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Table of Content-Volume 12 Issue 2 - November 2019


 

 

 

Association of tumour infiltrating lymphocytes with conventional clinicopathological prognostic factors of colorectal adenocarcinoma: A study from eastern India

 

Debjani Mallick1, SayanKundu2, Sudipta Chakrabarti3, Prosun Gayen4

 

1Associate Professor, 2Senior Resident, 3Professor, Department of Pathology, ESI PGIMSR and ESIC Medical College, Joka, Kolkata, West Bengal, INDIA.

4Alumnus, Department of Applied Statistics and Informatics, Indian Institute of Technology, Bombay. Powai, Mumbai 400 076,

Maharashtra, INDIA.

Email: sudiptach@gmail.com

 

Abstract               The anti tumour defense mechanisms plays an important role in tumour microenvironment. The composition of inflammatory cells present determine the immune responses. The aim of the present study was to evaluate both the stromal and intraepithelial tumour infiltrating lymphocytes (TIL) morphologically in the invasive front and the center of the tumor mass in patients with colorectal adenocaecinoma and determine their association with the other conventional clinic pathological features. This was a retrospective study of 150 patients of colorectal adenocaecinoma from 2014 to 2019 selected randomly. Data was collected for age, sex, location of the tumour, tumour size, tumour gross growth pattern, histological type, tumour depth, lymph node status, number of positive lymph nodes, lymphovascular invasion, perineural invasion and TNM staging. TIL was determined as per recommendations of International Working TIL group. TIL score of more than 50 percent were labeled as high TIL and TIL score less than 50 percent as low TIL. Univariate analysis was done for all the variables. Evaluation of TIL score was assessed with the variables. Absence of lymphovascular invasion, lymph node metastasis and metastasis in less than 5 lymph nodes were observed with high TIL (75%, 75%, 89% respectively).Conversely presence of low TIL were observed with presence of lymphovascular invasion, lymph node metastasis and metastasis in more than 5 lymph nodes (81%,76%,63% respectively). The statistically significant variables associated with TIL were lymphovascular invasion, lymph node metastasis and number of lymph node metastasis on the basis of Chi square test, information value and correlation coefficient. Hence evaluation of TIL infiltration is recommended in routine histopathological examination.

Key Word: Colorectal adenocarcinoma, Conventional prognostic parameters, tumour infiltrating lymphocytes

 

 

INTRODUCTION

The antitumour defense mechanisms plays an important role in tumour microenvironment. The composition of inflammatory cells present determine the immune responses1.Lymphocytes present in the tumourmicro environment, also known as Tumour Infiltrating Lymphocytes (TIL), identify the tumour antigens. Their antitumor effect may be either due to direct tumorlysis, or by release of chemotactic and pro‑inflammatory cytokines2.The role of TILs on tumor progression as well as clinical outcome have been reported in various types of cancer, including colorectal cancer.3,4 Galon et al. found that the density of TIL is even more valuable prognostic markers than the conventional TNM classification5. The aim of the present study was to evaluate both the stromal and intraepithelial TIL morphologically in the invasive front and the center of the tumor mass in patients with colorectal adenocaecinoma and determine their association with other conventional clinicopathological features. TIL populations were assessed both in the stroma and in the intraepithelial tumour tissue at the invasive front and center of the tumor by light microscopy.

 

MATERIAL AND METHODS

The present study was a retrospective study of 150 colorectal adenocarcinoma patients from 2014 to 2019 selected randomly. Data was collected for age, sex, location of the tumour, tumoursize, tumour gross growth pattern, histological type, tumour depth, lymph node status, number of positive lymph nodes, lymphovascular invasion, pernineural invasion and TNM staging. As per institutional protocol all the specimens were fixed with 10% neutral phosphate-buffered formalin. Paraffin-embedded, 4 μm-thick sections from representative areas of the tumour were stained by Haematoxylin and Eosin stain were studied.

Inclusion criteria included colectomy specimens of colorectal adenocarcinoma irrespective of location of tumour within the colon and its stage.

