Prognostic Value of Immunoscore in Colorectal Carcinomas

doi:10.21203/rs.3.rs-2234645/v1

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Prognostic Value of Immunoscore in Colorectal Carcinomas

https://doi.org/10.21203/rs.3.rs-2234645/v1

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Background

Immunoscore (IS), based on the evaluation of CD3 + and CD8 + densities in the center of the tumor (CT) and its invasive margin (IM), is currently considered as a potential prognostic factor, particularly in colorectal carcinomas (CRC). In the current study, we aimed to assess the prognostic value of IS in CRC stage I to IV, through a survival study.

Methods

It was a descriptive and retrospective study involving 104 cases of CRC. Data were collected over three years (2014–2016). An immunohistochemical study (anti-CD3, anti-CD8) by the Tissue Microarray (TMA) technique was carried out in the areas of "Hot Spot" in CT and IM. A percentage was assigned to each marker and within each region. Then, the density was classified as "Low" or "High", according to a cut-off fixed at the median of percentages. IS was calculated by the method described by Galon et al. The prognostic value of the IS was assessed through a survival study.

Results

The mean age of patients was 61.63 years. IS was low in 60.6% (n = 63). Our study had shown that low IS significantly deteriorates survival and a high IS enhances survival significantly (p < 0.001). We found a correlation between IS and T stage (p = 0.026). A multivariate showed that the predictive factors for survival were IS (p = 0.001) and age (p = 0.035).

Conclusion

IS is a potential prognostic factor. Its reproducibility and reliability allow its introduction into daily practice for better therapeutic management.

Immunoscore

Tumor-infiltrating lymphocytes

Colorectal carcinomas

Immunohistochemistry

Pronostic

Survival

Colorectal carcinoma (CRC) is a major public health problem. Its annual incidence is increasing representing the third most occurring cancer in the world and the second cause of death by cancer in 2020 [1].

CRC is a heterogeneous disease with different suggested prognostic factors [2]. The prognostic value of pTNM tumor staging classification of CRC has been known for decades. However, it is currently accepted that the clinical course can vary considerably between patients within the same histological stage pTNM [3,4]. This has directed research towards new prognostic factors, in particular those related to the tumor microenvironment, and more particularly, to tumor-infiltrating lymphocytes (TILs).

In the literature, the infiltration of the tumor by lymphocytes is predictive of cancer progression and would therefore represent a potential prognostic factor [2]. TILs would play a role in inhibiting tumor growth and would be associated with a better prognosis [5].

In this context, a new test called "Immunocore" (IS), based on the immunohistochemical evaluation of total T lymphocytes (CD3+) and cytotoxic T lymphocytes (CD8+) densities in the center of the tumor (CT) and its invasive margin (IM) has been proposed [6]. Studies have shown that this immune test is an independent prognostic factor, correlated with better survival and lower risk of recurrence [7]. The prognostic value of IS was validated in 2018 during an international consortium [8]. Thus IS seems to have a therapeutic impact, particularly for stage II and III CRC [8].

In Tunisia, the evaluation of IS is not yet carried out and a few studies on the prognostic significance of IS were conducted until now.

The objective of our study was to evaluate the prognostic value of IS in a Tunisian series of CRC.

One-hundred and four patients diagnosed with CRC at the Pathology department of Habib Thameur Hospital in Tunisia during a three-year period (2014–2016) were included in this trial.

Inclusion criteria

Our study included all patients with histologically proven colorectal carcinoma stage I to IV, who underwent surgical resection of the tumor during the study period (2014–2016) at Habib Thameur Hospital in Tunisia.

Exclusion criteria

We did not include in this study Patients who received neoadjuvant therapy that could influence the immune response in the tumor microenvironment.

Management of patients

Pre-operative assessment included a complete medical history, a physical examination, a colonoscopy with biopsies, and a computed tomographic scans. Gender, age, tumor location, tumor size, gross appearance, lymphovascular, or perineural invasion, histological classification according to the 2019 WHO classification, TNM stage according to the 8th edition of the American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC) and type of surgery were recorded.

Selection of ‘’Hot spot’’ areas

For each case of CRC, the ‘’Hot spot’’ areas of lymphocyte infiltration at the CT and at the IM were selected on HE slides. CT was defined as the central region containing the tumor stroma and cancer cell nests. IM corresponded to the invasive front. Only lymphocytes were counted. Granulocytes, macrophages, mast cells, and plasma cells were not considered. Areas of necrosis and fibrosis have been avoided.

