Annals of Oncology Advance Access published November 16, 2015 1
Strategies for clinical implementation of TNM-immunoscore in resected non-small cell lung cancer
T. Donnem1,2, T.K. Kilvaer1, S. Andersen1, E. Richardsen3,4, EE. Paulsen1,2, S. M. Hald2, S. Al-Saad3,4, O.T. Brustugun5, A. Helland5,6, M. Lund-Iversen7, S. Solberg8, B.H. Gronberg9,10, S.G.F. Wahl11, L. Helgeland12, O. Fløtten13, M. Pohl14, K. Al-Shibli15, T.M. Sandanger16, F. Pezzella17, LT. Busund3,4,†, R. M. Bremnes1,2,†
Department of Oncology, University Hospital of North Norway, Norway
2
Institute of Clinical Medicine, The Arctic University of Norway, Norway
3
Department of Clinical Pathology, University Hospital of North Norway, Norway
4
Institute of Medical Biology, The Arctic University of Norway, Norway
5
Department of Oncology, Oslo University Hospital, The Norwegian Radium Hospital, Oslo,
Norway 6
Department of Cancer Genetics, Oslo University Hospital, The Norwegian Radium Hospital,
Oslo, Norway 7
Department of Pathology, Oslo University Hospital, The Norwegian Radium Hospital, Oslo,
Norway 8
Department of Cardiothoracic Surgery, Oslo University Hospital, Rikshospitalet, Oslo,
Norway 9
The Cancer Clinic, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway
10
European Palliative Care Research Centre, Department of Cancer Research and Molecular
Medicine, Norwegian University of Science and Technology, Trondheim, Norway 11
Department of Pathology and Medical Genetics, St. Olavs Hospital - Trondheim University
Hospital, Trondheim, Norway © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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1
2 12
Department of Pathology, Haukeland University Hospital, Bergen, Norway
13
Department of Thoracic Medicine, Haukeland University Hospital, Bergen, Norway
14
Department of Oncology, Rigshospitalet, Copenhagen, Denmark
15
Department of Pathology, Nordland Hospital, Bodo, Norway
16
Department of Community Medicine, The Artic University of Tromso, Norway
17
Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe
Hospital, Oxford, UK
Oncology, The Arctic University of Norway / University Hospital of North Norway, 9038 Tromso, Norway, Telephone: +47 77645427 / +47 77626000, Fax: +47 77626779, E-mail: [email protected] †These
authors have contributed equally to this paper.
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Corresponding author: Professor Tom Donnem, Institute of Clinical Medicine / Department of
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ABSTRACT
Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates, and appears highly promising as a supplement to the TNM classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor infiltrating lymphocytes (TILs) have
potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In non-small cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC.
Key Words: Immunoscore, T-cells, TNM-I, NSCLC, lung cancer
Key message: This Review focuses on the TNM-Immunoscore (TNM-I) potential in nonsmall cell lung cancer (NSCLC) and proposes strategies for clinical implementation of a TNM-I in resected NSCLC.
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been proposed by an international working group. Regardless of promising results, there are
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Introduction Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide [1]. In addition, lung cancer affects also younger patients, and is responsible for as many lost lifeyears as colorectal, breast and prostate cancers combined [2]. Lung cancer is divided in smallcell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), the latter representing approximately 85% of all lung cancer patients [3]. About 20-25% of NSCLC are resectable (stage I-IIIA) at the time of diagnosis and potentially curable. There is a considerable
current 7th edition of the lung cancer staging system based on an initiative undertaken by the International Association for the Study of Lung Cancer (IASLC) [4]. In the TNM staging system there is, however, evidently room for improvement as clinical outcome vary significantly among patients with the same pathological stage (pStage). In resected breast and colorectal cancer patients there is a similar picture. The cancers may be potentially curable, but whereas the TNM classification is important in clinical decision making, the outcomes vary significantly within each TNM stage. Common for all these malignancies is the present interest in the immune contexture and the influence of tumor infiltrating immune cells on clinical outcome [5,6]. Different types of infiltrating immune cells have diverse effects on tumor progression and clinical outcome according to context and cancer type [5]. Interestingly, the most consistent positive prognostic impact has been shown for T-cells, especially cytotoxic T-cells, memory T-cells and T helper cells-1 [6]. Two commendable NSCLC-specific reviews on this topic were recently published [7,8]. These papers discuss the prognostic impact of different immune cells and the prognostic and potential predictive impact of immune checkpoint T-cell markers as programmed death -1 (PD1), PD-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4).
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difference in prognosis between stage I and stage IIIA patients (Figure 1) as shown in the
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In colorectal cancer, an impressive diagnostic development has been achieved by using assessment of infiltrating immune T cells in cancer tissue to create an “Immunoscore” as an essential important supplement to TNM, designated TNM-Immune (TNM-I) [9-11]. A worldwide task force has ongoing prospective multicenter studies aiming to implement TNMI in clinical decision making [9-11]. A similar international working group has evaluated the role of tumor infiltrating lymphocytes (TILs) in breast cancer and recently published their recommendations [12]. However, as shown in Table 1, there are major differences in the colorectal and the breast cancer approach. The diverse strategies in colorectal cancer and
specific. On the other hand, it may also reflect that there are several ways of evaluating potentially similar mechanisms. Further efforts to standardize and optimize the TNM-I are therefore warranted. In NSCLC, many comprehensive studies have evaluated the prognostic impact of TILs (Table 2). However, to our knowledge only one recently published study has focused on TNM-I in a NSCLC setting where the difference in prognostic impact across each pStage is emphasized [13]. A prospective multicenter NSCLC TNM-I study is planned and central issues with regard to implementing TNM-I into clinical decision making are discussed below.
Immune cell markers of interest Interestingly, the task force in colorectal cancer recommends specific TIL immune markers (CD3 and CD8), while the breast cancer working group endorses hematoxylin & eosin (H&E) staining and morphological evaluation of TILs for their immunoscore [9,11,12]. In colorectal cancer, CD45RO have also shown promising results, but due to background staining and loss of antigenicity in stored sections it was decided to use the two most successful stainings (CD3 and CD8) in the ongoing prospective multicenter colorectal study [11].
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breast cancer indicate that that the prognostic impact of TILs may be cancer type and context
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Based on experiences from colorectal cancer and breast cancer, CD3, CD8, CD45RO positive cells as well as TILs evaluated by H&E staining and morphology has been the focus in the search for candidate markers in NSCLC. CD4 has also been included as it has shown prognostic impact in NSCLC in several studies. CD4 T-helper cells differentiate into Th1, Th2, Th17 and regulatory T-cells (Tregs). Several studies have assessed prognostic roles of these T cell markers in lung cancer [6,14], but due to conflicting prognostic impact in both lung cancer and in several other malignancies, these have not been included in this review [6]. Using the following search criteria: i) Stage I-IIIA NSCLC patients, ii) studies published
assessments of TILs, we identified 15 eligible studies (Table 2) [13,15-28]. Five of these included CD3 [15,21,22,26,28], four CD4 [16,22,27,28], 11 CD8 [13,16-18,20-22,24,26-28], one CD45RO [28] and four evaluating TILs morphologically by H&E staining [19,21,23,25]. The most promising candidate marker so far seems to be CD8. The results are quite consistent showing that a high number of infiltrating CD8+ TILs is a positive prognostic factor regardless of tissue compartments evaluated, staining/scoring methods or endpoints used. There is some variation in the statistical strength (from being a strong independent prognostic variable to show significance in only univariate analyses or just indicating a trend), but none of the examined studies did show a negative prognostic impact of CD8+ TILs. A large comprehensive study by Suzuki et al did not show any prognostic trend for CD8, but they included exclusively NSCLC adenocarcinomas stage I [28]. Reassuringly, the two most recent studies which included both test and validation cohorts did show a strong independent positive prognostic impact of CD8+ TILs [13,26]. To our knowledge, merely our recent study have included the results in a TNM-I context. As presented in Figure 1, the stromal CD8+ density adds valuable additional prognostic impact within each pathological stage (pStage) [13].
