Macrophage Migration Inhibitory Factor Gene Polymorphism and some Immunological Aspects of Urothelial Carcinoma Patients

 This study was designed to shed light on the association between polymorphism of macrophage migration inhibitory factor (MIF) gene at -173 locus as risk factor of bladder cancer in Iraqi population by PCR-RFLP ,estimation the serum level of MIF with some cytokines (IL-6, IL-10 and TNF-α) by sandwich ELISA and  determine the expression of several marker (CD74, CD44, P 53  and P 21 ) by  immunohistochemical staining. Blood, urine and bladder biopsy  were collected from patients and healthy volunteer at AlYarmook Teaching Hospital and Al-Jaibachi Private Hospital from March to November
2014.  One hundred and thirty five subjects from Baghdad (104 male and 31 female) were divided into three investigated groups: first group was urinary bladder carcinoma (UBC) (60 male and 13 female) where bladder cancer increases with age, second group was urinary bladder disorders (UBD) (22 male and 10 female) and third group was healthy control (22male and 8 female). Results showed that, after clinical laboratory diagnosis of urine samples of three groups  64 (87.7%) of  UBC sample  gave positive culture and 9 (12.3%) samples were negative culture; 32(100%) urine samples of UBD were positive culture while all urine sample  of healthy control were negative. Gram staining and microscopic  examination of UBC and UBD urine showed that 9 (9.4 %) isolates were identified as Gram positive bacteria,
85 (88.5 %) isolates as Gram negative bacteria and only 2 (2.1%) isolates as fungi. In addition , These isolates were distributed as, 2 (2.1%) isolates were identified as Candida albicans, 1(1%)  Citrobacter spp, 1(1%) Streptococcus fecalis, 7 (7.3 %) Pseudomonas sp., 9(9.4%) Staphylococcus aureus , 14(14.6%) Klebsiella sp , 19(19.8%) Proteus sp., and 43(44.8%) isolates identified as E.coli  .
 According to histopathology’s diagnosis, all tumors of this study have been classified as urothelial cell carcinoma (UCC). Stage and grade for each tumor has been identified according to WHO (2004).  Forty (54.8%) tumors were characterized as high grade and 33(45.2%) as low grade. While according to stage of tumors, 12(16.4%) Ta, 28(38.4%) T1 invaded subepithelial connective tissue, 22(30.1%) T2 invaded muscle, 7(9.6) T3 invaded perivesical tissue and 4(5.5%) T4 invade other organ. Forty five (61.6%) out of the 73 patients had the bladder tumor for first time while 28(38.6%) patients were suffering from recurrent bladder tumor after chemotherapy.  Results revealed serum IL-6 levels were significantly higher in patients with UBC (90.14 pg/ml) than  in both of  UBD  (61.5 pm/ml)   and  healthy controls (8.4) pg/ml. Serum level of IL-10 was significantly higher in both patients with UBC (35.84pg/ml) and  UBD patients (27.95pg/ml) when compared with healthy subjects (19.26pg/ml). Serum level of TNF-α was
significantly higher for UBC and UBD patients when compared with mean level of control (36.19, 15.31 and  10.15 pg/ml) respectively. The mean level of serum MIF in UBC patients was significantly higher than that observed in UBD patients and healthy control (55.57 and 40.39 vs. 18.83) pg/ml, respectively. Positive significant correlation was found between four cytokines, and all of these were increased proportionally with advanced tumors stages and high grade. Genomic DNA were extracted from peripheral blood of all 135 subjects and  amplified by PCR with using specific primer for -173 locus that giving
the PCR product (366) bp that containing both the polymorphic and a non polymorphic AluI site. Results showed that G/G genotype gave two fragments (268- and 98-bp), C/C genotype gave (62-, 206- and 98-bp) and four fragments as (62-, 98-, 206- and 268-bp) of G/C genotype after digestion by Alu I. The frequency distributions of genotypes and alleles of the groups showed that  the GG and G allele were (61.9 and 78.77) % among the UBC cases and (56.3 and 75)% among the UBD cases, while represent (53.3and 70)% respectively among healthy cases. In addition,  and no significant variation was recorded between UBC and control with no significant departure from Hardy- Weinberg equilibrium. Results  also showed that MIF level in GG was (62.69) pg/ml which significantly higher in UBC and level was significantly lower in UBD and healthy (37.98 and 16. 61 pg/ml), respectively. In order to study the expression of CD74 molecules, staining was done by using anti-CD74 clone, then  it was compared between the CD74 expressions. Results exhibited positive staining 50(68.49 %) with different scores and 23(31.51%) gave negative staining with highly significant differences. while all tissues free from bladder tumors were expressed negative staining. Results showed highly positive CD44 immunohistochemical expressions in bladder tumor tissues, 64 (87.7%) gave positive result of staining with different score and 9(12.3%) gave negative results, while 9(90%) out of the ten normal urothelium gave positive expression with highly significant differences. Analysis of  the 73 bladder tumors revealed that nuclear p53 protein of 42(57.5%) gave positive staining which were exclusively nuclear and 31(42.5%) gave negatively staining result while expression of p53 was nil in all normal transitional epithelium specimens.  Results showed that 51(69.9%) out of 73 urinary bladder tumors gave positive nuclear staining for p21 with different scores while 22 (31.1%) and all ten normal urothlium  consistently demonstrated no p21  immunoreactivity, providing a negative staining pattern.Also, results showed a significant positive expressions of CD74, CD44, p53 and p21 in stages T2 and T1 with  high grade.