Assessment of the tumor microenvironment has grown in importance over the past decade, as strong evidence has emerged showing a significant correlation with patient survival in a number of different malignancies.1–3 In addition, the emergence of immunotherapeutic agents has prompted a reassessment of the tumor microenvironment as a potential biomarker of response to these agents.4 The presence of increased tumor-infiltrating lymphocytes (TILs), a marker of immune activation by a tumor, has consistently been proven to be an independent predictor of better prognosis across a range of malignancies, including colorectal carcinoma (CRC).1–3
Although the prognostic significance of TILs in CRC has been recognized for several decades, early studies mostly concentrated on the association with microsatellite unstable (MSI) tumors, often as a marker to identify which patients should undergo further screening for Lynch syndrome.5 Recently, attention has shifted to the prognostic value of TILs in all CRCs, regardless of mismatch repair (MMR) status. However, the data is significantly limited by both nonuniform scoring methodologies and inconsistent application in clinical practice.2,5
Several different systems are currently in widespread use to assess TILs. One widely used system is the Klintrup-Makinen Score, wherein the immune infiltrate is scored from 0 to 3, where score 0=no increase in inflammatory cells; score 1=a patchy increase of inflammatory cells at the invasive margin, but no destruction of invading cancer cell islets (tumor cell nests or glandular structures); score 2=a band-like infiltrate at the invasive margin with some destruction of cancer cell islets; and score 3=a very prominent inflammatory reaction, forming a cup-like zone at the invasive margin, and frequent and invariable destruction of cancer cell islets.6 In Australia, the Royal College of Pathologists of Australasia (RCPA) defines intraepithelial lymphocytes (IELs) as those that are in direct contact with tumor cells or are located directly between tumor cell clusters.7 A high density of lymphocytes, defined as ≥5/HPF), is considered significant. However, it is advised that the IEL counts are not necessary if mismatch repair deficiency (MMRd) or MSI is to be formally assessed. Although previous versions of the College of American Pathologists (CAP) protocol for reporting CRC have recommended assessment of TIL/IELs using a semiquantitative system (none; mild to moderate [0 to 2/HPF]; and marked [≥3/HPF]), this was primarily used to assist screening for Lynch syndrome, and, in the era of universal MMR/MSI screening, the current CAP protocol does not recommend the routine assessment of TILs/IELs.8
In 2014, the International TILs Working Group (ITWG) published a standardized approach for evaluating TILs on hematoxylin and eosin (H&E) sections in the context of breast cancer.9 Briefly, stromal TILs are scored as a percentage of the stromal areas between nests of carcinoma cells, and areas occupied by the malignant cells themselves are not included in the total assessed surface area.9 Subsequently, the International Immuno-Oncology Biomarkers Working Group has recommended the use of the same methodology in the evaluation of TILs in a range of other solid malignancies, including gastric, colorectal, and non–small cell lung carcinomas.2,3
To the best of our knowledge, no studies have evaluated the prognostic value of TILs in CRCs using this standardized ITWG scoring method in a sufficiently large cohort to justify implementation into clinical practice.
We, therefore, sought to:
- Validate the prognostic significance of the ITWG (also known as the International Immuno-Oncology Biomarkers Working Group) methodology for the assessment for TILs in a large unselected cohort of CRCs.
- Compare the prognostic significance of the ITWG scoring methodology for TILs with the RCPA-endorsed method of scoring IELs in clinical practice.
- Assess the interobserver concordance (κ score) of the ITWG methodology across a range of observers.
- Determine whether the ITWG scoring methodology has prognostic significance independent of MMR/MSI status.
MATERIALS AND METHODS
We searched the computerized database of the Department of Anatomical Pathology Royal North Shore Hospital, Sydney Australia, for all patients undergoing surgical resection for colorectal adenocarcinoma during the calendar years 2012 to 2015. Exclusion criteria included extracolonic and appendiceal location, tumors undergoing biopsy alone or treated endoluminal only, and histologic type other than adenocarcinoma, as defined by the World Health Organization 2019 system. When multiple tumors were present, the highest stage tumor was assessed. For the purpose of binary analysis in this study, the right colon was defined as cecum, ascending colon, hepatic flexure, and transverse colon, whereas the left colon was defined as splenic flexure, descending colon, sigmoid, and rectum. At the time of diagnosis, all patients had routine assessment of MMR status by immunohistochemistry with a 4-marker panel (PMS2, MLH1, MSH6, and MSH2), and the assessment of BRAFV600E mutation status using mutation-specific immunohistochemistry, which we have demonstrated to be consistently reliable in our hands.10,11 At the time of primary reporting, the number of IELs was assessed using the RCPA-endorsed system, as outlined above,7 as was the presence or absence of a Crohn’s-like peritumoral lymphoid reaction. Further data on TNM stage, histologic grade, and tumor location were obtained from the pathology reports. All-cause survival data were current as of August 1, 2019. Data on adjuvant chemotherapy were not available. The study was approved by the Northern Sydney Local Health District Human Research Ethics Committee.
