JNCI J Natl Cancer Inst (2016) 108(8): djw033 doi: 10.1093/jnci/djw033 First published online May 12, 2016 Editorial
EDITORIAL
Additional Support for the Introduction of Immune Cell Quantification in Colorectal Cancer Classification ro ^ me Galon Robert L. Ferris and Je Affiliations of authors: Cancer Immunology Program and Tumor Microvenvironment Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA (RLF); INSERM, Paris Descartes, Sorbonne Paris Cite , UMRS1138, Paris, France (JG); Sorbonne UMRS1138, Laboratory of Integrative Cancer Immunology, Paris, France (JG); Universite s, UPMC Univ Paris 06, UMRS1138, Centre de Recherche des Cordeliers, Paris, France (JG) Universite ro ^ me Galon (e-mail: [email protected]). Correspondence to: Robert L. Ferris (e-mail: [email protected]); or Je
Cancer is a complex and dynamic disease, and predicting clinical outcome is a major clinical challenge. Indeed, classification systems such as the traditional American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provide insufficient prognostic information (1). A likely explanation for the predictive accuracy of the traditional TNM classification system is its reliance on the assumption that disease progression is tumor cell–autonomous and fails to incorporate the effects of the host immune response. Indeed, tumor progression has more recently been considered to be the result of a balance between an invasive tumor and the host immune/inflammatory response (2). Mounting evidence suggests that an enhanced lymphocytic reaction may be an informative prognostic indicator in cancer staging. Colorectal cancer (CRC) is the fourth leading cause of cancer deaths worldwide. While clinical outcomes are largely dependent on stage at diagnosis and treatment, increasing evidence suggests that microsatellite instability (MSI) and host immune infiltration may also be highly informative prognostic indicators. Molecular genetic studies of CRC have identified high levels of MSI (MSI-H) in approximately 15% of CRCs. Histologic differences between MSI-H and microsatellite stable (MSS)/ microsatellite-low (MSI-L) tumors have been well described, and the MSI-H phenotype has been associated with a better prognosis than tumors with an MSS or MSI-L phenotype. While the underlying drivers for the MSI-H survival advantage are not fully understood, the prognostic benefit has been at least partially attributed to the pronounced lymphocytic infiltration in this subset of cancers. Tumor-infiltrating lymphocytes (TILs) can trigger lysis of cancer cells by recognizing enhanced expression of abnormal tumor antigens presented by HLA molecules. The presence of TILs is more common in MSI-H than microsatellite-stable (MSS) tumors, perhaps because of DNA mismatch repair deficiencies causing frameshift mutations, generating
potentially immunogenic neoantigens. This hypothesis remains to be fully demonstrated. Rozek et al., in this issue of the Journal (3), examine the relative roles of intratumoral TIL, peritumoral Crohn’s-like lymphoid reaction (CLR), MSI, and clinicopathological characteristics in survival for nearly 2400 CRC cases from northern Israel. It has been understood for years that patients with CRCs containing many TILs have a survival advantage over those that do not (4). In addition, the number of TILs discriminates between MSI-H and MSS CRCs. Further, CLR, characterized by peritumoral lymphoid aggregates, is a common feature of MSI-H tumors and has also been associated with improved prognosis. A pronounced host immune reaction is not unique to MSI-high cancers, but independent contributions of intratumoral and peritumoral lymphocytic responses to survival have not been fully characterized in the context of established prognostic indicators. The Rozek study evaluated the importance of TILs and the CLR as prognostic factors for CRC, in addition to age, sex, MSI, stage, and grade, in a large population-based, case-control study in northern Israel. Several ways to classify cancer rely on tumor cell characteristics, including morphology, molecular pathways, mutation status, cell origin, and gene expression-based methods, and allow the distinction of multiple, often overlapping, subtypes (Figure 1). Rozek et al. evaluated in a semi-quantitative manner the presence of TILs based on hematoxylin and eosin (H&E) slide evaluation. Fields of interest were selected and TILs were evaluated by a pathologist. Tumors with TIL/high powered field (HPF) of 2 or greater were associated with a statistically significant increase in CRC-specific (P < .0001) and overall survival (P < .0001) compared with tumors with TIL/HPF of less than 2. Similarly, tumors with a prominent CLR experienced better CRC-specific (P < .0001) and overall survival (P < .0001) as compared with those with no response. High TILs (hazard ratio [HR] ¼ 0.76, 95% confidence interval [CI] ¼ 0.64 to 0.89, P < .001)
Received: January 27, 2016; Accepted: January 28, 2016 © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected].