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between 2000- and March 2015, iii) more than 200 included patients and iv) in situ
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Another promising marker is CD3. As a pan T-cell marker it is not surprising that a high CD3+ TILs density would indicate a positive prognostic impact in the same way as evaluating TILs by H&E staining and morphology. CD4 T-helper cells show basically the same results as CD3 and CD8, but may be considered unspecific as it stains both regulatory (Tregs) and tumor suppressing T cells. Nevertheless, as a prognostic factor it is promising. To our knowledge CD45RO (memory T-cells) is only evaluated in one large NSCLC study by Suzuki et al.[28]. As this study included stage I adenocarcinomas patients only, and it showed highly significant impact in colorectal cancer, CD45RO deserves to be further elucidated in
TIL location and compartments within the tumor The presence of TILs related to compartments within the tumor is frequently a matter of debate and the terminology used is often ambiguous. Essentially, compartments mentioned are either intra-epithelial (often named “tumor” or “cancer nests”) or stromal (called “intratumoral stroma” or “tumor-related stroma”). These compartments may be located in the center or/and at the invasive margin of the tumor. In addition, lymphocytes may be located in adjacent tertiary lymphoid structures [6,16]. In colorectal cancer, the TNM-I score differentiates between central tumor and invasive margin, but the selected areas for TIL assessment (both in the central tumor and in the invasive margin) includes lymphocytes from both intra-epithelial and stromal compartments [9-11]. The breast cancer recommendations endorse solely the stromal compartment (i.e. area occupied by mononuclear inflammatory cells over the total intratumoral stroma) within the borders of the invasive margin [12]. In NSCLC prognostic studies (Table 2), the compartment of interest has often been described as intra-epithelial, stromal or a combination of these two. In some cases the intra-
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NSCLC [29,30].
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epithelial and the stromal compartment have been evaluated separately. However, whether the calculated lymphocytes are located in the invasive margin or in the central parts of tumor is generally not described. In our study [13], we considered the same approach as Galon et al. as both intra-epithelial and stromal CD8+ expression turned out as positive prognostic factors in our test cohort, but the stromal expression alone showed a better prognostic impact than the combination of these two closely related compartments. Planning a prospective study, we are considering using the total expression for the simplicity and potentially easier use of computer based analysis. Basically, we have to establish an approach fitting the NSCLC biology as well
work by Galon et al. To our knowledge, only our recently published study has addressed the issue of central tumor versus invasive margin in NSCLC TNM-I [13]. In one of our cohorts the localization of each tissue core was clearly noted and, interestingly, the prognostic impact of stromal CD8+ TILs appeared to be strongest when located in the invasive margin. When it comes to evaluation of different compartments (e.g. intraepithelial versus stromal) Schalper et al. has taken this issue a step further by using multiplexed quantitative fluorescence (QIF) [26]. QIF score of each fluorescence channel in the tumor and/or stromal compartment was calculated by dividing the target TIL marker pixel intensity by the area of desired compartment defined by the cytokeratin positivity (i.e. intra-epithelial compartment), absence of cytokeratin positivity (i.e. stromal compartment) or DAPI positive cells (e.g. including both intraepithelial and stromal compartments). They advocate for this method and state that differentiating between intra-epithelial and stromal staining is pivotal in optimizing the assessment of the prognostic impact of CD8+ TILs in NSCLC. This is in contrast to the colorectal TNM-I approach where scoring was a mix between these two compartments. Based on our findings, we believe that both approaches may have strengths and weaknesses. Our data indicate a stronger impact based on assessments in the stromal compartment for CD8+
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as possible. In this process some solutions may differ from the impressive TNM-I pioneer
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density, supporting Schalper et al, but localization (invasive margin) is definitively also an issue in NSCLC TNM-I. Combining these strategies may be a sound solution, but further research is warranted to clarify these important questions.
Tissue microarrays versus whole slides in the assessment of TNM-I Closely related to the compartment/localization issue is the debate whether tissue microarrays (TMAs) are adequately representative or inferior to whole slides when it comes to reflecting the infiltration of lymphocytes in the tumor. The colorectal world task force argues that for
well as the invasive part. However, they are evaluating a total of only four selected areas within the whole slide, not unlike a TMA approach. In the breast cancer recommendations they also initially preferred whole slides morphological assessment (H&E staining), but concluded that TMA may be a good option for future studies, particularly for the rapid evaluation of large cohorts using immunohistochemistry based assessments of TILs subsets. More research on this issue is needed in NSCLC. Several studies have shown that TMA results largely correlate with whole slide findings [31]. The numbers of cores, their size, and the localization they are taken from are of pivotal importance. It is, however, worth mentioning that in these correlation studies, the gold standard is usually whole slides. As a tumor is three- and not two-dimensional, one may ask how representative is a slide for the whole tumor. This is a highly relevant question also in a TNM-I setting and needs further attention. Another relevant matter is the thickness of the tissue sections. In cases where the score is based on a ratio between an actual marker and the number of other nucleated cells, this is not a problem. However, when the score is based on the number of marked cell per area this
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routine practice purposes whole slides is favorable as it includes areas from both the center as
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may be challenging. Most slides are 4-µm or 5-µm thick, but even this minor difference will in average lead to a result discrepancy of about 20 percent.
Challenges related to scoring – pathologist or computer? In colorectal cancer, Galon et al. advocate the use of digital scoring as computer-assisted image analyses provides important advantages [9,11]. Due to the fact that validation studies have demonstrated high concordance between digital and manual scoring the authors state that digital scoring is pivotal to facilitate routine pathology and to speed up the process of
approaches, while promising, have not been published in large series with consistent methodology and, hence, is an important area for further research. Digital imaging may further improve the reproducibility in the assessment of percentages of immune positive area compared to visual semiquantitative estimation. This is also the key message in a published study evaluating TILs in colorectal cancer, focusing on this issue [32]. However, in the same paper the authors also state that the semiquantitative scoring showed an excellent reproducibility (T-cell markers CD3 and FoxP3 were evaluated) and conclude that computer-assisted image analysis provides a valuable alternative to semiquantitative assessments in immunohistochemically stained sections. It is important to note that manual exact count is the gold standard that both the semiquantitative and the computer-based analysis are compared against. As mentioned, digital scoring analysis is less time-consuming when finally established. This is the case when the area to be evaluated is well defined and the computer counts the number of stained cells. In NSCLC, stromal and intra-epithelial compartments are very closely related. Consequently, evaluation of only one of these compartments is regarded challenging. Hence, in a digital setting a pathologist will manually have predefined the correct
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quantification. In contrast, the breast cancer recommendations conclude that digital scoring
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compartments. Thus in a prospective study one may as Galon et al. consider not to evaluate the TIL markers in the intra-epitelial and stromal compartment separately. Several wellperformed NSCLC studies have already used a computer-based scoring approach when evaluating TIL specific markers [17,18,20,24,26], and the previously mentioned comprehensive study by Schalper et al. addressed the issue of different compartments (intraepithelial versus stromal). Their method seems promising and further testing with a digital approach in prospective studies is needed before concluding on this matter. A major advantage with digital analyses (as for the more time-consuming exact count)
multivariate prediction models without any cut-offs. Continuous data are also much more flexible in an explorative study where cut-off is to be decided. Median, percentiles and “best p-value” are frequently used cut-off strategies, all with potential strengths and weaknesses. In any case, cut-offs must be predefined and validated in several cohorts prior to entering a prospective multicenter study and, finally, potentially be used in NSCLC TNM-I clinical decision making.
