reﬂections on the histopathology of tumor- ﬁltrating ...precursor of melanoma, lentigo maligna,...

Masters of Immunology

Reflections on the Histopathology of Tumor-Infiltrating Lymphocytes in Melanoma and theHost Immune ResponseMartin C. Mihm Jr1 and James J. Mul�e2

Abstract

In the past five decades, the role for lymphocytes in hostimmune response to tumors has been shown, at least in somepatients, to be a critical component in disease prognosis. Also,the heterogeneity of lymphocytes has been documented,including the existence of regulatory T cells that suppress theimmune response. As the functions of lymphocytes havebecome better defined in terms of antitumor immunity, spe-cific targets on lymphocytes have been uncovered. The appre-ciation of the role of immune checkpoints has also led to

therapeutic approaches that illustrate the effectiveness ofblocking negative regulators of the antitumor immuneresponse. In this Masters of Immunology article, we trace theevolution of our understanding of tumor-infiltrating lympho-cytes and discuss their role in melanoma prognosis from thevery basic observation of their existence to the latest mani-pulation of their functions with the result of improvementof the host response against the tumor. Cancer Immunol Res; 3(8);827–35. �2015 AACR.

Disclosure of Potential Conflicts of InterestM.C. Mihm is a consultant/advisory board member for Caliber Imaging Diagnostics and MelaSciences. No potential conflicts of interest

were disclosed by the other author.

Editor's DisclosuresThe following editor(s) reported relevant financial relationships. R. Vonderheide—None.

CME Staff Planners' DisclosuresThe members of the planning committee have no real or apparent conflicts of interest to disclose.

Learning ObjectivesLymphocytes comprise a critical component of the host immune response to tumors, and the presence of tumor-infiltrating lymphocytes

(TIL) correlates with disease prognosis. Identification of TILs in the tumor microenvironment and characterization of their role in

melanoma prognosis, as well as the strategies available for manipulating TIL functions, will facilitate the augmentation of host immune

response against tumors. Upon completion of this activity, the participant should gain insight from the discovery of TILs in the tumor

microenvironment to the histopathology of TILs in melanoma and their roles in host immune response.

Acknowledgment of Financial or Other SupportThis activity does not receive commercial support.

IntroductionThe importance of lymphocytes in tumors has been long

recognized. At first, it was considered that they were causative ofmalignancy. Their possible role in prognosis in melanoma and

other tumors has only been recognized in the past 40 to 50years. This Masters of Immunology article relates, often throughthe lens of personal experience, the history of the discovery andcharacterization of tumor-infiltrating lymphocytes (TIL) and

1Brigham and Women's Hospital, Dana-Farber Cancer Institute, Har-vard Medical School, Boston, Massachusetts. 2Translational Science,Melanoma Research and Treatment, Moffitt Cancer Center, Tampa,Florida.

Note: Collaborators with M.C. Mihm include April Armstrong, Raymond Barnhill,Alistair Cochran, Martin Cook, Claudio Clemente, A. Neil Crowson, GlennDranoff, Phyllis Gimotty, F. Stephen Hodi, John Kirkwood, Cynthia M. Magro,Donald Morton, George F. Murphy, Lloyd Old, Hubert Pehamberger,Adriano Piris, Richard J. Reed, Dirk Ruiter, Tobias Schatton, Richard Scolyer,Alain Spatz, John Thompson, Uma Rao, Joost van den Oord, and Klaus Wolff.Collaborators with J.J. Mulé include Jane Messina, Vernon Sondak, and JeffreyWeber.

CorrespondingAuthors:Martin C. Mihm, Brigham andWomen's Hospital, Dana-Farber Cancer Institute, 41 Avenue Louis Pasteur, Room 317B, Boston, MA 02115.Phone: 617-264-5910; E-mail: [email protected]; and James J. Mul�e, Trans-lational Science, Melanoma Research and Treatment, Moffitt Cancer Center,12902 Magnolia Drive, SRB-3, Room 23015, Tampa, FL 33612. Phone: 813-745-1536; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-15-0143

�2015 American Association for Cancer Research.

CancerImmunologyResearch

www.aacrjournals.org 827

on February 16, 2020. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

http://cancerimmunolres.aacrjournals.org/

the elucidation of their possible role in the control of humanmelanoma.

This work has been accomplished by many collaborations (seeNote on page 1, bottom right). We acknowledge the role ofvarious fellows and staff of the Massachusetts General Hospital,the Brigham and Women's Hospital, and the Moffitt CancerCenter, as well as the members of the WHO and EORTC Mela-noma Pathology groups for their aid, guidance, and support. Asthe focus will be on the histopathology aspects of this topic, wefurther wish to acknowledge the successful past and ongoingimmunotherapy clinical trials of the adoptive transfer oftumor-infiltrating lymphocytes in patients with advanced mela-noma, first pioneered at the NCI Surgery Branch (1, 2) andexpanded to other treatment institutions nationally (3, 4) andinternationally (5, 6), whichwill not be covered in our reflections.

Historical Aspects of Tumor-InfiltratingLymphocytes in Melanoma

The observation of lymphocytes and mononuclear cells asso-ciated with human cancer was first noted more than a centuryago, and these infiltrates were thought to be causative of thedisease (7). The concept of an immunologic response to malig-nant tumors in patients was probably first proposed by PaulEhrlich in 1907 and later expanded in 1909 (8). Coley injectedbacteria into tumors, so-called Coley toxin, with some tumorshrinkage recognized as a host response to tumors (9). Subse-quently, studies of various cancers, including osteogenic sarco-ma, neuroblastoma, carcinoma of the colon, and melanoma,have been performed and have elucidated the relationship ofhost immune response to tumors and patient survival. Mooreand Foote hypothesized for medullary carcinoma of the breastthat the "rather characteristic lymphoid infiltrate indicates somemaladjustment between tumor and host, and that the clinicalbehavior is a further indication thereof" (10). Already, by thetime of the publication of "Melanoma and Skin Cancer" in the1972 Proceedings of the International Congress on Skin Cancerin Sydney, Australia, descriptions of host immune response tomelanoma in the form of antimelanoma antibodies (11) andlymphocytotoxicity (12) to human melanoma and mouse mel-anoma cells had been documented. The injection of mincedmelanoma cells into 26 patients with metastatic melanomaresulted in 2 patients with complete tumor regression and5 patients with partial regression of tumors (13). In a similarstudy, Ryan and colleagues (14) reported a high index of lym-phocytotoxicity in patients with metastases injected with irradi-ated autologous melanoma cells. They also reported regressionof metastases after injections with Bacillus Calmette–Guerin(BCG). Histology of the regressing tumors exhibited markedinfiltration with lymphocytes.