Exclusion criteria was small biopsies from colon and rectum, colorectal neoplasm other than colorectal adenocaecinoma, colorectal cancer cases with neo adjuvant chemotherapy, extensive necrosis. Tumour depth was determined as per seventh AJCC TNM classification6.TIL was determined including both stromal and intraepithelial TILs at the invasive front and centre of the tumour mass as per recommendations of International Working TIL group7.Tumour bed was scanned and average TIL was scored in 400X microscopic fields. Stromal and intraepithelial TILs comprised all mononuclear cells excluding polymorphonuclear leucocytes. Stromal TIL score was determined by percentage of stromal areas occupied by mononuclear inflammatory cells. TIL score of more than 50 percent were labeled as high TIL and TIL score less than 50 percent as low TIL. Data was tabulated and analyzed statistically. Univariate analysis was done for all the variables. Different statistical measures like correlation coefficient, Chi Square test and information value were calculated for all the variables with TIL to test their significance level. P value was calculated by Wald Chi square test for each variable. A value of p <0.0001 was considered to indicate statistical significance. Bar diagram of correlation coefficient and information value was plotted for all the significant variables.SAS software was used to analyze the data.

RESULT

Mean age of the population under study was 58 +/-2 years. Patients were predominantly male (113 cases, 75%). Sigmoid colon(34 cases,,23%) and rectum (33 cases,22%) followed by left colon (33 cases,22%) were the most frequent location of the tumour. Tumour size more than 5cm occurred in78cases (52 %) with predominantly expanding type of growth grossly (90 cases,60%).Non mucinous adenocarcinoma was the most prevalent histological type (132cases,88% ) followed by the mucinous infiltrating type.(18cases,12%).Tumour depth varied from T1 to T3 in most of the cases (92cases,61%).Lymph node metastasis was seen in 83 cases (55%) but lymph node metastasis occurred in more than 5 lymph nodes in40cases(48%). Lymphovascular invasion was present in 87 cases (58%),perineural invasion occurred in 74 cases (59%) and were mostly seen in aggressive tumours. TNM stage II was the commonest category in the colon cancer patients in the present study. (Table 1)(Figure1) Absence of lymphovascular invasion, lymph node metastasis and metastasis in less than 5 lymph nodes were observed with high TIL (75%, 75%, 89% respectively).Conversely presence of low TIL were observed with presence of lymphovascular invasion, lymph node metastasis and metastasis in more than 5 lymph nodes (81%,76%,63% respectively).Evaluation of TIL score was assessed with the variables and the statistically significant variables were lymphovascular invasion, lymph node metastasis and number of lymph node metastasis on the basis of Wald Chi square test, information value and correlation coefficient. Bar diagram of the correlation coefficient and information value showed that the significance of association with TIL of the three statistically significant variables; lymphovascular invasion, lymph node metastasis and number of lymph node metastasis are in descending order. Though not statistically significant, high TIL was found in rectal tumours 25cases (41%). Age, sex, tumour location, tumour size, tumour growth, histological type, tumour depth and TNM stage were non-significant variables in the present study in association with TIL.(Table 2)(Figure2)(Figure3)


 

 

 

Table 1: Characteristics of colon cancer patients with respect to prognostic variables

Sl No.

Variable

Number of cases

Percentage

1.

Age in years

Less than 60

More than 60

 

74

76

 

49

51

2.

Sex

Male

Female

 

113

37

 

75

25

3.

Location

Right

Transverse

Left

Sigmoid

Rectum

 

24

22

33

34

33

 

18

15

22

23

22

4.

Tumour Size

Less than 5 cm

More than 5 cm

 

72

78

 

48

52

5.

Tumour growth

Expanding

Infiltrating

 

90

60

 

60

40

6.

Histological type

Non mucinous

Mucinous

 

132

18

 

88

12

7.

Tumour depth

T1-3

T4

 

92

58

 

61

39

8.

Lymph Node Metastasis

Present

Absent

 

83

67

 

55

45

9.

No. of lymph node metastasis

Less than 5

More than 5

 

43

40

 

52

48

10.

Lymphovascular Invasion

Present

Absent

 

87

63

 

58

42

11.

Perineural Invasion

Present

Absent

 

74

76

 

59

51

12.