1 of 3
2 of 3 | JNCI J Natl Cancer Inst, 2016, Vol. 108, No. 8
Figure 1. Ways to classify CRC.
and a prominent CLR (HR ¼ 0.71, 95% CI ¼ 0.62 to 0.80, P < .0001), but not MSI, were associated with a statistically significant reduction in all-cause mortality after adjustment for established prognostic factors. TILs and CLR were both prognostic indicators for CRC after adjusting for traditional prognostic indicators. It is not entirely unexpected to find that TILs and CLR are both prognostic indicators for CRC (4). The host immune response has generally been correlated with the survival-associated MSI phenotype, and several groups have established the relationship between TIL and MSI. Using digital pathology and T-cell density quantification of 594 CRC cases, Galon et al. showed that immune cell characteristics in CRCs have a better prognostic value than UICC-TMN staging and that tumor progression and invasion were, in fact, statistically dependent on the host-immune reaction (T-cell densities) (6,7). Memory T-cells were protective against early-metatstic invasion and were associated with improved clinical outcome (8). Densities of cytotoxic and memory T-cells were also associated with survival (disease-free survival [DFS], disease-specific survival, overall survival [OS]) in 602 early-stage (I/II) CRCs (9). Chang et al. showed in 150 CRCs that lymphocytic infiltrate was associated with a survival benefit regardless of MSI status, and no survival difference was found in patients with a host immune response between MSI-H and MSS tumors (10). The immune landscape from 28 different different immune cell populations was
quantified, and its impact on DFS and OS times was previously determined (11). The quantification of cytotoxic T-cells in specific tumor regions (core and invasive margin of the tumor), also known as the Immunoscore, has been proposed as a standardized reliable measurement of good immune prognostic markers (12,13). High overall and subtype-specific lymphocyte infiltration have been suggested as independent prognostic factors for overall survival in a variety of other cancers as well (5). Recently, the standardized Immunoscore assay also predicted survival in patients with brain metastasis (14). In this issue, Rosek et al. show that the presence of TILs is strongly associated with improved prognosis and then quantify the relationship to illustrate that each TIL/HPF is associated with approximately a 3% to 4% reduction in the risk of mortality. With respect to Crohn’s-like lymphoid aggregation, previous evidence has suggested its association with improved survival. A 2014 meta-analysis including 12 studies identified from Ovid MEDLINE and EMBASE demonstrated that the density of generalized tumor inflammatory infiltrate at the invasive margin is associated with better overall survival (HR ¼ 0.59, 95% CI ¼ 0.48 to 0.72), cancerspecific survival (HR ¼ 0.40, 95% CI ¼ 0.27 to 0.61), and diseasefree survival (HR ¼ 0.72, 95% CI ¼ 0.57 to 0.91) for CRC patients. Rozek et al. thus provide a comprehensive analysis of 2369 incident CRCs from northern Israel with nearly perfect follow-up that further supports the major impact of TILs on patient
R. L. Ferris and J. Galon | 3 of 3
survival. This study provides new evidence that simultaneously evaluates TILs and CLR as prognostic factors, integrating full molecular and clinical characterization. This large, population-based study of incident CRCs supports the inclusion of TILs and CLR into the development of new CRC staging guidelines. However, there are limitations in this study. Immunohistochemistry data for CD3þ, CD20þ, CD19þ, NKp46þ, CD4þ, CD8þ, CD45ROþ FoxP3þ, and IL17þ cells were not available. Thus, the authors did not examine the overall quantity of TILs subtypes (subsets of NK cells, B-cells, T-cells) that may also be important, in some cases having opposing function and impact on survival (5,15,16). Second, tumor areas are