The term "tumor-infiltrating lymphocytes" was used for thefirst time in the experience of one of us (M.C. Mihm) duringwork with Wallace H. Clark Jr. In the detailed study of malig-nant melanomas that was spearheaded by Clark, the anatomiclevels of invasion were proposed (15). The epidermis, thepapillary dermis, and the superficial vascular plexus were con-sidered reactive sites where cutaneous inflammatory processesoccurred. Clark considered these sites the sensitizing area ofthe skin and used the example of poison ivy to support hishypothesis. During the course of these studies (16), Clark wasstruck with the prominent inflammatory infiltrate (Fig. 1A)

that accompanied the superficial spreading variant of malig-nant melanoma in the radial growth phase (RGP), and we bothinvoked the possibility of a contact-like hypersensitivity reac-tion. He also noted that, in the lesion that we described as theprecursor of melanoma, lentigo maligna, there was often noinflammation until the lesion became microinvasive. Theseobservations and hypotheses led to the designation of level IIas a microinvasive melanoma that was probably controlled bythe host response through a sensitization of the host by thetumor cells (15). Another feature that was described both insuperficial spreading melanoma and in lentigo maligna mela-noma was regression in the RGP (17). This phenomenon wasidentified by the presence of partial areas of fibrosis withlymphocytes and melanophages flanked on one or both sidesby the tumor (Fig. 1B). This observation was considered to beevidence of a host immune response to the tumor. Levels III, IV,and V represented a new event in tumor progression that wasassociated with the acquisition of metastatic potential. How-ever, at level III, the tumor was still confined to the papillaryreticular dermal junction and also often had an inflammatoryinfiltrate, frequently at the base of the tumor. Levels IV and Vwere proposed to confer the most adverse prognosis, eitherbecause the tumor could overcome the barrier of the papillarydermal interface and successfully invade through the dermisand into the fat or because the host response had failed. In1967, a patient at the Massachusetts General Hospital (Boston,MA) Pigmented Lesion clinic presented with a metastatic nod-ule surrounded by a halo of hypopigmentation. A biopsyrevealed a nodule of metastatic melanoma diffusely infiltratedby lymphocytes as observed by Clark, who described thisresponse as TIL. This was the first time in the recollection ofone of us (M.C. Mihm) that this description of TILs was appliedto this type of host response in melanoma (Fig. 1C). Our hopewas to someday find a way to enhance the patient's lymphocyteresponse to these tumors.

In the meantime, Larsen and Grude (18) described increasedsurvival in patients with a dense lymphocytic infiltrate in contrastwith patients who showed a weak response. The dense infiltratewas found predominantly in patients whose melanomas wereconfined to the papillary dermis. Themore deeply invasive lesionshad little host response and a worse prognosis. Hansen andMcCarten reported that patient survival was related to the densityof the lymphoid infiltrate at the advancing edge of the tumor.Favorable prognosis was associated with a dense response (19).Day and colleagues (20) reported better survival in patients withprimary melanoma that had a moderate to marked infiltrate ofintratumoral and/or peritumoral lymphocytes compared withthose with no infiltrate.

Meanwhile, in Australia, McGovern and Lane Brown began tostudy lymphocytic infiltration in halo nevi and melanoma (21).Thompson reported an improved prognosis in patients with aprominent infiltrate at the advancing edge of the melanomanodule in a very limited study (22). McGovern studied theevolution of regression and considered a dense infiltrate oflymphocytes in a nodule of melanoma to represent early regres-sion (23). A panel of 11 pathologists and a surgeon, G.W. Milton,chaired by Vincent McGovern with one of us (M.C. Mihm) asrapporteur was convened at the Sydney Conference in 1972 toformalize the classification of melanoma and its reporting. Thegroup recognized that lymphoid infiltrates appeared to be impor-tant in primarymelanomabutwere notwell-enough studied to be

Mihm and Mul�e

Cancer Immunol Res; 3(8) August 2015 Cancer Immunology Research828



included in the routine pathology report (24). In a subsequentreview of prognosis inmelanoma,McGovern reported on varyingstudies that described improved prognosis to be associated withlymphocytes, especially in the base of the tumor nodule (25).

In the early 1970s, a great deal of interest was expressed invaccination of metastases. In collaboration with M. Lane Brown,Cosimi, and T.B. Fitzpatrick, we injected vaccinia virus into severalmelanoma nodules of the lower leg of a patient with multiplemetastatic lesions in the right lower limb. The injected noduleshadmarked inflammatory reactions and some nodules regressed.Allmelanomanodules,with orwithout vaccinia injection, reactedwith at least some swelling and erythema. These changes werepossible examples of the now-recognized abscopal response. Abiopsy of one of the injected lesions revealed histologically astriking infiltrate of mainly lymphocytes with somemononuclearcells and focal neutrophils. Direct interactions of the lymphocyteswith melanoma cells were observed with pyknosis of melanomacells. Roenigk and colleagues (26) reported on a similar responseof vaccinia virus injections into metastatic melanoma nodules.These changes certainly reinforced earlier suggestions for devel-

oping a therapy that uses the lymphocyte response to controlmelanoma.

In the United States, Morton and colleagues pursued studiesshowing the importance of the immune response in melano-mas that led him to publish a rationale for immunotherapy(27). One study showed significant sensitization of lympho-cytes by melanoma antigens (28), and further studies with BCGvaccination of tumor nodules recorded the importance of thelymphocytes in the reaction (29).

