الفهرس 
Aim of The WorkOnly 14 pages are availabe for public view
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Abstract
Colorectal carcinoma (CRC) is the third most common cancer diagnosed in both men and women globally. In Egypt, cancer rectum and colon represent 3.43% and 2.83% of total malignancies and 18.5% and 15.3% of total gastrointestinal tract tumors, respectively ranking the 3rd and the 4th after cancers of oral cavity and cancer stomach. Advances in molecular biology have helped to elucidate some of the genetic mechanisms leading to colorectal carcinogenesis. However, accumulating evidence suggests that tumor progression is governed not only by genetic changes intrinsic to cancer cells but also by tumor microenvironment (TME). Increased understanding of the immune TME has revealed novel immune-based biomarkers that helped in the development of new agents for blockade of immune checkpoint molecules to activate antitumor immunity.
Among the most promising immune checkpoint molecules are the programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA–4) pathways. PD-L1 and CTLA-4 interactions act on suppressing T-cell response and inducing tumor immune escape. Therefore, they help to create a suitable TME that enables the tumor cells of continuous proliferation. PD-L1 interacts with its receptor programmed death 1 (PD-1), transmitting a negative signal to a series of processes of T cell-mediated cellular immune responses including priming, growth, proliferation and functional maturation.
In the early phase of tumorigenesis, CTLA-4 is up-regulated and binds B7 molecule, resulting in reduced T cell proliferation, activation and decreased cytokine secretion. Additionally, CTLA-4 has been involved in mediating the immunosuppressive role of T regulatory cells.
To date, 7 drugs targeting CTLA-4/PD-L1 are approved for treatment of different types of cancers including melanoma, lung cancer, breast cancer, head and
neck cancer, bladder cancer, Merkel cell cancer, cervical cancer, hepatocellular cancer, gastric cancer, cutaneous squamous cell cancer, classic Hodgkin’s lymphoma and B-cell lymphoma.
However, a clear consensus has not been reached on the relationship of PD-L1, CTLA-4 with the clinicopathological features and prognosis of patients with colorectal cancer. This study aimed to evaluate the immunohistochemical expression of PD-L1 and CTLA-4 in CRC cases and its relationship with clinicopathological parameters and survival data.
This retrospective case control study involved 146 cases, divided into 21 normal specimens, 22 adenoma specimens and 103 CRC specimens retrieved from the archival material of Pathology Department, Faculty of Medicine, Menoufia University, during the period between 2015 and 2019. Hematoxylin and eosin (H&E) stained slides of the selected cases’ blocks were examined carefully to identify viable and representative areas of each sample. Tissue microarray technique was performed through sampling of three cores of reprensentative areas of the tumor and stroma from each included specimen.
The included 21 normal colonic specimens represented 14.3% of the study sample, while the included 22 colonic adenoma specimens represented 15.06% of the study sample. Fourteen specimens (63.6%) were of tubulovillous type, 5 specimens (22.7%) were of the tubular type and 3 cases (13.6%) were of villous type. Thirteen specimens (59.1%) showed low grade dysplasia, while 9 cases (40.9%) showed high grade dysplasia.
The included 103 CRC cases represented 70.5% of the study sample. Their age ranged between 25 and 85 years with a median of 57 years and a mean±SD of 55±13 years. Thirty seven cases (35.9%) were males, while 66 cases (64.1%) were females, with 0.56:1 as a male to female ratio. Thirty eight cases (36.9%) had the
tumor in proximal colon in comparison to 39 cases (37.9%) in distal colon and 26 cases (25.2%) in rectum.
The greatest dimension of tumor size in studied CRC specimens ranged between 2 and 21 cm with a mean±SD of 5.98±3.36 cm and a median of 6 cm. Fifty four cases (52.4%) presented as fungating masses, 32 cases (31.1%) were infiltrative and 17 cases (16.5%) were ulcerative lesions. Sixteen cases (15.5%) showed gross perforation. Twenty five cases (24.3%) were of early T stage (T1, T2), while 78 cases (75.7%) were of advanced T stage (T3, T4).
Fifty six cases (54.4%) showed negative lymph node (LN) involvement , while 47 cases (45.6%) showed positive LN involvement. Metastasis was reported in 8 cases of the studied CRC cases (7.8%) and it was absent in 60 cases (58.2%), while in 35 cases (34%) metastasis could not be assessed. Stage grouping was assessed in 68 cases in which data about metastasis was available, thirty eight cases (55.9%) were early stage (I, II), while 30 cases (44.1%) were of advanced stage (III, IV).
The number of investigated LNs ranged between 2 and 66 LNs with a mean±SD of 14.92±9.56 LNs and a median of 13 LNs. The number of positive LNs ranged between 1 and 22 LNs with a mean±SD of 1.78±3.5 LNs and a median of 2 LNs.