Quantification of TILs Using the ITWG System
A single H&E-stained slide showing deepest tumor invasion was evaluated by 1 pathologist (T.L.F.) who was blinded to all clinical and pathologic data. After 1 hour of intensive face-to-face training, the pathologist assessed the number of TILs in strict accordance with the ITWG guidelines.2,8,9
The ITWG methodology2,3 is summarized in Table 1 and illustrated in Figure 1. Briefly, the density of TILs was assessed within the stromal compartment of the tumor mass, and scored as a percentage of stromal area, rounded to the nearest 5%. Scores were based on the average across the whole slide (not hotspots). Only TILs within the border of invasive tumors were assessed, so that dysplastic and in situ areas (including growth confined to the lamina propria) and inflammation outside the tumor borders were disregarded. As per ITWG recommendations, all mononuclear cells (eg, lymphocytes and plasma cells) were included as TILs, whereas other inflammatory cells (ie, neutrophils/granulocytes) were excluded. Areas of necrosis, including the central “dirty necrosis” characteristic of CRC, were excluded from the assessment, and only stromal TILs were assessed, so that TILs within nests of epithelial cells were excluded. Examples of TILs assessment using the ITWG system are provided in Figure 2.
Assessment of Interobserver Concordance of TILs Using the ITWG System
Two additional pathologists (C.J.R. and K.J.-N.) independently scored a subset of 100 and 181 randomly selected cases. To evaluate the importance of adequate training, 1 pathologist was given face-to-face interactive training on the ITWG methodology for 1 hour in addition to written information, whereas the other pathologist was provided written information only. These 2 pathologists scored different subgroups of patients.
All statistical analyses were performed using SPSS, version 25.0 (IBM Inc., Armonk, NY). Overall survival was defined as the time from surgery until any-cause death (date of census: August 1, 2019). The continuous TILs data were categorized into 3 groups: low (0% to 10%), intermediate (15% to 50%), and high (55% to 100%). The associations between categories of TILs and clinicopathologic variables were compared using χ2 tests. Kaplan-Meier curves were constructed to compare survival across groups of TILs. The effect of TILs on overall survival was also estimated in subgroup analyses stratified by the TNM stage, MMR status, and BRAFV600E status. The same survival analyses were performed using the presence of high or low/absent IEL scores and the presence or absence of peritumoral Crohn’s-like lymphocytic response based on the RPCA-endorsed system obtained from the original histopathologic reports. Univariate and multivariate Cox proportional hazard regression analyses were used to evaluate the association between TILs, age, sex, TNM stage, histologic grade, tumor location, MMR status, BRAFV600E status, and overall survival. Finally, Cohen κ analyses were performed to determine the level of agreement between the pathologist who scored all 1034 patients (T.L.F., considered the gold standard for purpose of analysis) and the 2 other pathologists who scored TILs as a categorical variable. All statistical tests were 2 sided, and P-values ≤0.05 were considered significant.
The cohort included 1034 patients with resected primary CRCs and is summarized in Table 2. TILs were scored on all patients. MMR/MSI status was recorded for 1031 patients, of whom 1028 also had BRAFV600E mutation status recorded. The mean age at surgery was 73 years (range: 27 to 101 y). A total of 526 (50.9%) patients were female. During the follow-up period, 206 (19.9%) had death recorded. The mean duration of follow-up was 5 years. After categorizing the continuous TILs data using the ITWG system, 395 cases (38.2%) were classified as low TILs, 584 (56.5%) as intermediate TILs, and 55 (5.3%) as high TILs. Using the RPCA-endorsed system, increased intratumoral lymphocytes were absent in 940 cases (92%) and present in 86 cases (8%). A peritumoral Crohn’s-like lymphocytic response was categorized as absent in 755 cases (74%) and present in 260 cases (26%).
