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Immunoscore as a Predictor of Disease Recurrences and Patients’ Survival in Colon Cancer: A Clinicopathologic Study

BACKGROUND: For years, the American Joint Committee of Cancer/International Union against Cancer TNM staging system was the only accepted staging system for colorectal cancer. Different studies highlighted limitations in this staging system with the need to another staging approach that takes into consideration the individual patient immune response. Recently, the immunoscore was introduced; however, no accurate data regarding its sensitivity and specificity over the routinely used TNM staging system.


Introduction
Colorectal cancer (CRC) is now considered the second position in cancer-related mortality worldwide and is recognized to be a heterogeneous disease, with diverse prognostic outcome probabilities [1].
Staging of CRC is one of the most useful strategies for proper treatment planning. For decades, the American Joint Committee of Cancer/International Union against Cancer TNM staging system was highly recommended and the mostly accepted worldwide, with a high compliance rate in all medical centers and institutions [2].
However, despite its popularity, recent reviews have highlighted several limitations in this staging system [2], proved by that the prognosis can significantly vary among patients within the same stage. As well, tumor recurrences rate for cancer reaches up to 50% during the follow-up period regardless the stage, despite the optimal management, the facts that point to the potential influence of the individual tumor cells' biology [3].
The concept of this traditional staging system is based fundamentally on the assumption that tumor development and progression are basically cellautonomous processes which depend on the properties of the tumor cell itself and its degree of anaplasia. The prognosis, therefore, depends on the tumor cells progress inside the body at the time of surgery, the fact that ignore the variable and fascinating role of the host immune response and the active dynamic role of the tumor microenvironment [4].
In the past 10 years, the focus of cancer research has notably changed and the study of tumor microenvironment as a whole and tumor-associated inflammation specifically has taken the spotlight [5]. The comprehensive studies in tumor-associated inflammation throughout these years were extremely beneficial for cancer researches progress in different aspects and drive to the discovery of immunotherapy which proofed great results in different cancers as leukemia and cancer breast [6].
T lymphocytes, basically CD3 and CD8 positive cells, are considered the maestro of the body adaptive immune response, both cells play a fundamental role in the stage of immunosurveillance of the immunoediting [7].
In 2012, Galon et al. [8] proposed what called "immunoscore" IS and invited the scientific committees to further study its validity in different cancers. From that time and then, research teams all over the world started to explore IS and test its possible advantages over the routinely used staging system [9].
According to Galon et al. [10], the evaluation of "immunoscore" is calculated by counting CD8 and CD3 T lymphocytes both in the tumor core stroma and in the invasive margin (IM) fronts. The immunoscore is proposed to add significant data and provides important information regarding tumor cross-talk within its microenvironment that may tell a lot about the tumor behavior, the step that is mandatory to overcome the downsides in the routinely applied TNM staging system [10].
Many studies had examined the validity of immunoscore in different cancers as cancer liver and stomach [11], [12].
In cancer colon, immunoscore has been studied in correlation with different pathologic prognostic parameters [13]; however, correlation of IS with tumor recurrences and patients' survival is not frequently studied, and until now, the accuracy of IS in terms of sensitivity and specificity is not clear.
In this study, we are comparing the accuracy of cancer colon versus TNM staging system in predicting tumor recurrences and patient survival and well as in their association with other known prognostic factors as perineural invasion and tumor grade; aiming to provide definite sensitivity, septicity, and predictive values for both IS and TNM staging system in prognosis prediction, as evidence-based statistical documentation of its validity to clinical use.

Material and Methods
Fifty-three cases of colectomy for colon cancer were included in the study, collected retrospectively from cases in El Sheikh Zaid specialized Hospital, and other private centers from cases between 2015 and 2016: i.
Histopathology and data collection • The hematoxylin and eosin-stained sections from the tumor and dissected pericolic lymph nodes were examined for TNM staging. According to Edge and Compton, cases were staged as 1, 2, 3, and 4 [14]. For better data calculations, stages were grouped as two groups; low stage (Stages 1 and 2) and high stage (Stages 3 and 4) • Nodal deposits were evaluated according to the TNM routine methods as follows: N0: No nodes affected, N1: Positive tumor deposit in up to 2 lymph nodes, and N2: Positive deposits in more than two lymph nodes [14] • The presence of perineural invasion was evaluated as 1, the absence of perineural invasion in the examined tumor sections was expressed as 0 • Tumors' grades were evaluated as 1, 2, and 3 according to the routine guidelines.
ii. Prognostic data was represented by the recurrence-free survival RFS in 2 years' time follow-up period The occurrence of recurrence or mortality within the 2 years' time of the study was expressed as 1, while the absence of recurrence in the 2 years was evaluated as 0.
iii. Immunohistochemistry  iv. Image analysis morphometric study The morphometric analysis was performed at the pathology laboratory, at the Medical Research Centre of Excellence Unit, National Research Centre using the image analysis system Leica QWin DW3000 (LEICA Imaging Systems Ltd., Cambridge, England), which consists of Leica DM-LB microscope with JVC color video camera attached to a computer system. The slide to be examined was placed on the stage of the microscope. The light source was set to the required level. Successful adjustment of illumination was checked for the video monitor. The morphometric analysis was carried out on tumor tissue core and the invasion front in each slide stained with CD3 and CD8 using an objective lens with a magnification of ×20 [15].
The most representative five ×200 fields were assessed for each tumor section. Any areas of necrosis were as well as processing related artifacts were neglected [16].