In addition to the study of melanoma, one of us (M.C.Mihm) formed a very active collaboration with HaroldF. Dvorak in the study of delayed hypersensitivity reactionsas a component of allergic and tumor responses. Because of theinterest in hypersensitivity reactions, Dvorak and colleagues(30) studied four examples of RGP superficial spreading mel-anoma with electron microscopy and compared these withprior findings in human delayed hypersensitivity responses,including allergic contact dermatitis and human allograftskin rejection. This investigation revealed activation of lym-phocytes and macrophages, prominent vascular changes,

© 2015 American Association for Cancer Research

A

B C

Figure 1.Melanoma infiltrated with various immune cell types. A, superficial spreading melanoma with multiple lymphocytes (arrow L) infiltrate amidst melanoma cells(arrow) in papillary dermis (hematoxylin and eosin–stained light micrographs; �40) B, dense melanophages (MP) with scattered lymphocytes (blue) andfibrosis represent area of regression flanked by the tumor (arrowhead). C, nodule of metastatic melanoma with dense lymphoid infiltrate (L arrowhead). Note thestriking interaction of lymphocytes with melanoma cells and areas of melanoma cell nuclear pyknosis.

TILs in Melanoma

www.aacrjournals.org Cancer Immunol Res; 3(8) August 2015 829



basophils, and mast cells with a partial loss of mast cell granulecontent, as seen in delayed-type hypersensitivity reactions.There was evidence for killing of melanoma cells throughdirect contact with lymphocytes. The vascular changes includedendothelial cell swelling as well as vessels manifesting necrosis ofthe endothelium. The basal lamina showedmarked reduplicationtypical of recurrent injury such as chronic inflammation. Whilethese changes, other than the lymphocyte–melanocyte satellitosis,were not specific, they were compatible with the findings inhuman cutaneous hypersensitivity responses (30).

During these years, Clark, along with Elder and other colla-borators, started to study the patterns of lymphocyte infiltra-tion that resulted in the publication in 1989 of the mostcommonly used classification of TILs as brisk, non-brisk, andabsent (31). To qualify as a TIL, there had to be evidence oflymphocyte-melanoma cell interaction (Fig. 2). The study byClemente and colleagues (32) in the WHO Melanoma Pathol-ogy group confirmed the Clark data and demonstrated a highlysignificant survival advantage in patients with lesions with abrisk infiltrate, an intermediate survival for those with lesionswith non-brisk infiltrates, and a poor prognosis for those withmelanomas without an infiltrate (Fig. 3). However, neitherstudy described the variation in the density of the infiltratinglymphocytes. The WHO group subsequently studied the pres-ence of TILs in clinical stage II melanomas with positive lymphnodes from patients treated on a trial of IFNa, some of whomdid very well with a prolonged survival (33). These lymph nodemetastases were evaluated for TILs anonymously with the use ofthe grading system of brisk, non-brisk, and absent as applied tothe primary lesion. Fig. 3A–E are schematic diagrams illustratingthe different types of TILs. To qualify as brisk, all the metastatictumor deposits, whether in one or several lymph nodes, had toexhibit a diffuse infiltrate of lymphocytes interacting withtumor cells. The non-brisk category included those in whichthere was partial infiltration of the tumor by lymphocytes. The

results indicated that patients with the brisk immune infiltrateshad significantly better survival than those with absent inflam-mation or non-brisk inflammation. There was no evidence thatthe IFN therapy had any effect on the outcome. An additionalstudy of these patients indicated a matched clonality of lym-phocytes in the primary tumor and in the metastatic tumor withregard to T-cell receptor (TCR) usage; this finding suggested thepotential specificity of the host response (34). In a later study ofprognostic variables in 259 Eastern Cooperative OncologyGroup interferon trials, positive TILs had a beneficial survivaleffect, independent of therapy (35).

Over the past 20 years, withGlennDranoff and F. StephenHodi,we have studied the histopathology of the response to vaccinationwithautologousmelanoma cells engineered toproduceGM-CSFaswell as that due to antibodies blocking the immune checkpointantigen, CTLA-4, inmice and in humans (36, 37). Thiswork clearlydemonstrated the critical role of lymphocytes as they infiltrated thetumors in the response tomelanomametastases due to vaccinationor due to the release of the immune response with appropriate antiCTLA-4antibodies.During the same timeperiod, several additionalhistopathology studies of TILs in melanoma have been published.Some supported the antitumor significance of TILs in melanomaand T cells in sentinel lymph nodes (SLN). Others failed to showsignificant correlation, as reviewed by Schatton and colleagues(38).While thebasis for thesedifferences remains tobeestablished,one factor might relate to the heterogeneity of T-cell subsets. Theimportance of regulatory T-cell (Treg) inhibition of protectiveantimelanoma host responses, for example, has been documentedin murine studies (39). The importance of stimulatory and costi-mulatorymolecules indeterminingwhether lymphocyte activationor inhibition occurs has also been revealed. A more completedefinition of melanoma antigens, including mutated neoepitopes,has been achieved aswell. All of this new informationwill lead to amore comprehensive understanding of antimelanoma immunityincluding the critically different types of T cells (38, 40).


Figure 2.Vertical growth-phase melanoma with strikinglymphocyte (yellow L arrows)–melanoma cellsatellitosis (arrow M).

