Multiple biomarkers of colorectal tumor in a differential diagnosis model:A quantitative study

AIM:To evaluate the multiple biomarkers of colorectal tumor and their potential usage in early diagnosis of colorectal cancers. METHODS:Multiple biomarkers (DNA contents,AgNOR, PCNA,p53,c-erbB-2) in 10 normal colorectal mucosae,37 colorectal adenomas and 55 colorectal cancers were analyzed quantitatively in the computed processing imaging system. Discrimination patterns were employed to evaluate the significance of single and multiple indices in diagnosis of colorectal cancers. RESULTS:The mean values of the analyzed parameters increased in order of the normal mucosa,adenoma and adenocarcinoma,and this tendency reflected the progression of colorectal malignancy.The parameters including DNA index,positive rates,densities of AgNOR,c-erbB-2,and p53, shape and density of nucleus were relatively valuable for diagnoses.Then a diagnostic discrimination model was established.The samples were confirmed with the model, the sensitivity rates in cancer group and adenoma group were 96.36% and 89.19%,respectively.The value of proliferating cell nuclear antigen (PCNA) in early diagnosis of colorectal cancers was uncertain. CONCLUSION:The quantitative evaluation of some parameters for colorectal tumor can provide reproducible data for differential diagnosis.The established diagnostic discrimination model may be of clinicopathological value, and can make the early diagnosis of colorectal cancer possible.

The diagnostic value of multiple tumor markers in malignantovarian neoplasms

Objective:To study the diagnostic value of multiple tumor markers in malignant ovarian neoplasm.Methods:Sera obtained from 430 patients with ovarian masses (110 cases were malignant ovarian tumors,320 cases were benign ovarian tumors) before operation,and from 50 healthy women as control.Serologic examination of tumor markers included CA125,TSGF,SA,CEA,AFP,HCG and Fer.Results:The serum levels of CA125,TSGF,SA and Fer in patients with ovarian cancer were higher than those in patients with benign ovarian tumors (P<0.05),also in control group (P<0.05).In the diagnostic value of application for malignant ovarian neoplasm,CA125,TSGF and SA were better than the others.The sensitivity,specificity and accuracy in diagnosis of ovarian cancer were 86.4%,82.8%and 83.7% respectively for CA125 alone,78.2%,81.3%and 80.5% for TSGF alone,74.5%,81.9%and 80.0% for SA alone,whereas 95.5%,45.6%and 58.4% for multiple tumor markers combined in which 1 or more indices showed positive,93.6%,80.6%and 84.0% for that in which 2 or more indices showed positive,and 87.3%,90.3%and 89.5% for that in which 3 or more indices show positive.Conclusion:multiple tumor markers examination could improve the diagnosis of ovarian cancer,and examination of CA125,TSGF and SA combined is most ideal.

The prognostic molecular markers in hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

P53 immunohistochemical scoring:an independent prognostic marker for patients after hepatocellular carcinoma resection

AIM: To confirm if p53 mutation could be a routine predictive marker for the prognosis of hepatocellular carcinoma (HCC) patients. METHODS: Two hundreds and forty-four formalin-fixed paraffin-embedded tumor samples of the patients with HCC receiving liver resection were detected for nuclear accumulation of p53. The percent of P53 immunoreactive tumor cells was scored as 0 to 3+ in P53 positive region (【10% -, 10-30% +, 31-50% ++, 】50% +++). Proliferating cell nuclear antigen (PCNA) and some clinicopathological characteristics, including patients' sex, preoperative serum AFP level, tumor size, capsule, vascular invasion (both visual and microscopic), and Edmondson grade were also evaluated. RESULTS: In univariate COX harzard regression model analysis, tumor size, capsule status, vascular invasion, and p53 expression were independent factors that were closely related to the overall survival (OS) rates of HCC patients. The survival rates of patients with 3+ for P53 expression were much lower than those with 2+ or + for P53 expression. Only vascular invasion (P【0.05) and capsule (P【0.01) were closely related to the disease-free survival (DFS) of HCC patients. In multivariate analysis, p53 overexpression (RI 0.5456, P【0.01) was the most significant factor associated with the OS rates of patients after HCC resection, while tumor size (RI 0.5209, P【0.01), vascular invasion (RI 0.5271, P【0.01) and capsule (RI-0.8691, P【0.01) were also related to the OS. However, only tumor capsular status was an independent predictive factor (P【0.05) for the DFS. No significant prognostic value was found in PCNA-LI, Edmondson's grade, patients' sex and preoperative serum AFP level. CONCLUSION: Accumulation of p53 expression, as well as tumor size, capsule and vascular invasion, could be valuable markers for predicting the prognosis of HCC patients after resection. The quantitative immunohistochemical scoring for P53 nuclear accumulation might be more valuable for predicting prognosis of patients after HCC resection than the common qualitative analysis.

