الفهرس 
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Abstract
The prognosis of colorectal carcinoma (CRC) patients showed considerable
heterogeneity that greatly depends on their developmental pathways. Many
prognostic and predictive molecular markers, including MMR proteins, BRAFV600E
and KRAS are involved in those pathogenic pathways and guide individual
treatment plans.
The aim of this study was to evaluate the immunohistochemical expression
of MLH1, MSH2 and BRAFV600E in CRC patients for stratification of them into
distinct subgroups for a peculiar prognosis and future target therapy. Moreover, the
expression profile of those markers was correlated with the available
clinicopathological features.
This retrospective selective study included 81 specimens from Egyptian
patients with de novo CRC, obtained from the archive of Pathology Department,
Faculty of Medicine, Menoufia University during the period between January 2015
and August 2019.
The age of the studied cases ranged between 30 and 85 years with a median
of 58 years and a mean ± SD of 56.58±12.27 years. Forty eight out of 81 cases
(59.3%) were ≤58 years and the remaining 33 cases (40.7%) were >58 years old.
Thirty three (40.7%) of the studied CRC cases were males and the other 48 cases
(59.3%) were females, with 0.7:1 as a male to female ratio.
Forty five cases (55.6%) were presented with tumor in left colon in
comparison to 30 cases (37%) who had tumor located in right colon while the
remaining 6 cases (7.4%) had rectal tumor location. Two cases (2.5%) were
received as incisional colonoscopic biopsies while 79 (97.5%) were totally resected
(colectomy specimens).
Ten out of 50 cases with available data for recurrence (20%) developed
locoregional recurrence. Among the studied 56 cases for which data about distant
metastasis were available; 34 cases (60.7%) showed absence of metastasis to distant
sites and the remaining 22 cases (39.3%) were proved to be metastatic. Thirty five
out of 81 CRC cases (43.2%) died with the offending disease.
Fungating cauliflower mass was found in 50 CRC cases (61.7%) followed
by infiltrative mass in 21 cases (25.9%) and lastly 10 cases (12.3%) had malignantulcer. The greatest dimension of the studied tumor cases ranged between 2 and
12 cm with 7 cm as a median value. Seventy one cases (87.7%) were presented by
a large sized tumor >5cm while the remaining 10 cases (12.3%) were ≤5cm. Gross
perforation was found in 14 out of the studied 79 colectomy specimens (17.7%).
Regarding colectomy specimens (79 cases), 77 cases (97.5%) had free
surgical margin while 2 cases only (2.5%) had involved margin. Colorectal cancer
cases arising on top of adenoma was found in 13 cases (16%).
Histopathological examination of the studied CRC cases displayed that 65
cases (80.2%) were of adenocarcinoma type and 16 cases (19.8%) were of
mucinous type. Forty six of the studied CRC cases (56.8%) showed absence of
mucin. The remaining cases had either focal mucin (≤50%) in 19 cases (23.5%) or
extensive mucinous differentiation in 16 cases (19.8%). Low grade cases
represented by 59 cases (72.8%) and 22 cases (27.2%) were of high grade.
The studied colectomy specimens showed that 48 cases (60.8%) were of
T3 stage followed by T4 stage which was represented by 23 cases (29.1%), 8 cases
(10.1%) were of T2 stage and no cases exhibited T1 stage. Pathologic T2 and T3
stages were grouped together represented 56/79 cases (70.89%). Forty four cases
(55.69%) showed absence of lymph node metastasis (N0), 35 cases (44.31%)
showed nodal metastasis.
TNM staging system was used to categorize the studied 81 CRC cases into
stage group I & II represented by 38 cases (46.9%) and 43 cases (53.1%) were of
stage group III & IV. Thirty eight out of 81 CRC cases (46.9%) were of Dueks’ B
stage, 21 (25.9%) were of Dueks’ C stage and Dueks’ D stage was represented by
22 cases (27.2%). None of the studied cases belonged to Dueks’ A stage.