Regarding lymph node ratio (LNR), eighty eight cases (85.4%) were in the rN1 category, 10 cases (9.7%) were in the rN2 category while 5 cases (4.9%) were in the rN3 category.
Seventy two cases (69.9%) were conventional adenocarcinoma, 17 cases (16.5%) were adenocarcinoma with mucinous differentiation and 14 cases (13.6%) were mucinous adenocarcinoma.
Seventy nine cases (76.7%) were low grade, while 24 cases (23.3%) were high grade. Thirty one cases (30.1%) showed lymphovascular invasion and twenty cases
(19.4%) showed perineural invasion. Necrosis was detected in 22 cases (21.4%). Mitotic count/10 HPF ranged between 1 and 17 with a median of 7 and a mean±SD of 7.87±3.91.
Thirty four cases (33%) had a pushing tumor border configuration (TBC) while 69 cases (67%) had an infiltrating TBC. Forty five cases (43.7%) showed a low tumor budding (TB) score, 35 cases (34%) displayed an intermediate TB score and high TB score was seen in 23 cases (22.3%). High tumor- stroma ratio (TSR) was detected in 67 cases (65%) and low TSR was detected in 36 cases (35%). Intratumoral tumor infiltrating lymphocytes (TILs) percent ranged between 10% and 60% with a median of 20% and a mean±SD of 24.42%±13.29%. Cap like peritumoral lymphocytic reaction was detected in 72 cases (69.9%), while it was absent in 31 cases (30.1%).
Ten cases out of the 68 cases with available follow-up data (14.7%) died of their disease while 58 cases (85.3%) were alive till the end of follow-up period (at the end of December 2019). Fourteen cases out of the 68 cases with available follow-up data (20.6%) experienced tumor recurrence while 54 cases (79.4%) didn’t show recurrence. In normal specimens, PD-L1 epithelial immunoreactivity score (IRS) was high in three specimens (14.3%) and low in 18 specimens (85.7%). IRS of PD-L1+ lymphocytes was high in fourteen normal specimens (66.7%) and low in the 7 specimens (33.3%). CTLA-4 epithelial intensity score was high in four normal specimens (19%) and low in 17 specimens (81.0%). CTLA-4+ lymphocytes histoscore (H. score) was high in six specimens (28.6%) and low in 15 specimens (71.4%).
In adenoma specimens, PD-L1 epithelial IRS was high in twelve adenoma cases (54.5%) and low in 10 cases (45.5%). IRS of PD-L1+ lymphocytes was high in fourteen cases (63.6%) and low in 8 cases
(36.4%). CTLA-4 epithelial intensity score was high in eleven adenoma specimens (50%) and low in 11 cases (50%). CTLA-4+ lymphocytes H. score was high in thirteen adenoma specimens (49.9%) and low in 9 cases (40.9%).
In CRC cases, PD-L1 epithelial IRS was high in fifty seven CRC cases (55.3%) and low in 46 cases (44.7%). PD-L1+ TILs IRS was high in forty five cases (43.7%) and low in 58 cases (56.3%). CTLA-4 epithelial intensity score was high in sixty nine cases (67%) and low in 34 cases (33.0%). CTLA-4+ TILs H. score was high in forty nine cases (50.5%) and low in 51 cases (49.5%).
High PD-L1 epithelial expression was significantly in favor of CRC (P. value ˂0.001) and adenoma with high grade dysplasia (P. value ˂0.001) compared to normal specimens. High PD-L1 epithelial expression was significantly in favor of CRC (P. value =0.028) and adenoma with high grade dysplasia (P. value ˂0.001) compared to adenoma with low grade dysplasia. On the other hand, no significant statistical difference was detected between normal and adenoma with low grade dysplasia cases regarding PD-L1 epithelial expression. IRS of PD-L1+ lymphocytes didn’t show a significant statistical difference between the 4 studied groups.
High CTLA-4 epithelial expression was significantly in favor of CRC (P. value ˂0.001), adenoma with high grade dysplasia (P. value ˂0.001) and adenoma with low grade dysplasia (P. value =0.011) compared to normal specimens. On the other hand, no significant statistical difference was detected among adenoma with low grade dysplasia, adenoma with high grade dysplasia and CRC cases regarding CTLA-4 epithelial expression. High CTLA-4+ lymphocytes H. score was significantly in favor of adenoma
with low grade dysplasia compared to normal specimens (P. value =0.001). High CTLA-4+ lymphocytes H. score was significantly in favor of adenoma with low grade dysplasia compared to adenoma with high grade dysplasia (P. value =0.003). Furthermore, high CTLA-4+ lymphocytes H. score was significantly in favor of adenoma with low grade dysplasia compared to CRC cases (P. value =0.019).