v.
Immunoscore calculation Immunoscore was calculated according to Zhu et al. [17] considered the median of the values obtained from counting the CD3 and CD8 values as their cutoff value in segregation the low (0) and high (1) individual case immunoscore, and similarly, scores from 0 to 4 (by submission of the score of each marker both in tumors' cores and their invasion margins) were used (Figures 1 and 2).
For better data calculations, IS was grouped as two groups; low IS (0, 1, and 2) and high IS (3 and 4 In terms of accuracy, ROC curve analysis revealed that immune score was a significant discriminator for RFS (p < 0.05) where immune score showed an area under the curve = 0.879 with (95% confidence interval [CI] 0.788-0.971) (Figure 3).

•
The results of the immunoscore were as follows • IS 0: In 5 cases ( According to the statistical studies of this data, most of the cases of Stage I showed high IS (71.4%). In Stage II, high IS was seen in 63.2%. The percentage of high IS decreased to be 45.5% in cases of Stage   predict the patient prognosis (represented by RFS) with sensitivity = 28.6%, specificity = 37.5%, PPV = 23.1%, and NPV = 44.4%.

Discussion
Tumor microenvironment has become an important aspect in oncology research, focusing on the tumor cells interaction with its surroundings and considering the dynamic nature of the tumor cells-host immune system relationship [5].
The studies in tumor microenvironment lead eventually to the emergence of the what is called "Immunoscore" which was proposed to delineate the nature of the host immune reaction and represent an auspicious added value to the commonly used staging systems [13].
According to Galon et al., the immunoscore ("I") utilizes the counting of CD8 and CD3 T lymphocytes in the CT and the IM of the tumors to calculate a score value that ranges from (0), when low densities of both cell types are found in two examined regions, to (4), when high densities of both markers are found in both locations [8]. However, the exact determination of the thresholds (cutoff value) for low and high values differed in the subsequent studies [18].
The values' median and "best P value" were the most frequently used cutoff strategies, with variable overall potential advantages and disadvantages [9].
In this study, we calculated the IS of 53 cases of cancer colon by counting the CD3 and CD8 positive T lymphocytes in the tumor IM and its core using the image analysis system. The median value of the measures was used as the cutoff for determining the low and high scores for each marker before the final immunoscore calculation for each case. Immunoscore was compared with TNM staging in relation to tumor grade, perineural invasion, nodal status, and the most important RFS. Accuracy of the test was evaluated in terms of sensitivity, specificity, and predictive values for each test of them in (both immunoscore and TNM staging system) in respect to patient prognosis, represented by RFS in 2 years of follow-up.
According to our results, higher IS values were observed in low TNM stages and vice versa. However, despite the higher values of IS in lower TNM stages yet, the association showed no statistical significance.
Regarding the other prognostic factors, IS was strongly related to the nodal status as well as to the tumor grades (p = 0.041 and 0.013, respectively). However, no statistical association was seen between IS and the perineural invasion.
The association between the IS and the tumor stages was observed as well in some previous studies; Sinicrope et al. noticed higher IS in the low TNM stage category, with 91% of patients of low stages showed high IS, versus 68% of the patients of high stages. In their study, the values showed a significant statistical association [19].
In the present study, a strong association between IS of the cases and their RFS was observed (p < 0.001), as 85.7% of cases with no recurrences or disease-related mortality were of high IS category in comparison to 14.3% of low IS group.
In similar regards, Anitei et al. studied IS in a patient with and without pre-operative chemotherapy CT. They noticed the IS was associated with diseasefree survival and overall survival data. They suggested IS as an important risk factor and recommended its evaluation in an international multicenter study, although they advised against the measurement of IS after CT, as the later may cause significant changes in the immune cells' distribution within the tumor [2]. Galon et al. noticed that only 13% of cases that showed recurrences in their study was of high IS category. They related the results of IS with mismatch repair genes MSI and used both tests to define patients eligible for immunotherapy (immune checkpoint inhibitors, ICI), based on that patients with high T-cell infiltration, hence high IS, will eventually show a higher expression of PD-1 and PDL1, and therefore, they will be more likely to respond to ICI. They recommend more studies of IS accuracy and predictive values [20]. Galon et al. tested IS in patients of Stage II colon cancer with high-risk factors; in their results, they demonstrated that up to 69% of these patients showed a low rate of recurrences and that the recurrence was associated with higher IS values. They, therefore, concluded that IS may be a valuable test in neoadjuvant chemotherapy decision in Stage II patients [21].