χ2 tests were performed to evaluate associations between the TILs scores and age, sex, TNM stage, histologic grade, tumor location, MMR status, and BRAFV600E status. High TILs scores were significantly associated with advanced age (P=0.002), low TNM stage (P=0.0001), and low histologic grade (P=0.0001). No significant differences in TILs scores were observed for tumor location (P=0.566), sex (P=0.07), MMR status (P=0.641), or BRAFV600E status (P=0.583).
Overall survival was best in the high-TIL group (mean survival: 75 mo), followed by the intermediate-TIL group (mean survival: 67 mo), and then the low-TIL group (mean survival: 53 mo), P=0.0001 (Fig. 3A).
This survival benefit remained statistically significant in subgroup analyses for mismatch repair proficient (MMRp) cases (P=0.0001, Fig. 3B), MMRd cases (P=0.031, Fig. 3C), BRAFV600E wild-type cases (P=0.0001), and BRAFV600E-mutant cases (P=0.001). A survival analysis for combined stages II and III cases (n=782) was performed to investigate the potential value of TILs in aiding in treatment decisions in this subgroup. TILs were significantly correlated with overall survival within this group (P<0.0001). When restricted to (n=408) stage II cases only, TILs narrowly failed to show a statistically significant correlation with overall survival (P=0.072).
Age, tumor stage, histologic grade, tumor location, BRAFV600E status, and TILs were all significantly associated with overall survival in univariate analyses (Table 3). Multivariate analyses showed significant associations between decreased overall survival and low TILs (0% to 10%) (hazard ratio [HR]=1.891; 95% confidence interval [CI]: 1.415-2.527; P=0.0001), age >73 years (HR=2.711; 95% CI: 1.961-3.748; P=0.0001), tumor stage ≥3 (HR=2.795; 95% CI: 2.059-3.795; P=0.0001), right-sided location (HR=1.405; 95% CI: 1.040-1.899; P=0.027); MMRp status (HR=1.852; 95% CI: 1.190-2.884, P=0.006), and high histologic grade (HR=1.635; 95% CI: 1.199-2.231; P=0.002).
The presence of a peritumoral Crohn’s-like response assessed using standard reporting protocols was associated with a significantly better overall survival (mean survival: 68 mo) compared with cases reported as having an absent Crohn’s-like response (mean survival: 59 mo), P=0.001 (Fig. 3D). Although this association remained significant in MMRp cases (mean survival: 70 vs. 59 mo, P=0.001), it was not seen in the subgroup analyses for MMRd cases (mean survival: 65 vs. 59 mo, P=0.333) (Fig. 3E). There was no statistically significant association between increased IELs assessed using the RCPA method and overall survival (mean survival: 59 mo for the presence of IELs vs. 62 mo for absence of IELs; P=0.782) (Fig. 3F).
Cohen κ analysis showed good interobserver agreement between the gold-standard pathologist and the 1 pathologist who received face-to-face training, κ=0.753 (95% CI: 0.623-0.883), P=0.0001 (n=100). Without intensive training, the interobserver agreement was only moderate, κ=0.436 (95% CI: 0.313-0.559), P=0.0001 (n=181) with the gold-standard pathologist.
Given that approaches to scoring TILs have varied considerably both between and within tumor types, and across different institutions, a standardized methodology such as the ITWG has clear benefits. Although initially proposed and validated in breast cancer,9 this system has subsequently been evaluated in relatively small patient cohorts over a range of other tumor types.2,3,12,13 To the best of our knowledge, the only study to evaluate this methodology in CRC is that of Iseki et al,13 who reviewed a small cohort (n=160) of stage II and III tumors. In addition to their small sample size, their study was limited by the lack of subgroup analyses to control for MMR/BRAFV600E status, and the lack of κ scores to examine the interobserver agreement. These limitations have been overcome in this study, which provides evidence that the assessment of TILs using the ITWG system is a very strong predictor of all-cause survival across an unselected cohort of 1034 CRCs undergoing surgical treatment. This significant association between TILs, as assessed using the ITWG system, and all-cause survival was independent of many patient and tumor characteristics, including MMR deficiency, and maintained significance in a multivariable model that included sex, age, tumor site, histologic grade, MMR status, and BRAFV600E mutation status.