Mihm and Mul�e




Present and Future Histopathology Studiesof TILs in Melanoma

A recent article by Thomas and colleagues (41) described TILs in2,845 patients with a 5-year follow-up from the Population-BasedGenes, Environment and Melanoma (GEM) study. A definitesurvival benefit was found when comparing melanomas withbrisk and non-brisk TILs versus those with absent TILs (P <0.001). These investigators used the criteria established by Clarkand colleagues (31) in evaluating the infiltrates, and the reportincluded information on Breslow thickness, mitoses, and ulcera-tion, as well as the age and the site of the tumor. All cases includedthe American Joint Committee on Cancer (AJCC) tumor-stagingfeatures. The index cases were predominantly vertical growth-phase lesions but also included a small number of stage T1blesions according to the AJCC tumor determinations. The rates ofmelanoma-specific death were 30% and 50% less in tumors withnon-brisk and brisk TILs, respectively, compared with tumors withan absence of TILs. The brisk infiltrates were found more com-monly in tumors from young patients, with the absence ofinfiltrates found in tumors with increased mitoses and ulceration.Interestingly, in a larger population of patients from the GEMstudy, including the index cases, the RGP tumors were consistentlyassociated with a brisk infiltrate. This finding harkens back to thestudies of Clark and Dvorak and colleagues discussed above.During these years, the Sydney Melanoma group, now formalizedinto the Melanoma Institute of Australia under the leadership ofJohn Thompson, Jonathan Stretch, and Richard Scolyer, hasengaged in numerous melanoma studies. A recent benchmarkarticle on TILs and their relationship to sentinel lymph nodes and

on patient survival demonstrated that the presence of dense TILs inthe primary tumor was associated with 5.6% SLN positivity versus27.8% positivity in tumors with absent TILs. Furthermore, therewas 100% survival in patients with dense TIL infiltration (42).

Cintolo and colleagues (43) recently reported on 161 patientsand determined the influence of TILs and tumor regression inpatients evaluated by the standard parameters, including AJCCstaging criteria. The tumors had a median thickness of 5.27 mm.The favorable prognostic factors included female sex, site, non-axial, positive TILs, age < 60 years, no ulceration or histologicregression, no prior elective lymph node dissection, or no evi-dence of any regional nodal disease. The presence of TILs wasprognostically significant onlywhen therewas no RGP regression.The benefit of TILs was lost if RGP regression was present (P <0.001). These authors hypothesized that the presence of regres-sion indicated partial success of host response to the RGP lesionsbut failure as far as the deeper tumor was concerned. A recentlycomplete study of 1,240 patients with a 30-year follow-up by theEORTC Pathology Group suggests that certain patterns of TILshave a definite survival benefit (Cook M et al., manuscript inpreparation).

The Role of Chemokines, MononuclearDendritic Cells, and Immunosuppression inMelanoma Pathogenesis

The original concepts that lymphocytes could generate cancerhave long been based on the fact that chronic inflammation canbe associated with malignancy. One example is the association

A B

C

E

D

Figure 3.Brisk, non-brisk, and absent lymphocyticinfiltration in primary melanoma nodules.The three photomicrographs in the leftcolumn exhibit the brisk, non-brisk, andabsent patterns. The brisk pattern is definedby lymphocytes present throughout thetumor as shown by arrows (top left). Non-brisk is defined by scattered, focal groups oflymphocytes (arrow). The word "absent"above the bottom row of images is definedas "containing no lymphocyte infiltration inthe tumor." A–E (middle and right),schematic renderings of different types ofTIL patterns in primary melanoma. A, thebrisk lymphocytic responsemay involve theentire tumor nodule or B, be present alongthe advancing edge of the entire peripheralnodule. The focal nature of the non-briskpattern is sjpwm in C andD. In the renderingof the absent lymphocytic response, thereare either no lymphocytes in the tumor, or iflymphocytes are present (E), they do notinteract with melanoma cells. Panels A–Eare adapted from Fig. 4 of chapter 16 bySchatton et al., Tumor infiltratinglymphocytes and their significance inmelanoma prognosis. In: Thurin M,Marincola FM, editors. Moleculardiagnostics for melanoma. New York:Humana Press; 2014. p. 287–324.

TILs in Melanoma




of the inflammatory infiltrate triggered by Helicobacter pyloriinfection and gastric malignancy or the persistent inflammationof chronic prostatitis and prostate cancer. These observationspose the dilemma as to why some TILs in melanoma result inprolonged survival in some cases, whereas in other cases, thesame inflammatory infiltrate is associated with immunosup-pression. The answer, in part, to these observations may befound in understanding the role of inflammatory chemokines inlymphocyte function, as well as understanding the immuno-suppressive aspects of certain mononuclear dendritic cells andthe tumor milieu. At least 48 chemokines (in 4 groupings, i.e.,CC, CXC, C, and CX3C) and at least 20 chemokine receptorsare involved in the various types of response to stimuli thatresult in inflammation and/or are important in homeostasis(44). Taube and colleagues recently described how both mel-anoma cells and benign nevi, in the presence of immune hostresponses and secretion of IFNg , express the programmed deathligand-1 (PD-L1), whose interaction with the PD-1 receptorleads to lymphocyte exhaustion. The lymphocyte is the appar-ent source of IFN that then results in rendering the TILs'response effete. Blocking of this interaction by appropriateantibodies leads to enhancement of the immune response (45).

There are different types of dendritic cells in the skin and in thelymphnodes.On exposure to antigen, some result in sensitizationand others can lead to tolerance (46). Plasmacytoid dendritic cellsare abundant in positive SLN and appear to be associated withimmunosuppression (47). Finally, with regard to the issue ofmalignant transformation in themilieu of chronic inflammation,themedley of cytokines and chemokines that are expressed lead toaltered DNA, angiogenesis, and inactivation of suppressor genesamong others with resultant malignant change. Among the mostactive of cytokines are TNFa, IL1, IL6, and IL8 with support ofstromal fibroblasts. In fibroblasts and myofibroblasts, the reac-tion of, CXCL12 with CXCR4 results in angiogenesis associated

with tumor growth (48, 49). The future lies in unraveling thecomplexities of tumor–host and stromal interaction to betterunderstand the role of TILs in relation to tumor progression,control, and eradication, as well as establishing the definitive roleof the monocyte macrophages in enhancing and suppressing theimmune response (50).

Ectopic Lymph Node–like Structures orTertiary Lymphoid Structures withinMelanomas

The term "tumor-localized ectopic lymph node–like struc-tures" (TL-ELN) was introduced to one of us (J.J. Mul�e) for thefirst time in Seattle by Karl Erik and Ingegerd Hellstrom alongwith Ellsworth "Buster" Alvord during the studies of the capac-ity of immune lymphocytes to selectively accumulate in tumorsin vivo (Fig. 4; refs. 51, 52).