Future directions There are challenges related to translating promising prognostic immunoscore results into a TNM-I clinical setting. For the time being the world task force in colorectal cancer seems to be closest in reaching such a goal. The breast cancer working group recently has published some general recommendations. They acknowledge the immense complexity of lymphocyte assessment and suggest that closer molecular characterization of the TIL infiltrates may add both sensitivity and specificity to the predictive value of morphologically recognized TILs [12]. In NSCLC, the interest for TILs as a prognostic tool is increasing, but a shift from
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compared to a semi quantitative score is that a continuous data variable can be used directly in
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promising retrospective studies to larger prospective multicenter studies with focus on clinical use is warranted. So far stromal CD8 seems to be the most promising candidate in NSCLC as several studies have shown its positive prognostic impact and the fact that additional impact can be clearly demonstrated across each pStage (Figure 1). In addition CD3, CD4 and CD45RO are potentially candidate markers to be included in a prospective study, but validated results on the prognostic impact of these markers according to pStage are so far sparse. As there is a close connection between these markers and mechanisms related to new
checkpoint inhibitors targeting CTLA-4/PD1/PD-L1 are highly in need of valid predictive markers. PD-L1 expression by immunohistochemistry (IHC) has been found to predict response to PD1 and PD-L1 inhibitors, but patient with a low PD-L1 score may still respond to these antibodies [7]. As the TNM-I is a pathological TNM-I (pTNM-I), it may only be used as a predictor of treatment response in a postoperative setting. Yet, evaluating TNM-I as a predictive tool in an adjuvant immunotherapy setting is highly interesting. Nevertheless, both as a prognostic and potentially as a predictive tool, the IHC procedure and the scoring systems need to be standardized. The greatest challenge may be the heterogeneity of TILs in the tumor. Besides, the reproducibility and reliability have to be significantly improved as we move from predicting survival in large cohorts to testing sensitivity and specificity at a patient level. However, as the prognostic impact of TILs in NSCLC is promising the aim should be to develop a clinically useful NSCLC TNM-I by refining the current knowledge combined with prospective multicenter studies.
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targeted immunotherapy, there is a potential predictive value of TNM-I as well. Immune
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Acknowledgements We want to thank the Norwegian Cancer Society and Northern Norway Health Region Authority for financial support. Conflict of interest No conflict of interest.
Figure 1. From TNM to TNM-Immunoscore (TNM-I) in Non-Small Cell Cancer. The American Joint Committee of Cancer (AJCC) and the Union Internationale Contre le Cancer (UICC) define and periodically update the TNM classification system. In lung cancer, the current 7th edition of the lung cancer staging system is based on an initiative undertaken by the International Association for the Study of Lung Cancer (IASLC). (A) Five-year overall survival (OS) rates in 797 resected, stage I-IIIA, NSCLC patients from four different Scandinavian cohorts is comparable to IASLC data [4,13]. The combination of stromal CD8 expression (B) and pathological stage (C) survival data [13] results in a TNM-Immunoscore (D) adding significant prognostic impact across each pathological stage.
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Legend of figure
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15 12. Salgado R, Denkert C, Demaria S et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol 2015; 26: 259-71. 13. Donnem TP, Hald SM, Paulsen EE et al. Stromal CD8+ T Cell Density - A Promising Supplement to TNM staging in Non-Small Cell Lung Cancer. Clin Cancer Res 2015; 21: 2635-43. 14. Duan MC, Zhong XN, Liu GN et al. The Treg/Th17 paradigm in lung cancer. J Immunol Res 2014; 2014: 730380. 15. Al-Shibli K, Al-Saad S, Andersen S et al. The prognostic value of intraepithelial and stromal
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25. Ruffini E, Asioli S, Filosso PL et al. Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms. Ann Thorac Surg 2009; 87: 365-71. 26. Schalper KA, Brown J, Carvajal-Hausdorf D et al. Objective measurement and clinical significance of TILs in non-small cell lung cancer. J Natl Cancer Inst 2015; 107. 27. Sterlacci W, Wolf D, Savic S et al. High transforming growth factor beta expression represents an important prognostic parameter for surgically resected non-small cell lung cancer. Hum Pathol 2012; 43: 339-49. 28. Suzuki K, Kadota K, Sima CS et al. Clinical impact of immune microenvironment in stage I lung adenocarcinoma: tumor interleukin-12 receptor beta2 (IL-12Rbeta2), IL-7R, and stromal FoxP3/CD3 ratio are independent predictors of recurrence. J Clin Oncol 2013; 31: 490-8. 29. Pages F, Kirilovsky A, Mlecnik B et al. In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer. J Clin Oncol 2009; 27: 5944-51. 30. Pages F, Berger A, Camus M et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med 2005; 353: 2654-66. 31. Kallioniemi OP, Wagner U, Kononen J et al. Tissue microarray technology for high-throughput molecular profiling of cancer. Hum Mol Genet 2001; 10: 657-62.
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Table 1. At a glance - recommondations TNM-Imunnoscore (TNM-I) in colorectal cancer and breast cancer and status in non-small cell lung cancer (NSCLC) TNM-I. Breast cancer [12]
NSCLC
Lymphocyte markers
CD3 and CD8
Tumor infiltrating lymphocytes, TILs by H&E staining. All mononuclear cells should be stained
Stromal CD8 most conclusive candidate so far. But further studies needed on promising T-cell markers as CD3, CD4, CD45RO
Compartment
Both stromal and intraepithelial CD8 and CD3 positive cells included
Stromal compartment
Both stromal and intraepithelial compartment is promising. Potentially a combination of both
Invasive margin versus tumor center
Both invasive margin and central areas included
Borders of the invasive margin should be evaluated
Only one study has addressed this issue. For stromal CD8, invasive margin looks most promising
Score
Score based on a combination of CD3 and CD8 score in both central tumor and invasive margin
TILs should be assessed as a continuous parameter. No formally recommendation for a clinically relevant threshold(s) can be given at this stage
Too premature to conclude. Stromal CD8 looks promising – see Figure 1
TMA versus whole slides
Whole slides recommended, but selected areas from invasive margin and central tumor are scored
Full sections are preferred over biopsies whenever possible, but cores may be used in pre-therapeutic neoadjuvant setting
Promising results based on TMA, but whole slides may be preferred to better address localization - central tumor versus invasive margin
Manual versus digital scoring
Digital (computer based) scoring recommended
Manual scoring ok, digital scoring important in further research
Manual scoring shows clearly significant results across each pStage, but digital scoring may be more precise and less timeconsuming when established
Continuous versus semiquantitative variable
Scored as a continuous variable, but redefined as a semiquantitative variable
Ideally should be scored as a continuous variable (% stromal TILs). However, pathologists should report their scores in as much detail as the pathologist feel comfortable with
Semiquantitative scoring seems promising, but continuous variable may be preferred in the scoring to best decide cut-off
Formally recommended threshold
Immunscore established – score 1 to 4 based on the above mentioned combination
No
No
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Colorectal cancer [9, 11]
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Table 2. Characteristics of non-small cell cancer studies between 2000 and March 2015 with focus on in situ assessment of tumor infiltrating lymphocytes. Study
Year
N
Histology
pStage
Marker
Compartment
Invasive margin vs. tumor center addressed Yes
TMA vs. WS
- Scoring approach - Digital versus manual scoring - Cut-off
Donnem T et al [13]
2015
797
ADC, SCC, LCC
I-IIIA
CD8
Stromal
TMA
“Representative intratumoral areas” (mix intra-epithelial and stromal). Intra-epithelial, stromal and total.
