses in as many as537�70–90 %, but pathological complete response538�(pCR), defined as a complete or near complete539�absence of residual tumor, is only seen in 10–540�25 % of patients (Fisher et al. 1998; Smith et al.541�2002). pCR is a short term surrogate associated542�with a long-term favorable prognosis, especially543�in HER2-positive and TNBC (Cortazar and544�Geyer 2015), and is now accepted by regulatory545�agencies such as the United States Food and546�Drug Administration for accelerated approval of547�new agents in patients with localized breast548�cancer who are candidates for neoadjuvant

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549 chemotherapy (Prowell and Pazdur 2012).550 Patients with residual disease have a variable551 prognosis, but extensive residual disease in552 patients with TNBC and HER-positive breast553 cancer appear to have a high risk of recurrence554 (Symmans et al. 2007). There has therefore an555 interest in determining the relationship between556 TILs in pretreatment core biopsies as a predictive557 biomarker for pCR in patients with neoadjuvant558 therapy, and as a prognostic biomarker in559 patients with residual disease after neoadjuvant560 therapy because of the variable prognosis for this561 population. The results of these reports are562 summarized in Table 12.3.563 TILs and Predicting Response to Neoadjuvant564 Chemotherapy: Lymphocyte infiltration was565 analyzed in 1058 pre-treatment cancer tissues566 from the GeparDuo and GeparTrio cohorts, both567 of which were phase III, randomized trials568 assessing responses to combination neoadjuvant569 chemotherapy regimens. The presence of intra-570 tumoral lymphocytes, defined as >10 % stromal571 area infiltrated with lymphocytes, was an inde-572 pendent parameter for pCR in both the cohorts573 (Denkert et al. 2010). The pCR rates were 42 %574 and 40 %, respectively. Tumors with low TIL575 had pCR rates of 3 and 7 % respectively. In the576 GeparSixto neoadjuvant study assessing addition577 of carboplatin to an anthracycline-taxane com-578 bination in 580 patients, pCR rates were 76.2 %579 for LPBC (defined as >60 % of either intratu-580 moral or stromal TILs) compared to 52.2 % for581 non-LPBC (p = 0.01) in those with TNBC582 (Denkert et al. 2015).583 In a meta-analysis including 13 neoadjuvant584 studies and 3251 patients, TNBC with higher585 TILs in pretreatment biopsy correlated with586 higher pCR rates to neoadjuvant chemotherapy587 (Mao et al. 2014). Greater TIL density was588 associated with a higher pCR rate for neoadju-589 vant chemotherapy (OR: 3.93, 95 % CI: 3.26–590 4.73, p < 0.001), including iTILs (OR: 4.15,591 95 % CI: 2.95–5.84, p < 0.001) or sTILs (OR:592 3.58, 95 % CI: 2.50–5.13, p < 0.001). Pretreat-593 ment TILs had predictive values in ER negative,594 triple negative and HER2 positive breast cancer595 patients, but not in ER-positive disease. There-596 fore, TIL analysis on initial tumor samples serves

597�as an important predicting factor for pathologic598�response in TNBC.599�In a study of 180 stage II and III breast cancer600�patients, tumors with Foxp3 and CD8 infiltrates601�were associated with a high-pCR rate (p < 0.001602�and p = 0.007, respectively) in those who603�received neo-adjuvant weekly paclitaxel fol-604�lowed by 5-fluourouracil, epirubicin and605�cyclophosphamide (Oda et al. 2012). Foxp3606�infiltrate was a significant independent predictor607�of pCR (p = 0.014), but CD8 infiltrate was not.608�In another study with 153 tumor samples, high609�CD8+ TILs in pretreatment biopsy was found to610�be an independent predictor of response to611�neoadjuvant chemotherapy (Seo et al. 2013).612�These results demonstrate that subpopulations of613�lymphocytes may also be predictive of response614�to neoadjuvant chemotherapy, although further615�studies in larger populations are needed in order616�to determine whether this provides more accurate617�prognostic and predictive information than sim-618�ply evaluating sTILs by conventional hema-619�toxylin and eosin staining.620�TILs and Predicting Response to Tras-621�tuzumab: In the HER2-population, the response622�to trastuzumab and its association with TILs has623�been investigated in the neo-adjuvant setting. In624�the GeparQuattro trial, 156 patient with HER2+

625�breast cancer received neoadjuvant trastuzumab626�with chemotherapy (4 cycles of epirubicin/627�cyclophosphamide with docetaxel with or with-628�out capecitabine); each 10 % increment in TILs629�was associated with higher rates of pCR (ad-630�justed OR: 1.14, 95 % CI: 1.01–1.29) (Loi et al.631�2014a). The neoadjuvant trial GeparSixto,632�investigated the effect of adding carboplatin to an633�neoadjuvant anthracycline-taxane combination in634�580 patients with triple negative or HER2+ breast635�cancer (Denkert et al. 2015); trastuzumab and636�lapatinib were also given in patients with HER2+