Years later, Messina and colleagues (53) interrogated a novel12-chemokine gene expression signature (GES) on genomicarrays of stage IV melanoma metastases. They found this GES toaccurately predict the degree and type of lymphoid infiltrate,organized as TL-ELNs (53). TL-ELNs had been identified earlierby others in humannon–small cell lung cancers and in a fewothersolid tumors (reviewed in ref. 54). Harlin and colleagues (55)suggested that a lack of critical chemokines in a subset of mela-noma metastases might limit the migration of activated T cells,which in turn could limit the effectiveness of antitumor immu-nity. Of interest, Hong and colleagues (56) reported that CXCR3ligands and the CCL5 chemokine could determine T-cell infiltra-tion into cutaneous melanomas. In addition, certain chemother-apy drugs could induce the expression of these chemokines inhuman melanoma cell lines. These chemotherapy-induced che-mokines correlated with T-cell infiltration in melanomas, alsoresultant with tumor control and prolonged patient survival.


Figure 4.The image depicts in light microscopy, the Ki-67immunohistochemical staining of a section of a typicalTL-ELN (center) within a stage IV (nonlocoregional),visceral melanoma metastasis. The tissue was stainedusing the avidin–biotin complex method with retrievalunder high pH. Prediluted antibody to Ki-67 [VentanaMedical System, Inc.; anti–Ki-67 (30-9) primaryantibody is a rabbit monoclonal antibody (IgG)directed against C-terminal portion of Ki-67 antigen.]was used for the proliferative analysis of the TL-ELN.Lymphocytic proliferation within and surrounding thefollicle (brown staining cells) was found to containboth B and T lymphocytes, suggesting newly formedand/or activated TL-ELNs within the tumormicroenvironment. (Image generated byJane Messina, Departments of Anatomic Pathologyand Cutaneous Oncology at the Moffitt Cancer Center,Tampa, FL.)

Mihm and Mul�e




By immunohistochemistry, Messina and colleagues (53) dem-onstrated that TL-ELNs uniformly contained prominent CD20þ

B-cell follicles surrounded bymarginal zone areas of CD3þ T cells(comprising both CD4þ and CD8þ subsets). In addition, CD86þ

follicular dendritic cells, but not FoxP3þ Treg cells, were presentwithin these TL-ELNs. Stage IVmelanomametastases negative forthe GES were uniformly found to have either an absence or adiffuse pattern of lymphoid infiltrate. There was a highly statis-tically significant (P< 0.0001) and consistent association betweenamarked increase in overall patient survival, the value of themeanscore of the GES, and the presence of TL-ELNs in stage IVmelanoma metastases. These findings suggested an ongoingadaptive immune response within the melanoma microenviron-ment. More recently, van Baren and colleagues (57) have con-firmed lymphoid neogenesis in melanoma. Moreover, Cipponiand colleagues (58) performed an analysis of the repertoire ofrearranged immunoglobulin genes in B cells of themicrodissectedfollicles within melanoma metastases and demonstrated clonalamplification, somatic mutation, and isotype switching, all ofwhich suggested a local antigen-driven B-cell response. TL-ELNshave recently been described in primary cutaneous melanoma aswell (59), but this has not been observed by others in a verylimited number of specimens examined (58).

Future DirectionsThe recent progress in advanced melanoma therapy, both

targeted and immune response related, has emphasized theimportance of the relationship of melanoma and the hostresponse as well as the tumor microenvironment. The treatmentof BRAFV600-mutated melanoma with the BRAF-specific inhibitorBRAFi results not only in cessation of tumor growth but also indiminution in tumor size. This effect is also associated with anincrease of CD8þ TILs in the affected tumor nodule; the treatedtumor cells show an increase in MART-1 expression (60–62).Thus, targeted therapy is associated with TIL infiltration as amarker of its effect.

Furthermore, a study of the checkpoint blockade antigenPD-1 with one of its ligands, PD-L1, results in effective blockingof host response to the tumor as a result of exhaustion of thePD-1–bearing lymphocytes. PD-L1 can be found on a variety ofcell types, including tumor cells, stromal cells, and macro-phages. Treatment of patients with melanoma with a specifichumanized mouse monoclonal antibody directed against PD-1,pembrolizumab, or other antibodies directed against PD-L1 canresult in freeing of the host response and effective tumorregression. Tumeh and colleagues (63) reported that melano-mas that have a great number of CD8þ lymphocytes at theadvancing tumor margin are the ones that respond to thistreatment. Results from their study indicate that release of theblockade leads to a proliferation of the CD8þ cells that migrateinto the tumor and induce apoptosis and necrosis of tumorcells. In contrast, melanomas without CD8þ lymphocytes at theadvancing tumor edge do not show response and the tumorsprogress. Once again, antigen-specific TILs that infiltrate the

tumor show prognostic and therapeutic significance. TILs, bytheir presence in both of these instances, have led to betterunderstanding of the tumor–host interaction and, at least inpart, to the therapeutic response. The future lies in more intensestudy of the various inhibitors of TILs and of better ways toenhance their interaction not only with melanomas but alsowith other cancers. Special emphasis should be given to co-stimulatory and inhibitory molecules and the effect that TILsmay have on enhancing or thwarting their positive or negativeeffects, respectively (63). Moreover, the phenomenon of theabscopal effect in patients with melanoma following treatmentwith the combination of anti–CTLA-4 antibody and radiother-apy, although linked to the participation of the immune system,has not yet been thoroughly examined, particularly with respectto changes in the melanoma microenvironment as it pertains toTILs (64, 65).