TNM staging

I

II

III

IV

 

32

43

41

34

 

21

29

27

23

 

Table 2: Correlation of prognostic variables with TIL and their statistical significance

Sl no

Variable

TIL more than 50%

TIL less than 50%

P value

Correlation

with TIL

Number of cases

Percent

Number of cases

Percent

1.

Age

Less than 60

More than 60

 

26

35

 

43%

57%

 

48

41

 

54%

46%

0.1735

0.11

2.

Sex

Male

Female

 

48

13

 

79%

21%

 

65

24

 

73%

27%

0.4299

 

0.06

3.

Location

Right

Transverse

Left

Sigmoid

Rectum

 

8

9

9

10

25

 

13%

15%

15%

16%

41%

 

19

13

24

24

9

 

21%

15%

27%

27%

10%

0.0003

 

0.31

4.

Tumour Size

Less than 5 cm

More than 5 cm

 

28

33

 

46%

54%

 

44

45

 

 

49%

51%

0.6702

 

0.03

5.

Tumour growth

Expanding

Infiltrating

 

38

23

 

 

62%

38%

 

 

52

37

 

 

58%

42%

 

0.6347

 

0.03

6.

Histological type

Non mucinous

Mucinous

 

 

55

6

 

 

90%

10%

 

 

 

77

12

 

 

87%

13%

0.4995

 

0.05

7.

Tumour depth

T1-3

T4

 

42

19

 

69%

31%

 

 

50

39

 

 

56%

44%

 

0.1174

0.13

8.

Lymph Node Metastasis

Present

Absent

 

 

15

46

 

 

 

25%

75%

 

 

 

68

21

 

 

 

76%

24%

 

3.6066E-10

(<0.0001)

0.51

9.

No. of lymph node metastasis

 

Less than 5

More than 5

 

 

 

54

7

 

 

 

89%

11%

 

 

 

 

33

56

 

 

 

 

37%

63%

 

2.8879E-09

(<0.0001)

0.46

10.

Lymphovascular Invasion

Present

Absent

 

 

15

46

 

 

25%

75%

 

 

72

17

 

 

81%

19%

6.7151E-12

(<0.0001)

0.56

11.

Perineural Invasion

Present

Absent

 

 

26

35

 

 

 

43%

57%

 

 

 

48

41

 

 

54%

46%

 

0.1735

 

0.11

12.

TNM Staging

I

II

III

IV

 

19

22

11

9

 

31%

36%

18%

15%

 

13

21

30

25

 

15%

24%

34%

28%

0.0053

 

0.27


 

1

Figure 1: (a): The section showing invasive front of the rectal adenocarcinoma associated with stomal high TIL (HandE400X); (b): The section shows mucinous adenocarcinoma colon associated with low stromal TIL (HandE400X); (c): The section shows high TIL in the centre of the tumour ((HandE400X); (d): The section shows lymphovascular invasion associated with high TIL (HandE400X)

23

Figure 2                                                                                                Figure 3

Figure 2: Bar diagram showing information vale for all the significant variables associated with TIL

Figure 3: Bar diagram showing correlation coefficients for all the significant variables correlated with TIL