A particular area of clinical uncertainty is which stage II CRC patients would benefit from adjuvant therapy.14–19 There is no firm evidence that adjuvant chemotherapy improves overall survival for all stage II CRC patients,17,18 and, currently, the National Cooperative Cancer Network (NCCN) guidelines recommend adjuvant therapy only for stage II CRC with “high-risk features.”20 Despite ongoing investigation of many molecular markers, “high risk” has been difficult to define in the routine clinical setting.21 Unfortunately, in this study for (n=408) stage II CRCs, TILs just failed to reach statistical significance (P=0.072). It is possible that the use of adjuvant therapy in some cases could have affected these results, and our lack of access to these data should be considered a significant limitation of the study. Nevertheless, if other studies indicate that a low TIL score assessed using the ITWG system identifies “high-risk” patients with stage II CRC, a strong argument can be made to include TIL assessment using the ITWG method in the decision-making process for assessing these patients for adjuvant treatment. Indeed, it is promising that, for stage III (n=375, P<0.001), combined stages I and II as a group (n=613, P=0.02), and combined stages II and III as a group (n=782, P<0.0001), TILs assessed using the ITWG methodology remained prognostic.
Although the presence of a tumor-induced immune response has long been known to be associated with better survival in CRCs,1–3 it is important to note in this study that the predictive benefit of TILs assessed using the ITWG system was superior to the traditional approach of identifying a peritumoral Crohn’s-like inflammatory response or IELs using a cutoff of ≥5 lymphocytes per HPF. Although it was initially hypothesized that lymphocytes directly interacting with carcinoma cells might be more relevant, most studies have found stromal TILs to be a more reproducible parameter.9 This seems to be because intratumoral TILs are usually present in smaller numbers and are more heterogenous and difficult to observe on H&E-stained sections. Moreover, limiting evaluation to the stromal compartment means that the TIL counts will not be affected by the density and growth pattern of the carcinoma nests.9 In the present study, although the presence of a Crohn-like peritumoral lymphocytic response was found to be associated with survival benefit, this association was lost in the subgroup analysis of MMRd cases, an interesting finding given that this parameter was initially developed to help identify potential MMRd cases in the era before routine immunohistochemistry. Moreover, no survival benefit was found for cases with increased IELs assessed prospectively using the standard RCPA method. These findings support the superior prognostic value of the standardized ITWG methodology for scoring TILs over these other methodologies.
The ITWG system is easy to learn and quick to apply in clinical practice, as it simply involves the evaluation of stromal TILs, which are reported as a percentage of stromal area. Indeed, in our study, the ITWG system showed good interobserver concordance, κ=0.753 (95% CI: 0.623-0.883), P=0.0001, after just 1 hour of face-to-face training. However, it is noted that the concordance slipped to a moderate level, κ=0.436 (95% CI, 0.313-0.559), P=0.0001 (n=181), when only written materials were provided—illustrating the importance of appropriate education.
Studies using immunohistochemistry with or without digital quantitation of TILs have also demonstrated their prognostic value in CRC22–26 and have provided additional prognostic information compared with the semiquantitative systems described above.27 At present, it is unlikely that the costs associated with artificial intelligence-based or immunohistochemical scoring are justified by the additional prognostic information provided by these techniques when TILs can be readily assessed quickly and cheaply using the ITWG system on routine H&E sections. Nevertheless, a standardized and reproducible methodology for evaluating TILs on H&E sections in CRC may form the basis for the development of such artificial intelligence software in the near future.
MMRd CRCs are thought to be more immunogenic secondary to a higher mutation burden, leading to more neoantigen creation,28 and this increased immune response is thought to account for both increased IELs and a better prognosis compared with MMRp CRC.29 It is, therefore, surprising that, in this study, there was no statistically significant association between TILs, as assessed using the ITWG system, and MMR status (P=0.641). It is also interesting to note that MMR status did not predict survival in univariate analysis (P=0.479) but did become very significant in a multivariate model (P=0.006) because of an association between the positive prognostic factor of MMRd status and the adverse factors of advanced age and right-sided location. The early literature suggested that the primary reason to identify TILs was to triage genetic testing for Lynch syndrome by selecting patients for further MMR testing,30–35 and some protocols, including the CAP,8 no longer recommend TIL assessment in the era of universal screening for Lynch syndrome by MSI/MMR. However, our data clearly support the benefit of assessing both TILs and MMR/MSI status and question whether the number of TILs assessed using the ITWG system is a useful marker of MMR/MSI status.
In conclusion, this study provides strong support for the assessment of TILs in CRC using the ITWG system. This simple system shows good interobserver concordance after minimal training, has superior prognostic value when compared with other methods of scoring tumor-associated lymphocytic inflammation in clinical practice, and predicts all-cause survival independent of MMR/MSI status, TNM stage, and a range of other patient and tumor factors.
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