The existence of a functional connection between TL-ELNs andidentifying (and expanding) tumor-specific, therapeutic TILs isnow being examined in melanoma. Laser-capture microdissec-tionwithDNA/RNA isolation and sequencingmethodologies canalso be used to address questions of clonality and functionalrelationships of the resident T cells within the TL-ELNs, includingin-depth T-cell receptor (TCR) transcript repertoire analyses.Furthermore, the predictive nature of TL-ELNs in patients withmelanoma who have participated, or are participating, in TIL-based (3, 66) and/or checkpoint antibody–based clinical immu-notherapy trials is likely tobe examined retrospectively. Itwill alsobe of interest to determine whether the presence of TL-ELNsreflects an underlying pathogen infection or is related to baselinegene mutational load of the particular melanoma.

In summary, we have traced TILs and their relationship tomelanomas from their first identification under the microscopeover a century ago to a progressive understanding of theirrelationship to tumor control and prognosis. In the molecularbiology era, we find molecular-based therapies that directlyaffect patient survival. It is hoped that the great enthusiasm inthe scientific community engendered by these discoveries willlead not only to melanoma control but also to a possibleunderstanding of melanoma initiation and eradication byimmunotherapy alone or in combination with other promisingtherapeutics.

AcknowledgmentsThe authors wish to thank Claudio Clemente for the archival photomicro-

graphs in Fig. 3; Labib R. Zakka for the photomicrographs in Figs. 1 and 2, andfor his andAdrianoPiris' help inpreparing themanuscript; andStevenA.Conleyfor his overall assistance in assembling the figures and adapting the schema ofFigs. 1 and 3, respectively.

Grant SupportThis work was supported in part by the NCI–NIH (1 R01 CA148995;

P30CA076292; P50CA168536 to J.J. Mul�e), the V Foundation, a CancerResearch Institute grant (to M.C. Mihm), and the Dr. Miriam and SheldonG. Adelson Medical Research Foundation.

Published online August 4, 2015.

References1. Rosenberg SA, Yannelli JR, Yang JC, Topalian SL, Schwartzentruber DJ,

Weber JS, et al. Treatment of patients with metastatic melanoma usingautologous tumor infiltrating lymphocytes and interleukin-2. JNatl CancerInst 1994;86:1159–66.

TILs in Melanoma




2. Dudley ME, Wunderlich JR, Yang JC, Hwu P, Schwartzentruber DJ,Topalian SL, et al. A phase I study of nonmyeloablative chemotherapyand adoptive transfer of autologous tumor antigen-specific T lympho-cytes in patients with metastatic melanoma. J Immunother 2002;25:243–51.

3. Pilon-Thomas S, Kuhn L, Ellwanger S, Janssen W, Royster E, Marzban S,et al. Efficacy of adoptive cell transfer of tumor-infiltrating lymphocytesafter lymphopenia induction for metastatic melanoma. J Immunother2012;35:615–20.

4. Radvanyi LG, Bernatchez C, Zhang M, Fox PS, Miller P, Chacon J, et al.Specific lymphocyte subsets predict response to adoptive cell therapy usingexpanded autologous tumor-infiltrating lymphocytes in metastatic mela-noma patients. Clin Cancer Res 2012;18:6758–70.

5. Besser MJ, Shapira-Frommer R, Treves AJ, Zippel D, Itzhaki O, SchallmachE, et al. Minimally cultured or selected autologous tumor-infiltratinglymphocytes after a lympho-depleting chemotherapy regimen in meta-static melanoma patients. J Immunother 2009;32:415–23.

6. Ellebaek E, Iversen TZ, Junker N, Donia M, Engell-Noerregaard L,Met €O,et al. Adoptive cell therapywith autologous tumor infiltrating lymphocytesand low-dose Interleukin-2 in metastatic melanoma patients. J Transl Med2012;10:169.

7. Virchow R. Die Krankhaften Geschwulste: Dressig Vorlesungen, gehaltenwaehrend des Wintersemesters 1862–1863 an der Universitaet zu Berlin.In: Vorlesungen ueber Pathologie. Berlin, Germany: Verlag von AugustHirschwald; 1863.

8. Ehrlich P. Experimentelle studien an maestumoren. Zeitschrift f Krebs-forschung 1907;5:59–81.

9. Coley W. The treatment of malignant tumors by repeated inoculations ofErysipelas, with a report of ten original cases. Am JMed Sci 1989;105:487–511.

10. Moore OS Jr, Foote FW Jr. The relatively favorable prognosis of medullarycarcinoma of the breast. Cancer 1949;2:635–42.

11. Brownstein S, Sheikh KM, Lewis MG. Immunological studies inpatients with malignant melanoma of the uvea. Can J Ophthalmol1977;12:16–23.

12. Nairn RC. Mechanisms, detection and significance of immunity to skincancer. In: McCarthy WH, editor. Melanoma and skin cancer: proceedingsof the international cancer conference. Sydney, Australia: V.C.N. Blight;1972. p. 257–71.

13. Krementz ET, Goodwin DP, Samuels MS, Hornung MO, Benes EN. Immu-nologic approaches in the management of malignant melanoma. In:McCarthy WH, editor. Melanoma and skin cancer. Sydney, Australia:V.C.N. Blight; 1972. p. 473–95.

14. Ryan R, Krementz E, Carter R. Treatment of malignant melanoma: a reviewof the Tulane experience. Melanoma and skin cancer: proceedings of theinternational cancer conference. Sydney, Australia: V.C.N. Blight; 1972.p. 461–72.

15. Clark WH Jr, From L, Bernardino EA, Mihm MC. The histogenesis andbiologic behavior of primary human malignant melanomas of the skin.Cancer Res 1969;29:705–27.

16. Clark WH Jr, Ainsworth AM, Bernardino EA, Yang CH, Mihm MC Jr, ReedRJ. The developmental biology of primary human malignant melanomas.Semin Oncol 1975;2:83–103.

17. Clark WH Jr, Mihm MC Jr. Lentigo maligna and lentigo-maligna melano-ma. Am J Pathol 1969;55:39–67.

18. Larsen TE, Grude TH. A retrospective histological study of 669 cases ofprimary cutaneous malignant melanoma in clinical stage I. 3. The relationbetween the tumour-associated lymphocyte infiltration and age and sex,tumour cell type, pigmentation, cellular atypia, mitotic count, depth ofinvasion, ulceration, tumour type and prognosis. Acta Pathol MicrobiolScand A 1978;86A:523–30.