High PD-L1 epithelial IRS showed a statistically significant association with large tumor size (P=0.038), presence of perforation (P=0.023), pathologic T stage (P=0.007), infiltrative TBC (P˂0.0001), high TB score (P=0.004), low TSR (P=0.012), absence of cap like peritumoral lymphocytic reaction (P= 0.036), dead patients (P˂0.001) and cases showing recurrence (P˂0.001).
High PD-L1+ TILs IRS showed a significant association with absence of perforation (P=0.029), early pathologic T stage (P=0.019), pushing TBC (P˂0.0001), lower TB score (P=0.002), high TSR (P=0.017) and presence of cap like peritumoral lymphocytic reaction (P= 0.049).
Moreover, a significant inverse relationship has been detected between PD-L1 epithelial IRS and PD-L1+ TILs IRS in CRC cases (P >0.001).
High CTLA-4 epithelial intensity score showed a significant association with presence of an infiltrative TBC (P˂0.001), absence of cap like peritumoral lymphocytic reaction (P=0.05), positive LN involvement (P= 0.020) and dead patients (P= 0.002).
High CTLA-4+ TILs H. score showed a significant association with early pathologic T stage (P=0.044), low number of positive lymph nodes (P=0.046), pushing TBC (P=0.004), alive patients (P˂0.001) and cases showing no recurrence (P=0.009).
Furthermore, a significant inverse relationship was detected between CTLA-4 epithelial intensity score and CTLA-4+ TILs H. score in CRC cases (P >0.001).
In CRC cases, PD-L1 epithelial IRS showed a positive relationship with CTLA-4 epithelial intensity score (P< 0.001) and an inverse relationship with CTLA-4+ TILs H. score (P< 0.001). PD-L1+ TILs IRS showed a positive relationship with CTLA-4+ TILs H. score (P< 0.001) and an inverse relationship with CTLA-4 epithelial intensity score (P< 0.001).
Overall survival (OS) data was available for 68 cases of the studied CRC cases (66%). The follow-up period extended from January 2015 to December 2019. The survival time ranged from 1 to 52 months with a mean±SD of 21.47±13.52 months and a median of 18. Ten patients died of their disease through the period of follow-up (14.7%).
Univariate analysis for studied CRC cases revealed a statistical significant association between prolonged overall survival and absence of perforation (P=0.001), early T stage (P=0.021), negative LN involvement (P=0.008), absence of metastasis (P˂0.001), early stage grouping (P=0.002), less number of positive lymph nodes (P=0.035), low LNR category (P=0.003), adenocarcinoma with mucinous differentiation (P=0.046), pushing TBC (P=0.015), low TB score (P=0.001), high TSR (P˂0.001) and peritumoral lymphocytic cap- like reaction (P=0.011)
Moreover, prolonged OS was significantly associated with low PD-L1 epithelial IRS (P=0.001), high PD-L1+ TILs IRS (P˂0.001), low CTLA-4 epithelial intensity score (P=0.003) and high CTLA-4+ TILs H. score (P˂0.001).
Multivariate Cox regression analysis showed that presence of perforation was the most independent prognostic factor affecting OS (P=0.006) followed by PD-L1 epithelial IRS (P=.0.012) in the studied CRC cases. Other independent prognostic factors affecting OS for studied CRC cases included LNR (P=0.017), PD-L1+ TILs IRS (P=0.020), CTLA-4+ TILs H. score (P=0.036) and TBC (P=0.047).
In the present work, fourteen cases (20.6%) experienced tumor recurrence. Recurrence free survival (RFS) ranged between 1 and 52 months with a mean±SD of 19.59±14.31 and a median of 16 months.
Univariate survival analysis for studied CRC cases revealed that prolonged RFS was significantly associated with negative LN involvement (P=0.007), absence of metastasis (P=0.001), early stage grouping (P= 0.002), less number of positive lymph nodes (P=0.001), low LNR category (P=0.003), low grade tumors (P=0.03), absence of lymphovascular invasion (P=0.009), absence of perineural invasion (P=0.029), low mitotic count (P=0.005), pushing TBC (P=0.015), low TB score (P=0.001), high TSR (P˂0.001) and presence of peritumoral lymphocytic cap- like reaction (P=0.011).
Furthermore, univariate analysis of RFS showed the bad prognostic impact of high PD-L1 epithelial IRS (P=0.005), low PD-L1+ TILs IRS (P= 0.002), high CTLA-4 epithelial intensity score (P=0.012) and low CTLA-4+ TILs H. score (P=0.008).
Multivariate Cox regression analysis showed that TSR was the only independent prognostic factor affecting RFS (P= 0.010).