Immunohistochemical staining

An immunohistochemical study was carried out on the TMA slides, obtained from the recipient blocks, allowing the analysis on a single slide, of several samples. The study was carried out using the complex Avidin-Streptavidin technique using the BOND-MAX automaton (Leica Biosystems, Melbourne, Germany). Anti-CD3 and anti-CD8 antibodies (Leica Biosystems, Germany) were used for the study of T cells.

Evaluation of T lymphocytes densities

A semi-quantitative evaluation of T cells densities in the "Hot Spot" area on a surface of 0.95 mm² was carried out, following these procedures :

Measurement of an area of 0.95 mm² which corresponds to a field at magnification x 200.

For each region (CT and MI) and for each marker (anti-CD3 and anti-CD8), a photo where the lymphocyte infiltrate was maximal at magnification x 200 was taken.

A percentage of the lymphocyte infiltration was assigned to each photo (Fig. 1) :

The median of the infiltration percentages was calculated and retained as a Cut-off for the determination of densities (Table 1).

The density was considered “Low” when it was lower than the median of the percentages. It was considered “High” when it was above this median.

Table 1

Percentage of CD3 + and CD8 + lymphocyte densities in CT and IM and their median
	% CD3+		% CD8+
	CT	IM	CT	IM
Minimum	2	1	0	0
Maximum	65	65	35	30
Median	20	17,5	10	9

Computation of IS

We followed a method published by Galon et al [6]. This method has been validated in a task force to which 22 worldwide experts have participated. Indeed, for each marker (CD3 and CD8) and in each region studied (CT and IM), a score of 0 was assigned when the density was “Low” and a score of 1 when the density was “High”. The IS was obtained by the addition of these scores to give a minimum score of 0 and a maximum score of 4 (Fig. 2) :

I0 : low density of CD3 + and CD8 + in the CT and the IM.
I1 : high density of one of the markers in a single region (CT or IM).
I2 : corresponding to a score of 2.
I3 : corresponding to a score of 3.
I4 : high density of the two markers (CD3 + and CD8+) in the two regions (CT and IM).

Statistical analysis

We carried out a descriptive and analytical study. Data collection was stopped on 10/04/2021. Overall survival and predictive factors of survival were analyzed. Univariate analysis aimed to determine dependent prognosis factors. Qualitative variables were compared using the chi-square test or Fisher's exact test. Quantitative variables were compared by the independent Student-t test. Survival was analyzed by the Kaplan–Meier method and included postoperative deaths. The log-rank test was used to compare survival curves. Multivariate analysis aimed to determine independent prognosis factors. It was performed by linear regression by Cox proportional hazards stepwise procedure, including non redundant variables chosen by univariate analysis. All statistical analyses were performed using SPSS version 18.00. A p value 0.05 was considered statistically significant.

A total of 104 patients were included in the study. The clinicopathological features were detailed in Table 2. The average age was 61.6 years. A male predominance was noted (sex ratio = 1.36). The location of the tumor was colonic in 92.3% of cases (n = 96). The tumor size varied from 1.5 cm to 13 cm with an average of 5.52 cm. Histologically, adenocarcinoma not otherwise specified (NOS) was the most represented histological type (84.6% ; n = 88). The grade was low in 95.5% (n = 84). CRCs were mainly classified as pT3 and pT4 (88.4% ; n = 92). Vascular emboli and perineural invasion were present in 49% (n = 51) and 46% (n = 32) of cases, respectively. Lymph node metastasis was revealed in 43.3% (n = 45) of cases. Visceral metastasis was noted in 12.5% (n = 13) of cases. The pTNM stage was mostly II or III (75.9%), and IV in 12.5% of cases.

Table 2 Demographic and clinicopathological features of 104 primary Colorectal carcinomas

Variable

N (%)

Age (years)

• ≤ 40 ans

• 40 ans

9 (8,7%)

95 (91,3%)

Gender

• Male

• Female

60 (57,7%)

44 (42,3%)

Tumor size (cm)

• ≤ 4

• > 4

25 (24,1%)

79 (75,9%)

Tumor location

• Colonic

• Rectal

96 (92,3%)

8 (7,7%)

Histological type

• Adenocarcinoma NOS

• Mucinous Adenocarcinoma

• Rare subtypes

88 (84,6%)

12 (11,5%)

4 (3.9%)

Tumor differenciation

• Low grade

• High grade

84 (95,5%)

4 (4,5%)

Vascular emboli

• Absent

• Present

53 (51%)

51 (49%)

Perineural invasion

• Absent

• Present

72 (69,3%)

32 (30,7%)

pT stage

• p T1

• p T2

• p T3

• p T4

1 (1%)

11 (10,6%)

62 (59,6%)

30 (28,8%)

pN stage

• N0

• N+

59 (56,7%)

45 (43,3%)

pM stage

• M0

• M+

91 (87.5%)

13 (14.9%)

pTNM stage

• Stage I

• Stage II

• Stage III

• Stage IV

12 (11,6¨%)

46 (44,2%)

33 (31,7%)

13 (12,5%)

• I 0

• I 1

• I 2

• I 3

• I 4

20 (19,2%)

16 (15,4%

27 (26%)

17 (16,3%)

24 (23,1%)

• Low (≤ 2)

• High (>2)

63 (60,6%)

41 (39,4%)

Of the 104 patients, 16 patients were lost to follow-up. We were only able to judge the evolution in 88 patients. The average follow-up was 40.29 months.