19. Hansen MG, McCarten AB. Tumor thickness and lymphocytic infiltra-tion in malignant melanoma of the head and neck. Am J Surg 1974;128:557–61.

20. Day CL Jr, Lew RA, Mihm MC Jr, Sober AJ, Harris MN, Kopf AW, et al. Amultivariate analysis of prognostic factors for melanoma patients withlesions greater than or equal to 3.65 mm in thickness. The importance ofrevealing alternative Cox models. Ann Surg 1982;195:44–9.

21. McGovern VJ, Lane Brown M. Regression of nevi and malignant melano-ma. In: Kugelmass IN, editor. The nature of melanoma. Springfield, IL:Charles C. Thomas; 1969. p. 158–64.

22. Thompson P, editor The relationship of lymphocytic infiltration to prog-nosis in primary malignant melanoma of skin: proceedings of VIII inter-national pigment cell conference. Sydney: Blight, Government Printer;1972.

23. McGovern VJ. Spontaneous regression. In: Malignant melanoma: clin-ical and histological diagnosis. New York: Wiley & Sons Inc.; 1976.p. 86–149.

24. McGovern VJ, Mihm MC Jr, Bailly C, Booth JC, Clark WH Jr, Cochran AJ,et al. The classificationofmalignantmelanomaand its histologic reporting.Cancer 1973;32:1446–57.

25. McGovern VJ. Prognostic significance of histological features of malignantmelanoma.Melanoma: histological diagnosis and prognosis. Biopsy inter-pretation series. New York: Raven Press; 1983. p. 158–74.

26. Roenigk HH Jr, Deodhar S, St Jacques R, Burdick K. Immunotherapy ofmalignant melanoma with vaccinia virus. Arch Dermatol 1974;109:668–73.

27. Morton DL, Eilber FR, Joseph WL, Wood WC, Trahan E, Ketcham AS.Immunological factors in human sarcomas and melanomas: a rationalbasis for immunotherapy. Ann Surg 1970;172:740–9.

28. Roth JA, Grimm EA, Morton DL. A rapid assay for stimulation of humanlymphocytes by tumor-associated antigens. Cancer Res 1976;36:3001–10.

29. Eilber FR, Townsend CM Jr, Morton DL. Results of BCG adjuvant immu-notherapy formelanomaof the head andneck. Am J Surg 1976;132:476–9.

30. Dvorak AM, MihmMC Jr, Osage JE, Dvorak HF. Melanoma. An ultrastruc-tural study of the host inflammatory and vascular responses. J InvestDermatol 1980;75:388–93.

31. Clark WH Jr, Elder DE, Guerry Dt, Braitman LE, Trock BJ, Schultz D, et al.Model predicting survival in stage I melanoma based on tumor progres-sion. J Natl Cancer Inst 1989;81:1893–904.

32. Clemente CG, Mihm MC Jr, Bufalino R, Zurrida S, Collini P, CascinelliN. Prognostic value of tumor infiltrating lymphocytes in the verticalgrowth phase of primary cutaneous melanoma. Cancer 1996;77:1303–10.

33. Mihm MC Jr, Clemente CG, Cascinelli N. Tumor infiltrating lympho-cytes in lymph node melanoma metastases: a histopathologic prognos-tic indicator and an expression of local immune response. Lab Invest1996;74:43–7.

34. Clemente C, Rao S, Lupetti R, Tragni G, Pisarra P, Bersani I, et al. Immu-nohistochemical analysis of the T-cell receptor beta-chain variable regionsexpressed by T lymphocytes infiltrating primary human melanoma. LabInvest 1998;78:619–27.

35. Rao UN, Lee SJ, Luo W, Mihm MC Jr, Kirkwood HM. Presence oftumor-infiltrating lymphocytes and a dominant nodule within pri-mary melanoma are prognostic factors for relapse-free survival ofpatients with thick (t4) primary melanoma: pathologic analysis ofthe e1690 and e1694 intergroup trials. Am J Clin Pathol 2010;133:646–53.

36. Soiffer R, Lynch T, Mihm M, Jung KJ, Rhuda C, Schmollinger JC, et al.Vaccination with irradiated autologous melanoma cells engineered tosecrete human granulocyte–macrophage colony-stimulating factor gener-ates potent antitumor immunity in patients with metastatic melanoma.Proc Natl Acad Sci U S A 1998;95:13141–6.

37. Hodi FS, Butler M, Oble DA, Seiden MV, Haluska FG, Kruse A, et al.Immunologic and clinical effects of antibody blockade of cytotoxic Tlymphocyte-associated antigen 4 in previously vaccinated cancer patients.Proc Natl Acad Sci U S A 2008;105:3005–10.

38. Schatton T, Scolyer RA, Thompson JF, Mihm MC Jr. Tumor-infiltratinglymphocytes and their significance in melanoma prognosis. Methods MolBiol 2014;1102:287–324.

39. Cohen AD, Schaer DA, Liu C, Li Y, Hirschhorn-Cymmerman D, Kim SC,et al. Agonist anti-GITR monoclonal antibody induces melanoma tumorimmunity in mice by altering regulatory T cell stability and intra-tumoraccumulation. PLoS One 2010;5:e10436.

40. Oble DA, Loewe R, Yu P, Mihm MC Jr. Focus on TILs: prognostic signif-icance of tumor infiltrating lymphocytes in human melanoma. CancerImmun 2009;9:3.

41. Thomas NE, Busam KJ, From L, Kricker A, Armstrong BK, Anton-Culver H,et al. Tumor-infiltrating lymphocyte grade in primary melanomas isindependently associated with melanoma-specific survival in the popula-tion-based genes, environment and melanoma study. J Clin Oncol2013;31:4252–9.


Mihm and Mul�e



42. Azimi F, Scolyer RA, Rumcheva P, Moncrieff M, Murali R, McCarthy SW,et al. Tumor-infiltrating lymphocyte grade is an independent predictor ofsentinel lymph node status and survival in patients with cutaneousmelanoma. J Clin Oncol 2012;30:2678–83.