highly heterogeneous in TILs, and the selection of a high-power field of interest is, by essence, subjective. Furthermore, it is well known that semi-quantitative evaluation of H&E slides is not fully reliable (17), and with the method used, TIL/HPF of less than 2 as cutpoint is very sensitive, with errors in only one additional cell per field. It is possible that ethnic or geopgraphical differences in populations with unique diets, genetic predispotion, or triggers leading to CRC may yield variation in these results, which must be confirmed using tissues and large datasets from other populations. Multiple ways of classsifying CRC are used based on tumor cell characteristics; however, no immune classification is routine yet (Figure 1) (12). To be used globally in a routine manner, evaluation of a novel marker should have the following characteristics: routine, feasible, simple, rapid, robust, reproducible, objective, specific, quantitative, standardized, and powerful. TIL evaluation in the present study demonstrates its value but is not yet robust, reproducible, objective, specific, quantitative, or standardized enough to be included in clinical practice. Given the extensive literature that has demonstrated the prognostic impact of intratumoral immune cells, it is a hope that a standardized immune cell quantification such as Immunoscore (12) will soon be introduced into the classification of colorectal cancer to ultimately help save the lives of patients with cancer.
References 1. Nagtegaal ID, Quirke P, Schmoll HJ. Has the new TNM classification for colorectal cancer improved care? Nat Rev Clin Oncol. 2011;9:119–123. 2. Galon J, Angell HK, Bedognetti D, Marincola FM. The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures. Immunity. 2013;39:11–26. 3. Rozek LS, Schmit SL, Greenson JK, et al. Tumor-Infiltrating Lymphocytes, Crohn s-Like Lymphoid Reaction, and Survival From Colorectal Cancer. J Natl Cancer Inst. 2016;108(8):djw027. 4. Galon J, Fridman WH, Pages F. The adaptive immunologic microenvironment in colorectal cancer: a novel perspective. Cancer Res. 2007;67:1883–1886. 5. Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12:298–306. 6. Galon J, Costes A, Sanchez-Cabo F, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006;313:1960–1964. 7. Mlecnik B, Tosolini M, Kirilovsky A, et al. Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction. J Clin Oncol. 2011;29:610–618. 8. Pages F, Berger A, Camus M, et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med. 2005;353:2654–2666. 8. 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;27:5944–5951. 10. Chang EY, Dorsey PB, Frankhouse J, et al. Combination of microsatellite instability and lymphocytic infiltrate as a prognostic indicator in colon cancer. Arch Surg. 2009;144:511–515. 11. Bindea G, Mlecnik B, Tosolini M, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity. 2013;39:782–795. 12. Galon J, Mlecnik B, Bindea G, et al. Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours. J Pathol. 2014;232:199–209. 13. Galon J, Pages F, Marincola FM, et al. Cancer classification using the Immunoscore: a worldwide task force. J Transl Med. 2012;10:205. 14. Berghoff A, Fuchs E, Ricken G, et al. Density of tumor-infiltrating lymphocytes correlates with extent of brain edema and overall survival time in patients with brain metastases. Oncoimmunology. 2016:5. 15. Tosolini M, Kirilovsky A, Mlecnik B, et al. Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, th2, treg, th17) in patients with colorectal cancer. Cancer Res. 2011;71:1263–1271. 16. Yoon HH, Orrock JM, Foster NR, Sargent DJ, Smyrk TC, Sinicrope FA. Prognostic impact of FoxP3þ regulatory T cells in relation to CD8þ T lymphocyte density in human colon carcinomas. PLoS One. 2012;7:e42274. 17. Smits AJ, Kummer JA, de Bruin PC, et al. The estimation of tumor cell percentage for molecular testing by pathologists is not accurate. Mod Pathol. 2014;27:168–174.