No
TMA
- Percentages CD8+ / total amount of nucleated cells - Manual - Predefined three-tiered cut-off - CD8+ TILs per unit area (mm2) - Digital - Median cut-off
Kim MY et al [24]
2015
331
SCC
I-III
CD8, PD1, PDL1
Schalper KA et al [26]
2015
552
ADC, SCC, Other
I-IV
CD3, CD8, CD20
No
TMA
Alifano M et al [17]
2014
303
ADC, SCC, LCC, Others
I-IV
CD8, CD1A
Tumor nests (intra-epithelial) and stromal
No
WS
Goc J et al [18]
2014
458
ADC, SCC, Others
I-IV
DC, CD8
Tumor nests (intra-epithelial) and stromal
No
WS
Suzuki K et al [28]
2013
956
ADC
pStage I only
Tumor nests (intra-epithelial) and stromal
No
TMA
ADC, SCC, LCC
I-IV
FoxP3/C D3 ratio, CD4,CD8, CD45RO CD3, CD3/CD8 CD4/ CD25
Kayser G et al [22]
2012
232
Intra-epithelial and stromal
No
TMA
- Quantitative measurement of fluorescent signal (QIF) calculated by dividing the target TIL marker pixel intensity by the area of desired compartment. - Digital and manual - Cut-off: tertiles and categorized - Intra-tumoral density of CD8+ T-cells (Both stromal area and tumor nests gave significant associations, but they decided to report findings only from tumor cells nests) - Digital - “Minimum p-value approach”. Cut-off: 96 cells/mm2 - Absolute number positive cells/mm2 - Digital and manual -“Minimum p-value approach” Cut-off: Tumor nests: 114 cells/mm2 Stroma: 383 cells/ mm2 - Marked cells/field - Manual - Three-tiered score categorized in high and low - The absolute number of each IHC detectable subgroup/core - Manual - Median cut-off
Validation cohort
Prognostic impact (PI)
PI across each stage/TNM addressed Yes Significant within each pStage No
Yes
Independent positive PI
No
Significant positive PI in univariate with DFS as endpoint
Yes
CD8 independent positive PI in both cohorts. CD3 independent positive in one cohort
No
No
CD8 independent positive PI in total material and combined stage I-II
Partly
No
Both intra-epithelial and stromal CD8 significant positive PI in univariate
No
Yes
No marker significant PI in univariate
No
Stromal CD3 positive PI in univariate. Stromal CD4/CD25 independent PI
Included pStage I patients only No
Year
N
Histology
pStage
Marker
Compartment
Invasive margin vs. tumor center addressed
TMA vs. WS
- Scoring - Digital vs. manual - Cut-off
Ilie M et al [20]
2012
632
ADC, SCC, LCC, Others
I-III
CD8, CD8/ CD66b ratio
Intratumoral (mix Intraepithelial and stromal)
Central areas of the tumor
TMA
Sterlacci et al [27]
2012
383
I-IV
CD4 CD8
Intra-epithelial
No
TMA
Horne ZD et al [19]
2011
273
ADC, SCC, LCC ADC, SCC, LCC
1A
TILs
Combination tumor nests (intra-epithelial) and stromal
No
WS
Kilic A et al [23]
2011
219
ADC, SCC, Other
1A-1B
TILs
No
WS
Al-Shibli et al [15]
2010
335
ADC, SCC, LCC
I-IIIA
CD3, CD117, CD 138
Combination tumor nests (intra-epithelial) and stromal Intra-epithelial and stromal
No
TMA
Ruffini et al [25]
2009
1290
ADC, SCC, LCC, Others
I-IIIA
TILs
Intra-epithelial
No
WS
Al-Shibli et al [16]
2008
335
ADC, SCC, LCC
I-IIIA
CD8, CD4
Intra-epithelial and stromal
No
TMA
Johnson SK et al [21]
2000
710 (95)
I-III
TILs CD3 CD8
Intra-epithelial and stromal
No
WS
- Density as the number of positive cells per unit of tissue surface area - Digital - Median cut-off. The median ± SD was 110 ± 142 cells - Number of positive cells/area - Manual - Median cut-off - Subjective low (scattered), moderate and high (intense) TIL infiltration - Manual - Cut-off: TIL - : low infiltration, TIL+: modest and high infiltration - “Routine protocol by an expert academic pathologist; group 1, none to mild, or group 2, moderate to serve infiltrate” - Manual - Stroma, high if they represented > 50% of nucleated cells in stroma. Intraepithelial, high if they represented more than 1% of nucleated cells -Manual - TILs were evaluated morphologically, only in a subset of 21 patients IHC - Manual - Cut-off: Low: < 20% of cells, high: ≥ 20% of cells - Percentages marker / total amount of nucleated cells - Manual - Cut-off: CD4, intraepithelial ≥ 5% , CD4, stromal ≥ 25%, CD8, intraepithelial > 5%, CD8, stromal > 50% - In 710 patients: Morphologically TIL by H&E staining. Semiquantitative score. In subset of 95 patients: CD3 and CD8, intratumoral, low if < 10%, high if ≥ 10% cells per high power field - Manual and digital
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Study
Validation cohort
Prognostic impact (PI)
PI across each stage/TNM addressed
No
CD8 not significant PI in univariate. CD66b/CD8 ratio independent positive PI CD4/CD8 ratio positive PI for ADC, in stage IA only Significant positive PI with recurrence free survival as endpoint
No
No
Positive PI in univariate
Stage IA and IB only
No
Stromal CD3 independent positive PI. Stromal CD3 PI in pStage IIIA, trend in I and II Independent positive PI in the total cohort and in SCC. Univariate: pStage I, SCC Stromal CD4 and CD8 independent positive prognostic factors. Intraepithelial PI in univariate. TIL total cohort not significant. Subset 95 patients, intraepithelial CD3 positive PI in univariate
Partly
No
No
No
No
No
Partly
Stage IA only
Partly
No
No
Abbreviations: SCC, squamous cell carcinoma; LCC, large-cell carcinoma; ADC, adenocarcinoma; TILs, tumor infiltrating lymphocytes; PD1, programmed death -1; PDL1, PD-ligand 1; TMA, tissue microarrays; WS, whole slides; N, number of patients; PI, prognostic impact; DFS, disease-free survival.