The results of the international immunoscore project were concordant to our results regarding the association between IS values and patients' recurrences and survival. For example, in patients of Stage II tumors in their study (n = 1434), high IS was significantly associated with a low risk of recurrence and high overall survival rates (p < 0·05). Recurrence . They got RMST of 6.85 years (95% CI 6.54-7.17) in the high-immunoscore group and 5.25 years (4.79-5.71) in the low-immunoscore group. They, therefore, concluded that IS correlation with RMST provides clinically valid measurement of recurrence https://www.id-press.eu/mjms/index time for each immunoscore category and highlight the role of IS as a prognostic test [11].
Similarly, Kirilovsky et al. discussed the possible rationale basis of IS. From their study, they recommended IS as a predictor for CT and radiotherapy response as a predictor for immunotherapy response and a pivotal factor in recurrence and survival prediction [23].
Using the provided data in our results, ROC curve was used to define novel intrinsic values for IS in recurrence risk estimation and clinical prognosis prediction. According to our results, IS could effectively predict RFS of the 2 years follow-up of this study; the sensitivity was 85.7% compared with 28.6% in TNM staging system. The specificity was 78.1% versus 37.5% specificity of the TNM staging system. The positive predictive value PPV of IS was up to 72% in contrast to TNM PPV which was 23.1% and finally the negative predictive value of IS was 89.3% compared with 44.4% for TNM. According to these values, IS is superior as a screening and confirmatory test over TNM staging system. Although many studies had evaluated the accuracy of IS and TNM in the prediction of the clinical prognosis; yet, none of them -to the best of our knowledge -provided quantitative data about this point in terms of sensitivity, specificity, PPV and NPV.
The high sensitivity of IS in this study recommended it as a strong screening test for prognosis in colon cancer so that low IS should, therefore, consider as a poor prognostic factor. On the other hand, the high specificity of IS recommended it as a good confirmatory test that can confirm the data suggested by other risk factors profile.
Many studies recently recommended the approval of IS testing for all cases of colon cancer as prognostic profiling of the cases. In 2016, Kirilovsky et al. suggested what they call "TNMI" to integrate the use of IS as part of tumor staging [23].
New studies pointed to the importance of IS not only as a prognosis predictor but moreover as a predictor of the possible role of immunotherapy in colon cancer [24]. Moreover, similarly, Ogino and Giannakis pointed to the significance of tumor immune response study. They suggest that what they called "Tumor Immunity in the Micro Environment (TIME) classification" may become a new creditable trend incorporating the host immune response in cancer classification system. However, they found that the IS has not taken its proposed role in clinical use until now [25].
We agree with Donnem et al. that the validation of the IS measure methodology and pre-definition of the cutoff value used in its calculation is the most important and still missing points in the routine clinical application of IS [9].
Despite the effort provided by the international immunoscore team to validate IS as an important test in colon cancer patients, yet, still, the unavailability of a defined independent cutoff value to be used internationally in all centers and be internationally accepted, still a handicapping point against the routine use of IS in daily work. Collaborative work is needed to initiate robust predefined organ-specific cutoff values similar to the values used in Ki67 evaluation, for example.

Conclusion
Our study demonstrated the promising role of IS in prognostication of colon cancer, presented its association with different risk factors and quantitatively measured its accuracy as a predictor of survival in colon cancer.
Accordingly, we, therefore, recommend to integrate the computed technologies of image analysis and digital pathology in the individual patient's care and transfer the IS from the research zone to the clinical daily use, so that IS may be a proposed part of the pathology report, to enrich the clinical management with the immune system profile data that will dramatically influence the management in colon cancer and all other tumors.