637�disease, and bevacizumab to patients with638�TNBC, which included 25 % of patients who had639�LPBC (defined as >60 % of either iTILs or sILs).640�Overall, the pCR rate was significantly higher in641�the LPBC compared with the non-LPBC group642�(59.9 vs. 33.8 %, p = 0.001). pCR rate were643�significantly higher for the LPBC group in the644�absence of platinum (46.6 vs. 33.5 % p = 0.05)




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Table

12.3

Clin

ical

stud

iesof

TILsas

abiom

arkerin

neoadjuv

anttherapyof

breastcancer

Trial-A

utho

randreference

No.

ofsamples

Metho

d-

H&E/IHC

TILs

analyzed:

i-TILs,

s-TILsor

t-TILs

Lym

phocyte

predom

inantbreast

cancer

Neoadjuvant

Therapy

Survival

parameter

inHighTILs

subg

roup

All

types

Denkertet

al.

(201

0)10

58H&E

s-TILs

and

i-TILs

HighTILs(>60

%)

wereseen

in9.5%

patients

GeparDuo

coho

rt:

docetaxelwith

doxo

rubicinvs

doxo

rubicin/cyclop

hosphamide

follo

wed

bydo

cetaxel.

GeparTriocoho

rt:T

AC-6-8cycles

vsfour

cycles

ofTAC

follo

wed

byfour

cycles

ofvino

relbineandcapecitabine

∙HighTILs/LPB

CtumorshadpC

Rof

40%,comparedto

pCR

of5%

intumorswith

outanylymph

ocyte

infiltrate(<0.00

05)

Oda

etal.

(201

2)18

0IH

C-FO

XP3

andCD8

i-TILs

Medianvalues

used

ascut-off,

Intratum

oral

TILs

used

ascontinuo

usvariable

Sequ

entialweeklypaclitaxelfollo

wed

byFE

C∙HighpC

Rrate

of31

.3and25

.7%

was

associated

with

tumorshaving

FOXP3

andCD8infiltrates

(p<0.00

1andp=0.00

7,respectiv

ely)

∙Breasttum

orswith

both

FOXP3

and

CD8infiltrates

show

edthehigh

est

pCRrate

of33

.0%

whencompared

tothosewith

outlymph

ocyte

infiltrate

Yam

aguchi

etal.(201

2)68

H&E,IH

C-

CD3,

CD20

s-TILs

and

i-TILs

HighTIL

definedas

IHC

2and3by

visual

grading,

were

seen

in38

%patients

Anthracyclin

e-and

taxane-based

regimen

(4cycles

ofFE

Cand4cycles

ofdo

cetaxel)

∙HighTILswas

anindepend

ent

predictor

forpC

R(O

R:4.7,

95%

CI:2.2–

10.06)

Ladoire

etal.

(200

8)56

IHC

-CD3+,

CD8+

,FO

XP3

i-TILs

Lym

phocyte

infiltrates

were

graded

(0-3)perIH

C

Anthracyclin

e-basedregimen:FE

Cor

CEX;do

cetaxel

was

used

sequ

entially

with

anthracyclines-based

chem

otherapy

.Patientswith

HER-2–po

sitiv

e:treated

with

trastuzumab

with

docetaxeland

carbop

latin

∙HigherCD8infiltratebefore

and

afterchem

otherapy

was

associated

with

pCR

(p=0.03

7and

p=0.02

6,respectiv

ely)

∙After

neoadjuv

anttherapy,

FOXP3

+cells

decreasedin

patients

with

patholog

iccompleterespon

ses

(pCR),whereas

thesecells

remained

elevated

inno

n-respon

ders (c

ontin

ued)




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Table

12.3

(con

tinued)

Trial-A

utho

randreference

No.

ofsamples

Metho

d-

H&E/IHC

TILs

analyzed:

i-TILs,

s-TILsor

t-TILs

Lym

phocyte

predom

inantbreast

cancer

Neoadjuvant

Therapy

Survival

parameter

inHighTILs

subg

roup

Lee

etal.

(201

3)17

5IH

C-CD3+,

CD8+,

FOXP3

t-TILs

Usedin

acontinuo

usscalein

10%

increm

entsby

visual

gradingof

IHC

Anthracyclin

e-basedregimens(A

C),

anthracyclineandtaxane-based

regimens(docetaxel

anddo

xorubicin

orAC

follo

wed

bydo

cetaxel).

HER2-po

sitiv

etumors,weretreated

with

Herceptin-based

regimens

∙Higherlym

phocyteinfiltrates

ofCD8

+,CD3+

orFO

XP3

+was

asign

ificant

independ

entpredictorof

pCR

(OR:1.26

,p=0.02

4)

Seoet

al.