Strategies for clinical implementation of TNM-immunoscore in resected non-small cell lung cancer
T. Donnem1,2, T.K. Kilvaer1, S. Andersen1, E. Richardsen3,4, EE. Paulsen1,2, S. M. Hald2, S. Al-Saad3,4, O.T. Brustugun5, A. Helland5,6, M. Lund-Iversen7, S. Solberg8, B.H. Gronberg9,10, S.G.F. Wahl11, L. Helgeland12, O. Fløtten13, M. Pohl14, K. Al-Shibli15, T.M. Sandanger16, F. Pezzella17, LT. Busund3,4,†, R. M. Bremnes1,2,†
Department of Oncology, University Hospital of North Norway, Norway
2
Institute of Clinical Medicine, The Arctic University of Norway, Norway
3
Department of Clinical Pathology, University Hospital of North Norway, Norway
4
Institute of Medical Biology, The Arctic University of Norway, Norway
5
Department of Oncology, Oslo University Hospital, The Norwegian Radium Hospital, Oslo,
Norway 6
Department of Cancer Genetics, Oslo University Hospital, The Norwegian Radium Hospital,
Oslo, Norway 7
Department of Pathology, Oslo University Hospital, The Norwegian Radium Hospital, Oslo,
Norway 8
Department of Cardiothoracic Surgery, Oslo University Hospital, Rikshospitalet, Oslo,
Norway 9
The Cancer Clinic, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway
10
European Palliative Care Research Centre, Department of Cancer Research and Molecular
Medicine, Norwegian University of Science and Technology, Trondheim, Norway 11
Department of Pathology and Medical Genetics, St. Olavs Hospital - Trondheim University
Hospital, Trondheim, Norway © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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1
2 12
Department of Pathology, Haukeland University Hospital, Bergen, Norway
13
Department of Thoracic Medicine, Haukeland University Hospital, Bergen, Norway
14
Department of Oncology, Rigshospitalet, Copenhagen, Denmark
15
Department of Pathology, Nordland Hospital, Bodo, Norway
16
Department of Community Medicine, The Artic University of Tromso, Norway
17
Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe
Hospital, Oxford, UK
Oncology, The Arctic University of Norway / University Hospital of North Norway, 9038 Tromso, Norway, Telephone: +47 77645427 / +47 77626000, Fax: +47 77626779, E-mail: [email protected] †These
authors have contributed equally to this paper.
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Corresponding author: Professor Tom Donnem, Institute of Clinical Medicine / Department of
3
ABSTRACT
Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates, and appears highly promising as a supplement to the TNM classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor infiltrating lymphocytes (TILs) have
potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In non-small cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC.
Key Words: Immunoscore, T-cells, TNM-I, NSCLC, lung cancer
Key message: This Review focuses on the TNM-Immunoscore (TNM-I) potential in nonsmall cell lung cancer (NSCLC) and proposes strategies for clinical implementation of a TNM-I in resected NSCLC.
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been proposed by an international working group. Regardless of promising results, there are
4
Introduction Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide [1]. In addition, lung cancer affects also younger patients, and is responsible for as many lost lifeyears as colorectal, breast and prostate cancers combined [2]. Lung cancer is divided in smallcell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), the latter representing approximately 85% of all lung cancer patients [3]. About 20-25% of NSCLC are resectable (stage I-IIIA) at the time of diagnosis and potentially curable. There is a considerable
current 7th edition of the lung cancer staging system based on an initiative undertaken by the International Association for the Study of Lung Cancer (IASLC) [4]. In the TNM staging system there is, however, evidently room for improvement as clinical outcome vary significantly among patients with the same pathological stage (pStage). In resected breast and colorectal cancer patients there is a similar picture. The cancers may be potentially curable, but whereas the TNM classification is important in clinical decision making, the outcomes vary significantly within each TNM stage. Common for all these malignancies is the present interest in the immune contexture and the influence of tumor infiltrating immune cells on clinical outcome [5,6]. Different types of infiltrating immune cells have diverse effects on tumor progression and clinical outcome according to context and cancer type [5]. Interestingly, the most consistent positive prognostic impact has been shown for T-cells, especially cytotoxic T-cells, memory T-cells and T helper cells-1 [6]. Two commendable NSCLC-specific reviews on this topic were recently published [7,8]. These papers discuss the prognostic impact of different immune cells and the prognostic and potential predictive impact of immune checkpoint T-cell markers as programmed death -1 (PD1), PD-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4).
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difference in prognosis between stage I and stage IIIA patients (Figure 1) as shown in the
5
In colorectal cancer, an impressive diagnostic development has been achieved by using assessment of infiltrating immune T cells in cancer tissue to create an “Immunoscore” as an essential important supplement to TNM, designated TNM-Immune (TNM-I) [9-11]. A worldwide task force has ongoing prospective multicenter studies aiming to implement TNMI in clinical decision making [9-11]. A similar international working group has evaluated the role of tumor infiltrating lymphocytes (TILs) in breast cancer and recently published their recommendations [12]. However, as shown in Table 1, there are major differences in the colorectal and the breast cancer approach. The diverse strategies in colorectal cancer and
specific. On the other hand, it may also reflect that there are several ways of evaluating potentially similar mechanisms. Further efforts to standardize and optimize the TNM-I are therefore warranted. In NSCLC, many comprehensive studies have evaluated the prognostic impact of TILs (Table 2). However, to our knowledge only one recently published study has focused on TNM-I in a NSCLC setting where the difference in prognostic impact across each pStage is emphasized [13]. A prospective multicenter NSCLC TNM-I study is planned and central issues with regard to implementing TNM-I into clinical decision making are discussed below.
Immune cell markers of interest Interestingly, the task force in colorectal cancer recommends specific TIL immune markers (CD3 and CD8), while the breast cancer working group endorses hematoxylin & eosin (H&E) staining and morphological evaluation of TILs for their immunoscore [9,11,12]. In colorectal cancer, CD45RO have also shown promising results, but due to background staining and loss of antigenicity in stored sections it was decided to use the two most successful stainings (CD3 and CD8) in the ongoing prospective multicenter colorectal study [11].
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breast cancer indicate that that the prognostic impact of TILs may be cancer type and context
6
Based on experiences from colorectal cancer and breast cancer, CD3, CD8, CD45RO positive cells as well as TILs evaluated by H&E staining and morphology has been the focus in the search for candidate markers in NSCLC. CD4 has also been included as it has shown prognostic impact in NSCLC in several studies. CD4 T-helper cells differentiate into Th1, Th2, Th17 and regulatory T-cells (Tregs). Several studies have assessed prognostic roles of these T cell markers in lung cancer [6,14], but due to conflicting prognostic impact in both lung cancer and in several other malignancies, these have not been included in this review [6]. Using the following search criteria: i) Stage I-IIIA NSCLC patients, ii) studies published
assessments of TILs, we identified 15 eligible studies (Table 2) [13,15-28]. Five of these included CD3 [15,21,22,26,28], four CD4 [16,22,27,28], 11 CD8 [13,16-18,20-22,24,26-28], one CD45RO [28] and four evaluating TILs morphologically by H&E staining [19,21,23,25]. The most promising candidate marker so far seems to be CD8. The results are quite consistent showing that a high number of infiltrating CD8+ TILs is a positive prognostic factor regardless of tissue compartments evaluated, staining/scoring methods or endpoints used. There is some variation in the statistical strength (from being a strong independent prognostic variable to show significance in only univariate analyses or just indicating a trend), but none of the examined studies did show a negative prognostic impact of CD8+ TILs. A large comprehensive study by Suzuki et al did not show any prognostic trend for CD8, but they included exclusively NSCLC adenocarcinomas stage I [28]. Reassuringly, the two most recent studies which included both test and validation cohorts did show a strong independent positive prognostic impact of CD8+ TILs [13,26]. To our knowledge, merely our recent study have included the results in a TNM-I context. As presented in Figure 1, the stromal CD8+ density adds valuable additional prognostic impact within each pathological stage (pStage) [13].