The 1-year, 3-year, 5-year survival rates of CRC patients were 86,3%, 63,9% and 51,2%.

IS was I0 in 19.2% (n = 20), I1 in 15.4% (n = 16), I2 in 26% (n = 27), I3 in 16.3% (n = 17) and I4 in 23.1% (n = 24). Two groups were identified: those with a low IS (≤ 2: I0-I2) and those with high IS (> 2: I3 and I4). A predominance of low IS (60.6%, n = 63) was noted.

Univariate analysis showed a strong association between the different groups of IS (from I0 to I4) and the Overall survival (OS) (p = 0.001). Similarly, low IS (I0-I2) was correlated with shorter survival and high IS (I3 and I4) was correlated with longer survival (p < 0.001). Survival curves illustrating the association between IS and OS are shown in Fig. 3.

The other prognostic factors : age, tumor size, histologic grade, pT stage, pM, pTNM stage, vascular invasion, and perineural invasion tended to reduce survival without significant difference (Table 3).

Table 3 Univariate analysis of factors influencing the overall survival in Colorectal Carcinomas

Variable

N (%)

5 year survival

Gender

• Male

• Female

52 (59.1%)

36 (40,9%)

58.1%

62.5%

0.257

Age (years)

• ≤ 40

• > 40

9 (10.2%)

79 (89.8%)

85.7%

55%

0.109

Tumor size

• ≤ 4 cm

• > 4 cm

22 (25%)

66 (75%)

63.6%

58.3%

0.883

Histological subtype

• ADK NOS

• Mucinous ADK

75 (84,6%)

9 (11.5%)

58.5%

66.7%

0.187

Histological grade

• Low grade

• High grade

71 (94.7%)

4 (5.3%)

57.9%

66.7%

0.414

Vascular invasion

• Absent

• Present

43 (48.9%)

45 (51.1%)

69.2%

47.6%

0.197

Perineural invasion

• Absent

• Present

62 (70.5%)

26 (29.5%)

58.3%

63.6%

0.312

pT stage

• T1

• T2

• T3

• T4

1 (1.2%)

9 (10.2%)

50 (56.8%)

28 (31.8%)

71.4%

50%

76.9%

0.280

• N0

• N1

50 (56.8%)

38 (43.2%)

60.7%

57.9%

0.357

• M0

• M1

63 (71.6%)

10 (11.4%)

62.9%

75%

0.339

pTNM stage

• I

• II

• III

• IV

10 (11.4%)

39 (44.3%)

29 (32.9%)

10 (11.4%)

62.5%

60%

53.3%

75%

0.211

• I0

• I1

• I2

• I3

• I4

13 (14.8%)

22 (25%)

16 (18.2%)

24 (27.2%)

50%

40%

60%

54.5%

68.4%

0.001

• Low

• High

48 (54.5%)

40 (45.5%)

52.9%

63.3%

<0.001

Multivariate Cox proportional hazards models included variables associated with survival in univariate analysis, age, degree of parietal invasion, presence of vascular emboli, stage and IS showed that IS was the strongest independent prognostic factor (p = 0.001), follow-up by age (p = 0.035) (Table 4).

Table 4

Multivariate analysis of overall survival in Colorectal Carcinomas
Variable	HR	IC95%	p
Immunoscore	4.17	1.972 _ 8.816	< 0.001

A significant association was found between advanced pT stage and low IS (p = 0.026). On the other hand, IS was not correlated with age, tumor size, tumor grade, vascular emboli, perineural invasion, metastasis and pTNM stage (Table 5).