EDITORIAL
Additional Support for the Introduction of Immune Cell Quantification in Colorectal Cancer Classification ro ^ me Galon Robert L. Ferris and Je Affiliations of authors: Cancer Immunology Program and Tumor Microvenvironment Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA (RLF); INSERM, Paris Descartes, Sorbonne Paris Cite , UMRS1138, Paris, France (JG); Sorbonne UMRS1138, Laboratory of Integrative Cancer Immunology, Paris, France (JG); Universite s, UPMC Univ Paris 06, UMRS1138, Centre de Recherche des Cordeliers, Paris, France (JG) Universite ro ^ me Galon (e-mail: [email protected]). Correspondence to: Robert L. Ferris (e-mail: [email protected]); or Je
Cancer is a complex and dynamic disease, and predicting clinical outcome is a major clinical challenge. Indeed, classification systems such as the traditional American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provide insufficient prognostic information (1). A likely explanation for the predictive accuracy of the traditional TNM classification system is its reliance on the assumption that disease progression is tumor cell–autonomous and fails to incorporate the effects of the host immune response. Indeed, tumor progression has more recently been considered to be the result of a balance between an invasive tumor and the host immune/inflammatory response (2). Mounting evidence suggests that an enhanced lymphocytic reaction may be an informative prognostic indicator in cancer staging. Colorectal cancer (CRC) is the fourth leading cause of cancer deaths worldwide. While clinical outcomes are largely dependent on stage at diagnosis and treatment, increasing evidence suggests that microsatellite instability (MSI) and host immune infiltration may also be highly informative prognostic indicators. Molecular genetic studies of CRC have identified high levels of MSI (MSI-H) in approximately 15% of CRCs. Histologic differences between MSI-H and microsatellite stable (MSS)/ microsatellite-low (MSI-L) tumors have been well described, and the MSI-H phenotype has been associated with a better prognosis than tumors with an MSS or MSI-L phenotype. While the underlying drivers for the MSI-H survival advantage are not fully understood, the prognostic benefit has been at least partially attributed to the pronounced lymphocytic infiltration in this subset of cancers. Tumor-infiltrating lymphocytes (TILs) can trigger lysis of cancer cells by recognizing enhanced expression of abnormal tumor antigens presented by HLA molecules. The presence of TILs is more common in MSI-H than microsatellite-stable (MSS) tumors, perhaps because of DNA mismatch repair deficiencies causing frameshift mutations, generating
potentially immunogenic neoantigens. This hypothesis remains to be fully demonstrated. Rozek et al., in this issue of the Journal (3), examine the relative roles of intratumoral TIL, peritumoral Crohn’s-like lymphoid reaction (CLR), MSI, and clinicopathological characteristics in survival for nearly 2400 CRC cases from northern Israel. It has been understood for years that patients with CRCs containing many TILs have a survival advantage over those that do not (4). In addition, the number of TILs discriminates between MSI-H and MSS CRCs. Further, CLR, characterized by peritumoral lymphoid aggregates, is a common feature of MSI-H tumors and has also been associated with improved prognosis. A pronounced host immune reaction is not unique to MSI-high cancers, but independent contributions of intratumoral and peritumoral lymphocytic responses to survival have not been fully characterized in the context of established prognostic indicators. The Rozek study evaluated the importance of TILs and the CLR as prognostic factors for CRC, in addition to age, sex, MSI, stage, and grade, in a large population-based, case-control study in northern Israel. Several ways to classify cancer rely on tumor cell characteristics, including morphology, molecular pathways, mutation status, cell origin, and gene expression-based methods, and allow the distinction of multiple, often overlapping, subtypes (Figure 1). Rozek et al. evaluated in a semi-quantitative manner the presence of TILs based on hematoxylin and eosin (H&E) slide evaluation. Fields of interest were selected and TILs were evaluated by a pathologist. Tumors with TIL/high powered field (HPF) of 2 or greater were associated with a statistically significant increase in CRC-specific (P < .0001) and overall survival (P < .0001) compared with tumors with TIL/HPF of less than 2. Similarly, tumors with a prominent CLR experienced better CRC-specific (P < .0001) and overall survival (P < .0001) as compared with those with no response. High TILs (hazard ratio [HR] ¼ 0.76, 95% confidence interval [CI] ¼ 0.64 to 0.89, P < .001)
Received: January 27, 2016; Accepted: January 28, 2016 © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected].