(201

3)15

3IH

C-CD4+

,CD8+

,and

FOXP3

+TILs

i-TILs

HighCD4TIL

defined

as≥59

.67/HPF

,high

CD8as

≥16

.33/HPF

andhigh

FOXP3

as≥6.33

/HPF

AC,A

Dor

ACfollo

wed

bydo

cetaxel

∙HighCD4+,

CD8+,

and

FOXP3

+TILswereassociated

with

ahigh

pCR.

∙OnlyCD8+TILswerean

independ

entpredictiv

efactor

for

pCR

(OR:9.78

6;95

%CI:2.12–

45.14),irrespectiv

eof

breastcancer

subtyp

e

Aruga

etal.

(200

9)87

IHC-FO

XP3

+s-TILs

and

i-TILs

HighTILsdefinedas

6.6c/HPF

,wereseen

in50

.5%

patients

Anthracyclin

e(FECor

EC)with

orwith

outTaxane(D

ocetaxel

orPaclitaxel)

∙Prog

nosiswas

betteram

ongpatients

with

low

numbers

ofFO

XP3

-positive

cells

intumor.Theyhadabetter

recurrence-freesurvival,with

risk

ratio

of5.81

(95%

CI:1.09–10

7.5)

comparedto

high

FOXP3

-positive

cells

Liu

etal.

(201

2)13

2IH

C-FO

XP3

s-TILs

and

i-TILs

Medianwas

takenas

thecutoffand

samples

abov

ethe

medianwas

considered

high

Anthracyclin

e-basedregimens:FE

Cor

CEX.Fo

llowinganthracycline,

docetaxelwas

givento

43pts

∙Decreased

peritumoralTregs

werean

independ

entpredictorforpC

R∙Highintratum

oral

Tregs

afterchem

otherapy

was

associated

with

unfavo

rableprog

nosis-

OS

(p=0.00

1)andPF

S(p

=0.00

6)

(con

tinued)




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Table

12.3

(con

tinued)

Trial-A

utho

randreference

No.

ofsamples

Metho

d-

H&E/IHC

TILs

analyzed:

i-TILs,

s-TILsor

t-TILs

Lym

phocyte

predom

inantbreast

cancer

Neoadjuvant

Therapy

Survival

parameter

inHighTILs

subg

roup

TNBC

Dieci

etal.

(201

4)27

8H&E

s-TILs

and

i-TILs

HighTILsdefined

as>6

0%,wereseen

in9.7%

patients

Anthracyclin

ewith

orwith

outtaxane

∙HighTILsversus

low

TILs(>60

%)

–5yr

OS—

91versus

55%

–HR:0.19

,95

%CI:0.06

–0.6

Ono

etal.

(201

2)92

H&E,IH

Cs-TILs

and

i-TILs

HighTILs(>50

%)

wereseen

in73

%patients

Regim

enswith

anthracycline,

taxane

orbo

th∙HighTILsversus

Low

TILs(>50

%)

–pC

R=37

versus

16%,p=0.05

Denkertet

al.

(201

5)31

4H&E

s-TILs

and

i-TILs

HighTILs(>60

%)

wereseen

in28

.3%

patients

Anthracyclin

eandtaxane,with

orwith

outcarbop

latin

∙In

theLPB

C/HighTILsgrou

p,the

additio

nof

carbop

latin

toanthracycline-plus-taxaneshow

eda

high

pCR

of74

%

HER2

+Loi

etal.

(201

4a)

156

H&E

t-TILs

TILsused

asa

continuo

usvariable,

nocut-offused

Trastuzum

abwith

chem

otherapy

(epirubicin/cyclop

hosphamidewith

docetaxelwith

orwith

out

capecitabine)

∙Fo

reach

10%

increm

entin

TILs,

high

erratesof

pCRwereseen

after

neoadjuv

anttherapy

∙Fo

rHighversus

Low

TILs

–AdjustedOR:1.14

,95

%CI:

1.01–1.29

Denkertet

al.

(201

5)26

6H&E

s-TILs

and

i-TILs

HighTILs(>60

%)

wereseen

in19

.9%

patients

Anthracyclin

eandtaxane,with

orwith

outcarbop

latin

∙In

theLPB

C/HighTILsgrou

p,the

additio

nof

carbop

latin

toanthracycline-plus-taxaneshow

eda

pCR

of78

%

Salgadoet

al.