Table 5

Association between Immunoscore and other prognostic factors
Variable	IS Low	IS High	p
Age (years)			0.736
• ≤ 40 • > 40	5 58	4 37
Tumor size			0.242
• ≤ 4 cm • > 4 cm	18 45	7 34
Histological grade			0.638
• Low grade • High grade	52 2	32 2
pT stage			0.026
• T1 • T2 • T3 • T4	1 5 44 13	0 6 18 17
Vascular invasion			0.316
• Absent • Present	35 28	18 23
Perineural invasion			0.285
• Absent • Present	41 22	31 10
pM			1
• Absent • Present	45 8	29 5
pTNM stage			0.876
• I • II • III • IV	6 29 20 8	6 17 13 5

We calculated the IS in 104 patients with CRC following the recommendations published by Galon et al [6] but we have replaced the automated quantification method to determine the densities of T cells lymphocytes with a semi-quantitative method. We found that the immune response varied considerably between patients ; Indeed, 19.2% of patients had an IS I0, 15.4% had an IS I1, 26% had an IS I2, 16.3% had an IS I3 and 23.1% had an IS I4.

Two groups of patients were individualized: those with a low IS (≤ 2: I0-I2) and those with high IS (> 2: I3 and I4). A predominance of the group with a low IS (60.6%, n = 63) compared to the other group (39.4%, n = 41) was revealed.

The univariate analysis showed that there was a significant correlation between the different IS groups and 5-year survival (p = 0.001). Thus, a low IS was correlated with short survival, and a high IS was correlated with longer survival (p < 0.0001).

The IS was correlated with the degree of parietal invasion (p = 0.026).

For decades, the pTNM stage was thought to be the best prognostic factor in CRC. However, the clinical outcome can vary in patients with the same stage. The pTNM stage focuses on tumor cells and doesn’t take into account the host immune response. Recent studies support the role of immune cells in tumor progression, particularly, lymphocytes infiltrating tumors are demonstrated to have a protective role against tumor cells [5].

Of all immune cells involved in the tumor microenvironment, CD3, CD8, and CD45RO lymphocytes had the best prognostic impact [9,10]. In the present study, CD3 and CD8 were chosen as markers because of the stability of these antigens and the good quality of staining. CD45RO was excluded because of background staining and loss of antigenicity in stored sections as proven in other studies [11]. Besides, the prognostic value of IS in different patient groups was better in a combined analysis of two regions: CT and IM compared to a single region analysis [12]. So, IS is a test based on the evaluation of densities of both lymphocytes CD3 and CD8 in two specific regions: CT and IM [13].

The determination of IS is automated in most studies using software dedicated to IS. In Tunisia, we didn’t have software to enumerate lymphocytes in tumors. A semi-quantitative study was carried out and validated by significant results. To the best of our knowledge, this is the first Tunisian study that confirmed the prognostic value of IS using a semi-quantitative method. Therefore, it represents an alternative tool for quantification of IS, particularly in countries that do not have the software. However, This semi-quantitative method remains less precise than the automated method.

Based on the analysis of a cohort of 104 patients with CRC, we demonstrated the prognostic value of IS on Overall Survival (OS) : high IS was associated with longer OS while low IS was associated with shorter OS. These findings support the conclusions of many previous studies implicating immune cells in CRC. The majority have confirmed an association between IS and OS or Disease Free Survival (DFS) [8,15–23].

Pagès et al [8] have proved that the prognostic value of IS was superior to the pTNM staging system. They proposed the introduction of IS as a new component of the classification of cancers designated TNM-Immune (TNM-I).

IS has also a therapeutic role in CRC. On one hand, patients with CRC stage II, having a low IS, will be justified for adjuvant chemotherapy which would be useless for the group with a high IS [24]. On the other hand, patients with CRC stage III and a high IS will be justified for shorter adjuvant chemotherapy (3 months) compared to the group with a low IS (6 months) [24, 25]. Thus, the IS allows personalized therapeutic management of patients with CRC stage II and III.

IS is a simple, rapid, and reproducible immune test with a strong prognostic value and therapeutic impact. The introduction of this parameter in daily practice becomes mandatory.

Ethical Approval

The study was approved by the Ethics Committee of Habib Thameur Hospital
of Tunisia (approval number : HTHEC-2021-24). No signed informed consent was obtained by patients due to retrospective collect of data. Patient's confidentiality was respected according to ethical guidelines. All methods were carried out following relevant guidelines
and regulations.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

HA and HI, conceptualization, formal analysis, data curation, methodology, and writing the main manuscript. KFand JR, formal analysis, visualization, investigation and software. ZH and SM, data curation and software. CDA, methodology. BBE and CDA, validation. The authors read and approved the final manuscript.

Funding

There is no fund supporting this study.

Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available due to department policy but are available from the corresponding author on reasonable request.

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No competing interests reported.

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Prognostic Value of Immunoscore in Colorectal Carcinomas

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Inclusion criteria

Exclusion criteria

Management of patients

Selection of ‘’Hot spot’’ areas

Immunohistochemical staining

Evaluation of T lymphocytes densities

Statistical analysis

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

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