DISCUSSION

The present study evaluates the Tumour Infiltrating Lymphocytes in colorectal adenocaecinoma and its association with other conventional clinic pathological prognostic factors. After review of literature it was found that the present study was probably the only such study from India and especially from Eastern region of India. The clinical relevance of evaluation of the presence of intraepithelial and stromal TILs in colorectal cancer is that it might determine the degree of involvement of the immune response in colorectal cancer patients8.The first line of defense against cancer metastasis is the immune stimulation at the invasive front of the tumor. If inadequate, the selective clones of cancer cells invade the blood and lymphatic vessels, and perineural spaces, and spread to the surrounding tissues and to other organs9. The mean age at diagnosis in the present study was 58 +/- 2 years. Peedikayil et al also found mean age of 58.4 years in their study of colorectal cancer in Indian patients. They found 74% of the tumours located distal to the splenic flexure10. This was concordant to the present study where the commonest occurrence of the tumour were sigmoid colon, rectum and left colon together comprising 68% of the total tumours. 67.7% of the patients were male and 32.3% female in the study of Peedikayil et al10.The present study also showed male predominance (75%). The findings are concordant due to the fact that both the studies are conducted amongst the Indian population. Tumour size was non-significant variable with respect to TIL in the study of Matsutani et al. (pvalue:0.866) and Huh et al (p value: 0.87).11,12.In the present study also tumoursize was not statistically significant with respect to TIL (p value:0.67, Correlation coefficient : 0.03). Tumour growth was expanding type in most of the cases in present study (60%) but had poor correlation with TIL (0.03). Similarly, in the study of Jakubowska et al, expanding growth occurred in 83% of cases but the variable was non-significant with respect to its correlation with TIL8. Histological type was predominantly non mucinous though not statistically significant in the study of Jakubowska et al similar to the present study8.Most of the tumours occurred in T1 to T3 stage of tumour depth as per TNM classification but the variable was non-significant in its correlation with TIL in the study of Matsutani et al11.The present study also had 61% of the tumours in stage T1 to T3 but was non-significant in its association with TIL based on P value and correlation coefficient. In study of Jakubowsk et al, a decline in the stromal TIL was observed with the involvement of blood vessel, lymphatic tubes, perineural space and the presence of lymph node metastases. This determined that TIL has pivotal role in generating the immune response8. Similarly in the present study, low TIL score was seen in presence of lymphovascular invasion, lymph node metastasis and even more when more than 5 lymph node metastasis were present. However perineural invasion did not show significant association with TIL in the present study. On the other hand Pagès et al demon­strated that the high TIL at the invasive margin was associated with a lack of the char­acteristics of metastatic processes like vascular emboli, lymphatic invasion and perineural invasion in colorectal cancer13.The present study also showed high TIL score in absence of lymphovascular invasion, lymph node metastasis and lymph node metastasis in less than 5 lymph nodes. The present study showed strong association of number of metastatic lymph nodes with TIL. Lower TIL was observed with increased number of lymph node metastasis. However Jakubowska et al did not find statistically significant association with number of metastatic lymph nodes8. The degree of maturity of fibrotic cancer stroma depends of various factors like matrix components, the cells involved in matrix synthesis and also the inflammatory cells including lymphocytes, which produce and activate cytokines. Hence the increase in the number of TILs resulted in stromal maturity14. It has been demonstrated that the maturation of fibrotic cancer stroma limits tumor aggressiveness and it’s absence leads to an increase in spontaneous metastasis15.Wagner et al demonstrated that the role of TIL in colorectal metastases to the liver which in turn depends on the migration and activation of helper CD4+ T cells as well as stimulation of cytotoxic properties of CD8+ T cells16. Also, Schweiger et al confirmed a significant role of TILs of colorectalcases with respect to metastasis to the lung17. Nazemalhosseini-Mojarad et al showed that the presence of intratumoral TIL CD8+ lymphocytes are significantly associated with differentiation (p = 0.004), node, metastases (TNM) stage (p = 0.001), and microsatellite instability (p = 0.001) [18]. Huh et al also found that the TIL grade was a significant prognostic factor only for the patients with stage III tumor (specifically in the N2 category) but not for the patients with stage I and II tumors for both the overall survival and disease free survival.12 Jakubowska et al observed that a low proportion of stromal TILs in the center of the tumor was associated with an increase in the severity of colorectal cancer as classified by Dukes and TNM staging systems8. However in the present study, TNM stage was studied and higher TIL was observed with stages I and II compared to stages III and IV but the difference was not statistically significant. This discordance may be due to the fact that the present study did not segregate the subpopulations of TIL. It has been observed that patients with high TIL infiltration may respond to the administration of ipilimumab antibodies which acts against cytotoxic T lymphocyte associated protein. This results in reducing the immune inhibition thereby promoting an immune response against the tumor.19

 

CONCLUSION

The results of the present study showed that the TILs in colorectal adenocaecinoma are strongly associated with lymphovascular invasion, lymph node metastasis and the number of lymph node metastasis. Hence the evaluation of TIL infiltration should be included in routine histopathological examinations.

 

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