1 of 3
2 of 3 | JNCI J Natl Cancer Inst, 2016, Vol. 108, No. 8
Figure 1. Ways to classify CRC.
and a prominent CLR (HR ¼ 0.71, 95% CI ¼ 0.62 to 0.80, P < .0001), but not MSI, were associated with a statistically significant reduction in all-cause mortality after adjustment for established prognostic factors. TILs and CLR were both prognostic indicators for CRC after adjusting for traditional prognostic indicators. It is not entirely unexpected to find that TILs and CLR are both prognostic indicators for CRC (4). The host immune response has generally been correlated with the survival-associated MSI phenotype, and several groups have established the relationship between TIL and MSI. Using digital pathology and T-cell density quantification of 594 CRC cases, Galon et al. showed that immune cell characteristics in CRCs have a better prognostic value than UICC-TMN staging and that tumor progression and invasion were, in fact, statistically dependent on the host-immune reaction (T-cell densities) (6,7). Memory T-cells were protective against early-metatstic invasion and were associated with improved clinical outcome (8). Densities of cytotoxic and memory T-cells were also associated with survival (disease-free survival [DFS], disease-specific survival, overall survival [OS]) in 602 early-stage (I/II) CRCs (9). Chang et al. showed in 150 CRCs that lymphocytic infiltrate was associated with a survival benefit regardless of MSI status, and no survival difference was found in patients with a host immune response between MSI-H and MSS tumors (10). The immune landscape from 28 different different immune cell populations was
quantified, and its impact on DFS and OS times was previously determined (11). The quantification of cytotoxic T-cells in specific tumor regions (core and invasive margin of the tumor), also known as the Immunoscore, has been proposed as a standardized reliable measurement of good immune prognostic markers (12,13). High overall and subtype-specific lymphocyte infiltration have been suggested as independent prognostic factors for overall survival in a variety of other cancers as well (5). Recently, the standardized Immunoscore assay also predicted survival in patients with brain metastasis (14). In this issue, Rosek et al. show that the presence of TILs is strongly associated with improved prognosis and then quantify the relationship to illustrate that each TIL/HPF is associated with approximately a 3% to 4% reduction in the risk of mortality. With respect to Crohn’s-like lymphoid aggregation, previous evidence has suggested its association with improved survival. A 2014 meta-analysis including 12 studies identified from Ovid MEDLINE and EMBASE demonstrated that the density of generalized tumor inflammatory infiltrate at the invasive margin is associated with better overall survival (HR ¼ 0.59, 95% CI ¼ 0.48 to 0.72), cancerspecific survival (HR ¼ 0.40, 95% CI ¼ 0.27 to 0.61), and diseasefree survival (HR ¼ 0.72, 95% CI ¼ 0.57 to 0.91) for CRC patients. Rozek et al. thus provide a comprehensive analysis of 2369 incident CRCs from northern Israel with nearly perfect follow-up that further supports the major impact of TILs on patient
R. L. Ferris and J. Galon | 3 of 3
survival. This study provides new evidence that simultaneously evaluates TILs and CLR as prognostic factors, integrating full molecular and clinical characterization. This large, population-based study of incident CRCs supports the inclusion of TILs and CLR into the development of new CRC staging guidelines. However, there are limitations in this study. Immunohistochemistry data for CD3þ, CD20þ, CD19þ, NKp46þ, CD4þ, CD8þ, CD45ROþ FoxP3þ, and IL17þ cells were not available. Thus, the authors did not examine the overall quantity of TILs subtypes (subsets of NK cells, B-cells, T-cells) that may also be important, in some cases having opposing function and impact on survival (5,15,16). Second, tumor areas are