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between 2000- and March 2015, iii) more than 200 included patients and iv) in situ
7
Another promising marker is CD3. As a pan T-cell marker it is not surprising that a high CD3+ TILs density would indicate a positive prognostic impact in the same way as evaluating TILs by H&E staining and morphology. CD4 T-helper cells show basically the same results as CD3 and CD8, but may be considered unspecific as it stains both regulatory (Tregs) and tumor suppressing T cells. Nevertheless, as a prognostic factor it is promising. To our knowledge CD45RO (memory T-cells) is only evaluated in one large NSCLC study by Suzuki et al.[28]. As this study included stage I adenocarcinomas patients only, and it showed highly significant impact in colorectal cancer, CD45RO deserves to be further elucidated in
TIL location and compartments within the tumor The presence of TILs related to compartments within the tumor is frequently a matter of debate and the terminology used is often ambiguous. Essentially, compartments mentioned are either intra-epithelial (often named “tumor” or “cancer nests”) or stromal (called “intratumoral stroma” or “tumor-related stroma”). These compartments may be located in the center or/and at the invasive margin of the tumor. In addition, lymphocytes may be located in adjacent tertiary lymphoid structures [6,16]. In colorectal cancer, the TNM-I score differentiates between central tumor and invasive margin, but the selected areas for TIL assessment (both in the central tumor and in the invasive margin) includes lymphocytes from both intra-epithelial and stromal compartments [9-11]. The breast cancer recommendations endorse solely the stromal compartment (i.e. area occupied by mononuclear inflammatory cells over the total intratumoral stroma) within the borders of the invasive margin [12]. In NSCLC prognostic studies (Table 2), the compartment of interest has often been described as intra-epithelial, stromal or a combination of these two. In some cases the intra-
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NSCLC [29,30].
8
epithelial and the stromal compartment have been evaluated separately. However, whether the calculated lymphocytes are located in the invasive margin or in the central parts of tumor is generally not described. In our study [13], we considered the same approach as Galon et al. as both intra-epithelial and stromal CD8+ expression turned out as positive prognostic factors in our test cohort, but the stromal expression alone showed a better prognostic impact than the combination of these two closely related compartments. Planning a prospective study, we are considering using the total expression for the simplicity and potentially easier use of computer based analysis. Basically, we have to establish an approach fitting the NSCLC biology as well
work by Galon et al. To our knowledge, only our recently published study has addressed the issue of central tumor versus invasive margin in NSCLC TNM-I [13]. In one of our cohorts the localization of each tissue core was clearly noted and, interestingly, the prognostic impact of stromal CD8+ TILs appeared to be strongest when located in the invasive margin. When it comes to evaluation of different compartments (e.g. intraepithelial versus stromal) Schalper et al. has taken this issue a step further by using multiplexed quantitative fluorescence (QIF) [26]. QIF score of each fluorescence channel in the tumor and/or stromal compartment was calculated by dividing the target TIL marker pixel intensity by the area of desired compartment defined by the cytokeratin positivity (i.e. intra-epithelial compartment), absence of cytokeratin positivity (i.e. stromal compartment) or DAPI positive cells (e.g. including both intraepithelial and stromal compartments). They advocate for this method and state that differentiating between intra-epithelial and stromal staining is pivotal in optimizing the assessment of the prognostic impact of CD8+ TILs in NSCLC. This is in contrast to the colorectal TNM-I approach where scoring was a mix between these two compartments. Based on our findings, we believe that both approaches may have strengths and weaknesses. Our data indicate a stronger impact based on assessments in the stromal compartment for CD8+
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as possible. In this process some solutions may differ from the impressive TNM-I pioneer
9
density, supporting Schalper et al, but localization (invasive margin) is definitively also an issue in NSCLC TNM-I. Combining these strategies may be a sound solution, but further research is warranted to clarify these important questions.
Tissue microarrays versus whole slides in the assessment of TNM-I Closely related to the compartment/localization issue is the debate whether tissue microarrays (TMAs) are adequately representative or inferior to whole slides when it comes to reflecting the infiltration of lymphocytes in the tumor. The colorectal world task force argues that for
well as the invasive part. However, they are evaluating a total of only four selected areas within the whole slide, not unlike a TMA approach. In the breast cancer recommendations they also initially preferred whole slides morphological assessment (H&E staining), but concluded that TMA may be a good option for future studies, particularly for the rapid evaluation of large cohorts using immunohistochemistry based assessments of TILs subsets. More research on this issue is needed in NSCLC. Several studies have shown that TMA results largely correlate with whole slide findings [31]. The numbers of cores, their size, and the localization they are taken from are of pivotal importance. It is, however, worth mentioning that in these correlation studies, the gold standard is usually whole slides. As a tumor is three- and not two-dimensional, one may ask how representative is a slide for the whole tumor. This is a highly relevant question also in a TNM-I setting and needs further attention. Another relevant matter is the thickness of the tissue sections. In cases where the score is based on a ratio between an actual marker and the number of other nucleated cells, this is not a problem. However, when the score is based on the number of marked cell per area this
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routine practice purposes whole slides is favorable as it includes areas from both the center as
10
may be challenging. Most slides are 4-µm or 5-µm thick, but even this minor difference will in average lead to a result discrepancy of about 20 percent.
Challenges related to scoring – pathologist or computer? In colorectal cancer, Galon et al. advocate the use of digital scoring as computer-assisted image analyses provides important advantages [9,11]. Due to the fact that validation studies have demonstrated high concordance between digital and manual scoring the authors state that digital scoring is pivotal to facilitate routine pathology and to speed up the process of
approaches, while promising, have not been published in large series with consistent methodology and, hence, is an important area for further research. Digital imaging may further improve the reproducibility in the assessment of percentages of immune positive area compared to visual semiquantitative estimation. This is also the key message in a published study evaluating TILs in colorectal cancer, focusing on this issue [32]. However, in the same paper the authors also state that the semiquantitative scoring showed an excellent reproducibility (T-cell markers CD3 and FoxP3 were evaluated) and conclude that computer-assisted image analysis provides a valuable alternative to semiquantitative assessments in immunohistochemically stained sections. It is important to note that manual exact count is the gold standard that both the semiquantitative and the computer-based analysis are compared against. As mentioned, digital scoring analysis is less time-consuming when finally established. This is the case when the area to be evaluated is well defined and the computer counts the number of stained cells. In NSCLC, stromal and intra-epithelial compartments are very closely related. Consequently, evaluation of only one of these compartments is regarded challenging. Hence, in a digital setting a pathologist will manually have predefined the correct
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quantification. In contrast, the breast cancer recommendations conclude that digital scoring
11
compartments. Thus in a prospective study one may as Galon et al. consider not to evaluate the TIL markers in the intra-epitelial and stromal compartment separately. Several wellperformed NSCLC studies have already used a computer-based scoring approach when evaluating TIL specific markers [17,18,20,24,26], and the previously mentioned comprehensive study by Schalper et al. addressed the issue of different compartments (intraepithelial versus stromal). Their method seems promising and further testing with a digital approach in prospective studies is needed before concluding on this matter. A major advantage with digital analyses (as for the more time-consuming exact count)
multivariate prediction models without any cut-offs. Continuous data are also much more flexible in an explorative study where cut-off is to be decided. Median, percentiles and “best p-value” are frequently used cut-off strategies, all with potential strengths and weaknesses. In any case, cut-offs must be predefined and validated in several cohorts prior to entering a prospective multicenter study and, finally, potentially be used in NSCLC TNM-I clinical decision making.