(201

5a)

387

H&E

s-TILs

TILsused

asa

continuo

usvariable,

nocut-offused

Trastuzum

ab,lapatin

ibor

combinatio

n×6weeks

follo

wed

byweeklytaxo

l×12

,follo

wed

byFE

C

∙Every

1%

increase

inTILswas

associated

with

a3%

decrease

inrate

ofevent(pCR

andEFS

)–HR:0.97,

95%

CI:0.95–0.99

∙TILsgreaterthan

5%

were

prog

nostic

forpC

R–OR:2.60

,95

%CI:1.26

–5.39

(con

tinued)




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Table

12.3

(con

tinued)

Trial-A

utho

randreference

No.

ofsamples

Metho

d-

H&E/IHC

TILs

analyzed:

i-TILs,

s-TILsor

t-TILs

Lym

phocyte

predom

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650650650650650650 and presence of platinum (>75 vs. 38.1 %,651 p < 0.0005). Higher TIL density has also been652 associated with higher pCR rates after neoadju-653 vant HER2-directed therapy plus chemotherapy654 (Salgado et al. 2015a; Denkert et al. 2014, 2015).655 In a pooled meta-analysis of 13 published656 studies with 3555 patients, high level of TILs in657 pretreatment biopsy indicated higher pCR rates658 to neoadjuvant chemotherapy in TNBC and659 HER2+ breast cancer. The correlation of pCR660 with TILs was not seen in hormone receptor661 positive [HR+/HER2−] disease (Mao et al. 2014).662 High CD8+ T-lymphocytes in samples pre- (OR:663 3.36; 95 % CI: 1.15–9.85) or post-neoadjuvant664 chemotherapy (OR: 4.71; 95 % CI: 1.29–17.27)665 was associated with a higher pCR. In the HER2+

666 group, high TILs not only predict a favorable667 response to neoadjuvant trastuzumab, but also to668 chemotherapy. Study of TILs and its response to669 chemotherapy in 368 pretreatment tissues from670 two ER negative cohorts (EORTC 10994 and671 BIG 00-01) (West et al. 2011), showed that high672 level of CD8+ TIL was an independent predictor673 of anthracycline response.674 TILs and Predicting Response to Endocrine675 Therapy: The status of TILs following endocrine676 therapy is not clearly defined. In a study of patients677 with ER+ breast cancer treated with neoadjuvant678 steroidal aromatase inhibitor (AI) therapy, chan-679 ges in CD8+ T cells/Foxp3+ or T regulatory cells680 ratio before and after therapy correlated with681 response. A significant increase in the CD8+/Treg682 ratio was detected after hormonal therapy in683 responders (p = 0.028) but not in nonresponders684 (Chan et al. 2012). Thus, the CD8+/Treg ratio in685 surgical pathology specimens can be a potential686 surrogate marker for predicting responses to687 neoadjuvant endocrine therapy.

688�TILs in Residual Cancer after Neoadjuvant689�Chemotherapy and Prognosis: In a retrospective690�study of 304 TNBC patients with residual disease691�after primary neoadjuvant chemotherapy, the pres-692�ence of TIL in residual tumor was associated with693�better prognosis (Dieci et al. 2014). Both sTILs and694�iTILs were strong prognostic factors for metastases695�free survival and OS. The 5-year OS rate was 91 %696�for high TILs and 55 % for lowTILs subgroup (HR:697�0.19, 95 %CI: 0.06–0.61). The prognostic impact of698�TILs was most significant in patients with large699�tumor burden (>2 cm) or lymph node metastases.

700701�12.8 Conclusion

702�The key points described in this chapter are703�summarized in Table 12.4. First, TIL density is a704�prognostic biomarker, and also a predictive bio-705�marker for response to neoadjuvant chemother-706�apy in TNBC and HER2 positive breast cancer.707�Second, characterization of sTIL density is a708�more practical and reproducible biomarker than709�iTILs, and expert-based guidelines have been710�developed for characterization of TIL density.711�Third, TIL recruitment and function is influenced712�by various factors in the microenvironment.713�Further research is needed to evaluate in detail714�the composition of TIL, and their subclasses in715�the tumor microenvironment. In case of T cells716�their composition, T cell receptor repertoire,717�neoantigens being identified by the T cells and718�the presence of costimulatory or coinhibitory719�molecules needs to be further elucidated. Addi-720�tional research is also needed in order to deter-721�mine TILs provide prognostic in metastatic722�breast cancer, or predict better response to vac-723�cines or immune checkpoint blockade.

Table 12.4 Summary of key points and major conclusions

Key points

1. TIL density is a prognostic and predictive biomarker for TNBC and HER2+ breast cancer2. Stromal TIL are a better predictor of clinical outcomes than intratumoral TIL3. Expert-based guidelines have been developed for characterization of TIL density4. TIL recruitment and function is influenced by various factors in the microenvironment including cytokines,

regulatory cells and coinhibitory ligands expressed by the tumor




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724725 References

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Tumor Infiltrating Lymphocytes 12 as a Prognostic and ...U NCORRECTED PROO F 1 23 Tumor Infiltrating Lymphocytes 12 4 as a Prognostic and Predictive 5 Biomarker in Breast Cancer 6

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