highly heterogeneous in TILs, and the selection of a high-power field of interest is, by essence, subjective. Furthermore, it is well known that semi-quantitative evaluation of H&E slides is not fully reliable (17), and with the method used, TIL/HPF of less than 2 as cutpoint is very sensitive, with errors in only one additional cell per field. It is possible that ethnic or geopgraphical differences in populations with unique diets, genetic predispotion, or triggers leading to CRC may yield variation in these results, which must be confirmed using tissues and large datasets from other populations. Multiple ways of classsifying CRC are used based on tumor cell characteristics; however, no immune classification is routine yet (Figure 1) (12). To be used globally in a routine manner, evaluation of a novel marker should have the following characteristics: routine, feasible, simple, rapid, robust, reproducible, objective, specific, quantitative, standardized, and powerful. TIL evaluation in the present study demonstrates its value but is not yet robust, reproducible, objective, specific, quantitative, or standardized enough to be included in clinical practice. Given the extensive literature that has demonstrated the prognostic impact of intratumoral immune cells, it is a hope that a standardized immune cell quantification such as Immunoscore (12) will soon be introduced into the classification of colorectal cancer to ultimately help save the lives of patients with cancer.
References 1. Nagtegaal ID, Quirke P, Schmoll HJ. Has the new TNM classification for colorectal cancer improved care? Nat Rev Clin Oncol. 2011;9:119–123. 2. Galon J, Angell HK, Bedognetti D, Marincola FM. The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures. Immunity. 2013;39:11–26. 3. Rozek LS, Schmit SL, Greenson JK, et al. Tumor-Infiltrating Lymphocytes, Crohn s-Like Lymphoid Reaction, and Survival From Colorectal Cancer. J Natl Cancer Inst. 2016;108(8):djw027. 4. Galon J, Fridman WH, Pages F. The adaptive immunologic microenvironment in colorectal cancer: a novel perspective. Cancer Res. 2007;67:1883–1886. 5. Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12:298–306. 6. Galon J, Costes A, Sanchez-Cabo F, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006;313:1960–1964. 7. Mlecnik B, Tosolini M, Kirilovsky A, et al. Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction. J Clin Oncol. 2011;29:610–618. 8. Pages F, Berger A, Camus M, et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med. 2005;353:2654–2666. 8. 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;27:5944–5951. 10. Chang EY, Dorsey PB, Frankhouse J, et al. Combination of microsatellite instability and lymphocytic infiltrate as a prognostic indicator in colon cancer. Arch Surg. 2009;144:511–515. 11. Bindea G, Mlecnik B, Tosolini M, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity. 2013;39:782–795. 12. Galon J, Mlecnik B, Bindea G, et al. Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours. J Pathol. 2014;232:199–209. 13. Galon J, Pages F, Marincola FM, et al. Cancer classification using the Immunoscore: a worldwide task force. J Transl Med. 2012;10:205. 14. Berghoff A, Fuchs E, Ricken G, et al. Density of tumor-infiltrating lymphocytes correlates with extent of brain edema and overall survival time in patients with brain metastases. Oncoimmunology. 2016:5. 15. Tosolini M, Kirilovsky A, Mlecnik B, et al. Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, th2, treg, th17) in patients with colorectal cancer. Cancer Res. 2011;71:1263–1271. 16. Yoon HH, Orrock JM, Foster NR, Sargent DJ, Smyrk TC, Sinicrope FA. Prognostic impact of FoxP3þ regulatory T cells in relation to CD8þ T lymphocyte density in human colon carcinomas. PLoS One. 2012;7:e42274. 17. Smits AJ, Kummer JA, de Bruin PC, et al. The estimation of tumor cell percentage for molecular testing by pathologists is not accurate. Mod Pathol. 2014;27:168–174.