Future directions There are challenges related to translating promising prognostic immunoscore results into a TNM-I clinical setting. For the time being the world task force in colorectal cancer seems to be closest in reaching such a goal. The breast cancer working group recently has published some general recommendations. They acknowledge the immense complexity of lymphocyte assessment and suggest that closer molecular characterization of the TIL infiltrates may add both sensitivity and specificity to the predictive value of morphologically recognized TILs [12]. In NSCLC, the interest for TILs as a prognostic tool is increasing, but a shift from
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compared to a semi quantitative score is that a continuous data variable can be used directly in
12
promising retrospective studies to larger prospective multicenter studies with focus on clinical use is warranted. So far stromal CD8 seems to be the most promising candidate in NSCLC as several studies have shown its positive prognostic impact and the fact that additional impact can be clearly demonstrated across each pStage (Figure 1). In addition CD3, CD4 and CD45RO are potentially candidate markers to be included in a prospective study, but validated results on the prognostic impact of these markers according to pStage are so far sparse. As there is a close connection between these markers and mechanisms related to new
checkpoint inhibitors targeting CTLA-4/PD1/PD-L1 are highly in need of valid predictive markers. PD-L1 expression by immunohistochemistry (IHC) has been found to predict response to PD1 and PD-L1 inhibitors, but patient with a low PD-L1 score may still respond to these antibodies [7]. As the TNM-I is a pathological TNM-I (pTNM-I), it may only be used as a predictor of treatment response in a postoperative setting. Yet, evaluating TNM-I as a predictive tool in an adjuvant immunotherapy setting is highly interesting. Nevertheless, both as a prognostic and potentially as a predictive tool, the IHC procedure and the scoring systems need to be standardized. The greatest challenge may be the heterogeneity of TILs in the tumor. Besides, the reproducibility and reliability have to be significantly improved as we move from predicting survival in large cohorts to testing sensitivity and specificity at a patient level. However, as the prognostic impact of TILs in NSCLC is promising the aim should be to develop a clinically useful NSCLC TNM-I by refining the current knowledge combined with prospective multicenter studies.
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targeted immunotherapy, there is a potential predictive value of TNM-I as well. Immune
13
Acknowledgements We want to thank the Norwegian Cancer Society and Northern Norway Health Region Authority for financial support. Conflict of interest No conflict of interest.
Figure 1. From TNM to TNM-Immunoscore (TNM-I) in Non-Small Cell Cancer. The American Joint Committee of Cancer (AJCC) and the Union Internationale Contre le Cancer (UICC) define and periodically update the TNM classification system. In lung cancer, the current 7th edition of the lung cancer staging system is based on an initiative undertaken by the International Association for the Study of Lung Cancer (IASLC). (A) Five-year overall survival (OS) rates in 797 resected, stage I-IIIA, NSCLC patients from four different Scandinavian cohorts is comparable to IASLC data [4,13]. The combination of stromal CD8 expression (B) and pathological stage (C) survival data [13] results in a TNM-Immunoscore (D) adding significant prognostic impact across each pathological stage.
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Legend of figure
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16 22. Kayser G, Schulte-Uentrop L, Sienel W et al. Stromal CD4/CD25 positive T-cells are a strong and independent prognostic factor in non-small cell lung cancer patients, especially with adenocarcinomas. Lung Cancer 2012; 76: 445-51. 23. Kilic A, Landreneau RJ, Luketich JD et al. Density of tumor-infiltrating lymphocytes correlates with disease recurrence and survival in patients with large non-small-cell lung cancer tumors. J Surg Res 2011; 167: 207-10. 24. Kim MY, Koh J, Kim S et al. Clinicopathological analysis of PD-L1 and PD-L2 expression in pulmonary squamous cell carcinoma: Comparison with tumor-infiltrating T cells and the
25. Ruffini E, Asioli S, Filosso PL et al. Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms. Ann Thorac Surg 2009; 87: 365-71. 26. Schalper KA, Brown J, Carvajal-Hausdorf D et al. Objective measurement and clinical significance of TILs in non-small cell lung cancer. J Natl Cancer Inst 2015; 107. 27. Sterlacci W, Wolf D, Savic S et al. High transforming growth factor beta expression represents an important prognostic parameter for surgically resected non-small cell lung cancer. Hum Pathol 2012; 43: 339-49. 28. Suzuki K, Kadota K, Sima CS et al. Clinical impact of immune microenvironment in stage I lung adenocarcinoma: tumor interleukin-12 receptor beta2 (IL-12Rbeta2), IL-7R, and stromal FoxP3/CD3 ratio are independent predictors of recurrence. J Clin Oncol 2013; 31: 490-8. 29. Pages F, Kirilovsky A, Mlecnik B et al. In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer. J Clin Oncol 2009; 27: 5944-51. 30. Pages F, Berger A, Camus M et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med 2005; 353: 2654-66. 31. Kallioniemi OP, Wagner U, Kononen J et al. Tissue microarray technology for high-throughput molecular profiling of cancer. Hum Mol Genet 2001; 10: 657-62.
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status of oncogenic drivers. Lung Cancer 2015; 88: 24-33.
17 32. Vayrynen JP, Vornanen JO, Sajanti S et al. An improved image analysis method for cell counting lends credibility to the prognostic significance of T cells in colorectal cancer. Virchows Arch 2012; 460: 455-65.
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Table 1. At a glance - recommondations TNM-Imunnoscore (TNM-I) in colorectal cancer and breast cancer and status in non-small cell lung cancer (NSCLC) TNM-I. Breast cancer [12]
NSCLC
Lymphocyte markers
CD3 and CD8
Tumor infiltrating lymphocytes, TILs by H&E staining. All mononuclear cells should be stained
Stromal CD8 most conclusive candidate so far. But further studies needed on promising T-cell markers as CD3, CD4, CD45RO
Compartment
Both stromal and intraepithelial CD8 and CD3 positive cells included
Stromal compartment
Both stromal and intraepithelial compartment is promising. Potentially a combination of both
Invasive margin versus tumor center
Both invasive margin and central areas included
Borders of the invasive margin should be evaluated
Only one study has addressed this issue. For stromal CD8, invasive margin looks most promising
Score
Score based on a combination of CD3 and CD8 score in both central tumor and invasive margin
TILs should be assessed as a continuous parameter. No formally recommendation for a clinically relevant threshold(s) can be given at this stage
Too premature to conclude. Stromal CD8 looks promising – see Figure 1
TMA versus whole slides
Whole slides recommended, but selected areas from invasive margin and central tumor are scored
Full sections are preferred over biopsies whenever possible, but cores may be used in pre-therapeutic neoadjuvant setting
Promising results based on TMA, but whole slides may be preferred to better address localization - central tumor versus invasive margin
Manual versus digital scoring
Digital (computer based) scoring recommended
Manual scoring ok, digital scoring important in further research
Manual scoring shows clearly significant results across each pStage, but digital scoring may be more precise and less timeconsuming when established
Continuous versus semiquantitative variable
Scored as a continuous variable, but redefined as a semiquantitative variable
Ideally should be scored as a continuous variable (% stromal TILs). However, pathologists should report their scores in as much detail as the pathologist feel comfortable with
Semiquantitative scoring seems promising, but continuous variable may be preferred in the scoring to best decide cut-off
Formally recommended threshold
Immunscore established – score 1 to 4 based on the above mentioned combination
No
No
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Colorectal cancer [9, 11]
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Table 2. Characteristics of non-small cell cancer studies between 2000 and March 2015 with focus on in situ assessment of tumor infiltrating lymphocytes. Study
Year
N
Histology
pStage
Marker
Compartment
Invasive margin vs. tumor center addressed Yes
TMA vs. WS
- Scoring approach - Digital versus manual scoring - Cut-off
Donnem T et al [13]
2015
797
ADC, SCC, LCC
I-IIIA
CD8
Stromal
TMA
“Representative intratumoral areas” (mix intra-epithelial and stromal). Intra-epithelial, stromal and total.