Necrosis was present in 25 out of 81 CRC cases (30.9%). Mitotic count/10
HPF ranged between 0 and 56 with a median of 8 and a mean ± SD of 11.06±10.57.
Apoptotic count/10 HPF ranged between 2 and 90 with a median of 16 and a mean
± SD of 20.81±17.75.
Lymphovascular invasion was appreciated in 19 of the studied 81 CRC
cases (23.5%). Fifteen CRC cases (18.5%) showed evidence of perineural invasion.
Regarding IHC results, colorectal cancer cases were divided into mismatch
repair proficient (MMRP) and mismatch repair deficient (MMRD) cases using
MLH1 & MSH2 immunostaining. Thirty five out of 81 CRC cases (43.2%) were
MMRP and 46 cases (56.8%) were MMRD. Mismatch repair deficient (MMRD)group showed higher presentation of exophytic gross morphology (fungating mass)
(P=0.04) and large sized tumor (p=0.02).
Forty two CRC cases (51.9%) exhibited BRAFV600E mutation. On the other
hand, 39 cases (48.1%) were categorized as wild cases. No significant difference
was noted between wild and mutant BRAFV600E colorectal cancer cases regarding
the studied clinicopathological variables. Our results also revealed a slight increase
of BRAFV600E mutation in MMRP group (P=0.08).
Among the PCR tested 63 CRC cases; mutation analysis for exons 2, 3 & 4
of KRAS gene revealed that mutant cases constituted 19.05% (12 cases) while 51
cases (80.95%) were wild. A significant association was found between NRAS gene
mutation status and BRAFV600E staining results as all CRC cases with NRAS
mutation were also BRAFV600E mutant (P=0.03).
KRAS mutant cases exhibited extensive mucinous differentiation (P=0.04),
lymph node metastasis (P=0.006) together with advanced TNM (P=0.02) and
Dukes’ stages (P=0.04).
When IHC results of MMR and BRAFV600E proteins were combined
together to classify CRC cases into 4 groups in the current work; no significant
association was detected with the studied clinicopathological parameters.
Additionally, 59 cases (93.65%) exhibited wild NRAS status while only 4 cases
(6.35%) were mutant.
Additionally, when CRC cases were grouped according to KRAS mutation
status together with either MMR or BRAFV600E protein expression, KRAS mutant
cases showed high incidence of lymph node positivity (p=0.03 & 0.02 respectively)
as well as advanced stage at time of presentation (p=0.05 & 0.04 respectively)
regardless MMR or BRAFV600E protein expression status.
Finally, grouping of studied CRC cases regarding MMR and BRAFV600E
protein expression together with KRAS mutational status divided the cases into 6
groups [MMRP with wild both (8/63), MMRP with either KRAS or BRAFV600E
mutation (12/63), MMRP with mutant both (4/63), MMRD with either KRAS or
BRAFV600E mutation (19/63), MMRD with wild both (20/63) & MMRD with
mutant both (0/63)]. Comparison with clinicopathological parameters revealed thatperineural invasion was predominant in MMRD cases associated with either wild
both KRAS & BRAFV600E or having mutation of one of them (p=0.03).
Regarding univariate overall survival analysis, the survival time mean±SD
was 15.89±10.98 months. Shortened overall survival was associated with male
gender (P=0.03), advanced T stage (P=0.03) or advanced N stage (P=0.01). The
gender was the most independent prognostic factor affecting overall survival for
studied CRC cases (P=0.04) by multivariate cox regression analysis.
Regarding univariate event free survival analysis, event free survival time
ranged between 1 and 41 months with a mean±SD of 13.96±10.46 months and a
median of 12 months. TNM stage group III & IV (P=0.04) and presence of vascular
invasion (P=0.01) were predictors of poor event free survival in the studied CRC
cases. Lymphovascular invasion was the most independent prognostic factor
affecting event free survival for studied CRC cases (p=0.03).