43. Cintolo JA, Gimotty P, Blair A, Guerry D, Elder DE, Hammond R, et al.Local immune response predicts survival in patients with thick (t4)melanomas. Ann Surg Oncol 2013;20:3610–7.

44. Zlotnik A, Yoshie O. Chemokines: a new classification system and theirrole in immunity. Immunity 2000;12:121–7.

45. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL,et al. Colocalization of inflammatory response with B7-h1 expres-sion in human melanocytic lesions supports an adaptive resis-tance mechanism of immune escape. Sci Transl Med 2012;4:127ra37.

46. Flacher V, Tripp CH, Mairhofer DG, Steinman RM, Stoitzner P, IdoyagaJ, et al. Murine Langerinþ dermal dendritic cells prime CD8þ T cellswhile Langerhans cells induce cross-tolerance. EMBO Mol Med 2014;6:1638.

47. Shu S, Cochran AJ, Huang RR,MortonDL,Maecker HT. Immune responsesin the draining lymph nodes against cancer: implications for immuno-therapy. Cancer Metastasis Rev 2006;25:233–42.

48. Rollins BJ. Inflammatory chemokines in cancer growth and progression.Eur J Cancer 2006;42:760–7.

49. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow?Lancet 2001;357:539–45.

50. Mantovani A, Locati M. Tumor-associated macrophages as a paradigm ofmacrophage plasticity, diversity, and polarization: lessons and open ques-tions. Arterioscler Thromb Vasc Biol 2013;33:1478–83.

51. Mule JJ, Jones FR, Hellstrom I, Hellstrom KE. Selective localization ofradiolabeled immune lymphocytes into syngeneic tumors. J Immunol1979;123:600–6.

52. Mule JJ, Hellstrom I,HellstromKE.Cell surface phenotypes of radiolabeledimmune long-lived lymphocytes that selectively localize in syngeneictumours. Am J Pathol 1982;107:142–9.

53. Messina JL, Fenstermacher DA, Eschrich S, Qu X, Berglund AE, Lloyd MC,et al. 12-Chemokine gene signature identifies lymphnode-like structures inmelanoma: potential for patient selection for immunotherapy? Sci Rep2012;2:765.

54. Coppola D, Mule JJ. Ectopic lymph nodes within human solid tumors.J Clin Oncol 2008;26:4369–70.

55. Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, et al.Chemokine expression in melanoma metastases associated with CD8þT-cell recruitment. Cancer Res 2009;69:3077–85.

56. Hong M, Puaux AL, Huang C, Loumagne L, Tow C, Mackay C, et al.Chemotherapy induces intratumoral expression of chemokines in cutane-ous melanoma, favoring T-cell infiltration and tumor control. Cancer Res2011;71:6997–7009.

57. van Baren N, Baurain JF, Coulie PG. Lymphoid neogenesis in melanoma:What does it tell us? Oncoimmunology 2013;2:e22505.

58. Cipponi A,MercierM, Seremet T, Baurain JF, Theate I, van denOord J, et al.Neogenesis of lymphoid structures and antibody responses occur inhuman melanoma metastases. Cancer Res 2012;72:3997–4007.

59. Ladanyi A, Sebestyen T,Mohos A, Liszkay G, Somlai B, Toth E, et al. Ectopiclymphoid structures in primary cutaneous melanoma. Pathol Oncol Res2014;20:981–5.

60. Wilmott JS, LongGV,Howle JR, Haydu LE, Sharma RN, Thompson JF, et al.Selective BRAF inhibitors induce marked T-cell infiltration into humanmetastatic melanoma. Clin Cancer Res 2012;18:1386–94.

61. Wilmott JS, Scolyer RA, LongGV,Hersey P. Combined targeted therapy andimmunotherapy in the treatment of advancedmelanoma. Oncoimmunol-ogy 2012;1:997–9.

62. Frederick DT, Piris A, Cogdill AP, Cooper ZA, Lezcano C, Ferrone CR,et al. BRAF inhibition is associated with enhanced melanoma anti-gen expression and a more favorable tumor microenvironmentin patients with metastatic melanoma. Clin Cancer Res 2013;19:1225–31.

63. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al.PD-1 blockade induces responses by inhibiting adaptive immune resis-tance. Nature 2014;515:568–71.

64. Grimaldi AM, Simeone E, Giannarelli D, Muto P, Falivene S, Borzillo V,et al. Abscopal effects of radiotherapy on advanced melanoma patientswho progressed after ipilimumab immunotherapy. Oncoimmunology2014;3:e28780.

65. Postow MA, Callahan MK, Barker CA, Yamada Y, Yuan J, Kitano S, et al.Immunologic correlates of the abscopal effect in a patient withmelanoma.N Engl J Med 2012;366:925–31.

66. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ,et al. Durable complete responses in heavily pretreated patients withmetastatic melanoma using T-cell transfer immunotherapy. Clin CancerRes 2011;17:4550–7.


TILs in Melanoma



2015;3:827-835. Cancer Immunol Res Martin C. Mihm, Jr and James J. Mulé Lymphocytes in Melanoma and the Host Immune ResponseReflections on the Histopathology of Tumor-Infiltrating

Updated version

http://cancerimmunolres.aacrjournals.org/content/3/8/827

Access the most recent version of this article at:

Cited articles

http://cancerimmunolres.aacrjournals.org/content/3/8/827.full#ref-list-1

This article cites 58 articles, 19 of which you can access for free at:

Citing articles

http://cancerimmunolres.aacrjournals.org/content/3/8/827.full#related-urls

This article has been cited by 6 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerimmunolres.aacrjournals.org/content/3/8/827To request permission to re-use all or part of this article, use this link



http://cancerimmunolres.aacrjournals.org/content/3/8/827.full#ref-list-1

http://cancerimmunolres.aacrjournals.org/content/3/8/827.full#related-urls

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]



reﬂections on the histopathology of tumor- ﬁltrating ...precursor of melanoma, lentigo maligna,...

Documents