No
TMA
- Percentages CD8+ / total amount of nucleated cells - Manual - Predefined three-tiered cut-off - CD8+ TILs per unit area (mm2) - Digital - Median cut-off
Kim MY et al [24]
2015
331
SCC
I-III
CD8, PD1, PDL1
Schalper KA et al [26]
2015
552
ADC, SCC, Other
I-IV
CD3, CD8, CD20
No
TMA
Alifano M et al [17]
2014
303
ADC, SCC, LCC, Others
I-IV
CD8, CD1A
Tumor nests (intra-epithelial) and stromal
No
WS
Goc J et al [18]
2014
458
ADC, SCC, Others
I-IV
DC, CD8
Tumor nests (intra-epithelial) and stromal
No
WS
Suzuki K et al [28]
2013
956
ADC
pStage I only
Tumor nests (intra-epithelial) and stromal
No
TMA
ADC, SCC, LCC
I-IV
FoxP3/C D3 ratio, CD4,CD8, CD45RO CD3, CD3/CD8 CD4/ CD25
Kayser G et al [22]
2012
232
Intra-epithelial and stromal
No
TMA
- Quantitative measurement of fluorescent signal (QIF) calculated by dividing the target TIL marker pixel intensity by the area of desired compartment. - Digital and manual - Cut-off: tertiles and categorized - Intra-tumoral density of CD8+ T-cells (Both stromal area and tumor nests gave significant associations, but they decided to report findings only from tumor cells nests) - Digital - “Minimum p-value approach”. Cut-off: 96 cells/mm2 - Absolute number positive cells/mm2 - Digital and manual -“Minimum p-value approach” Cut-off: Tumor nests: 114 cells/mm2 Stroma: 383 cells/ mm2 - Marked cells/field - Manual - Three-tiered score categorized in high and low - The absolute number of each IHC detectable subgroup/core - Manual - Median cut-off
Validation cohort
Prognostic impact (PI)
PI across each stage/TNM addressed Yes Significant within each pStage No
Yes
Independent positive PI
No
Significant positive PI in univariate with DFS as endpoint
Yes
CD8 independent positive PI in both cohorts. CD3 independent positive in one cohort
No
No
CD8 independent positive PI in total material and combined stage I-II
Partly
No
Both intra-epithelial and stromal CD8 significant positive PI in univariate
No
Yes
No marker significant PI in univariate
No
Stromal CD3 positive PI in univariate. Stromal CD4/CD25 independent PI
Included pStage I patients only No
Year
N
Histology
pStage
Marker
Compartment
Invasive margin vs. tumor center addressed
TMA vs. WS
- Scoring - Digital vs. manual - Cut-off
Ilie M et al [20]
2012
632
ADC, SCC, LCC, Others
I-III
CD8, CD8/ CD66b ratio
Intratumoral (mix Intraepithelial and stromal)
Central areas of the tumor
TMA
Sterlacci et al [27]
2012
383
I-IV
CD4 CD8
Intra-epithelial
No
TMA
Horne ZD et al [19]
2011
273
ADC, SCC, LCC ADC, SCC, LCC
1A
TILs
Combination tumor nests (intra-epithelial) and stromal
No
WS
Kilic A et al [23]
2011
219
ADC, SCC, Other
1A-1B
TILs
No
WS
Al-Shibli et al [15]
2010
335
ADC, SCC, LCC
I-IIIA
CD3, CD117, CD 138
Combination tumor nests (intra-epithelial) and stromal Intra-epithelial and stromal
No
TMA
Ruffini et al [25]
2009
1290
ADC, SCC, LCC, Others
I-IIIA
TILs
Intra-epithelial
No
WS
Al-Shibli et al [16]
2008
335
ADC, SCC, LCC
I-IIIA
CD8, CD4
Intra-epithelial and stromal
No
TMA
Johnson SK et al [21]
2000
710 (95)
I-III
TILs CD3 CD8
Intra-epithelial and stromal
No
WS
- Density as the number of positive cells per unit of tissue surface area - Digital - Median cut-off. The median ± SD was 110 ± 142 cells - Number of positive cells/area - Manual - Median cut-off - Subjective low (scattered), moderate and high (intense) TIL infiltration - Manual - Cut-off: TIL - : low infiltration, TIL+: modest and high infiltration - “Routine protocol by an expert academic pathologist; group 1, none to mild, or group 2, moderate to serve infiltrate” - Manual - Stroma, high if they represented > 50% of nucleated cells in stroma. Intraepithelial, high if they represented more than 1% of nucleated cells -Manual - TILs were evaluated morphologically, only in a subset of 21 patients IHC - Manual - Cut-off: Low: < 20% of cells, high: ≥ 20% of cells - Percentages marker / total amount of nucleated cells - Manual - Cut-off: CD4, intraepithelial ≥ 5% , CD4, stromal ≥ 25%, CD8, intraepithelial > 5%, CD8, stromal > 50% - In 710 patients: Morphologically TIL by H&E staining. Semiquantitative score. In subset of 95 patients: CD3 and CD8, intratumoral, low if < 10%, high if ≥ 10% cells per high power field - Manual and digital
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Study
Validation cohort
Prognostic impact (PI)
PI across each stage/TNM addressed
No
CD8 not significant PI in univariate. CD66b/CD8 ratio independent positive PI CD4/CD8 ratio positive PI for ADC, in stage IA only Significant positive PI with recurrence free survival as endpoint
No
No
Positive PI in univariate
Stage IA and IB only
No
Stromal CD3 independent positive PI. Stromal CD3 PI in pStage IIIA, trend in I and II Independent positive PI in the total cohort and in SCC. Univariate: pStage I, SCC Stromal CD4 and CD8 independent positive prognostic factors. Intraepithelial PI in univariate. TIL total cohort not significant. Subset 95 patients, intraepithelial CD3 positive PI in univariate
Partly
No
No
No
No
No
Partly
Stage IA only
Partly
No
No
Abbreviations: SCC, squamous cell carcinoma; LCC, large-cell carcinoma; ADC, adenocarcinoma; TILs, tumor infiltrating lymphocytes; PD1, programmed death -1; PDL1, PD-ligand 1; TMA, tissue microarrays; WS, whole slides; N, number of patients; PI, prognostic impact; DFS, disease-free survival.