Relationship between phenotypes of cell-function differentiation and pathobiological behavior of gastric carcinomas

AIM: To reveal the correlation between the functional differentiation phenotypes of gastric carcinoma cells and the invasion and metastasis by a new way of cell-function classification.METHODS:Surgically resected specimens of 361 gastric carcinomas(GC) were investigated with enzyme-, mucin-, and tumor-related marker immunohistochemistry. According to the direction of cell-function differentiation, stomach carcinomas were divided into five functionally differentiated types. RESULTS: (1) Absorptive function differentiation type (AFDT): there were 82 (22.7%) patients including 76 (92.7%) aged 45 years. Sixty-nine (84.1%) cases belonged to the intestinal type. Thirty-eight (46.3%) expressed CD44v6 and 9 (13.6%) of 66 male patients developed liver metastasis.The 5-year survival rate of patients in this group (58.5%) was higher than those with the other types (P【0.01). (2) Mucin secreting function differentiation type (MSFDT): 54 (15%) cases. Fifty-three (98.1%) tumors had penetrated the serosa, 12 (22.2%) expressed ER and 22 (40.7%) expressed CD44v6. The postoperative 5-year survival rate was 28.6%. (3) Absorptive and mucin-producing function differentiation type (AMPFDT): there were 180 (49.9%) cases, including 31 (17.2%) aged younger than 45 years. The tumor was more common in women (62, 34.4%,) and expressed more frequently estrogen receptors (ER) (129, 81.7%) than other types (P【0.01). Ovary metastasis was found in 12 (19.4%) out of 62 female subjects. The patients with this type GC had the lowest 5-year survival rate (24.7%) among all types. (4) Specific function differentiation type (SFDT): 13 (3.6%) cases. Nine (69.2%) tumors of this type derived from APUD system, the other 4 (30.7%) were of different histological differentiation. Sixty per cent of the patients survived at least five years. (5) Non-function differentiation type (NFDT): 32 (8.9%) cases. Nineteen (59.4%) cases had lymph node metastases but no one with liver or ovary metastasis. The 5-year survival rate was 28.1%. CONCLUSION: This new cell-function classification of GC is helpful in indicating the characteristics of invasion and metastasis of GC with different cell-function differentiation phenotypes. Further study is needed to disclose the correlation between the cell-functional differentiation phenotypes and the relevant genotypes and the biological behavior of gastric carcinoma.

DNA ploidy and c-Kitmutation in gastrointestinal stromal tumors

AIM: To investigate the prognostic significance of c-Kitgen emutation and DNA ploidy in gastointestinal stromal tumors (GISTs).METHODS: A total of 55 cases of GISTs were studied for the expression of c-Kit by immunohistochemistry, and the c-Kit gene mutations in exons 9, 11, 13, and 17 were detected by polymerase chain reaction-single strand confirmation polymarphism (PCR-SSCP) and denaturing high performance liquid chromatography (D-HPLC) techniques. DNA ploidy was determined by flow cytometry.RESULTS: Of the 55 cases of GISTs, 53 cases (96.4%) expressed c-Kit protein. The c-Kit gene mutations of exons 11 and 9 were found in 30 (54.5%) and 7 cases (12.7%),respectively. No mutations were found in exons 13 and 17.DNA aneuploidy was seen in 10 cases (18.2%). The c-Kit mutation positive GISTs were larger in size than the negative GISTs. The aneuploidy tumors were statistically associated with large size, high mitotic counts, high risk groups, high cellularity and severe nuclear atypia, and epithelioid type.There was a tendency that c-Kit mutations were more frequently found in aneuploidy GISTs.CONCLUSION: DNA aneuploidy and c-Kit mutations can be considered as prognostic factors in GISTs.

Peripheral and mesenteric serum levels of CEA and cytokeratins,staging and histopathological variables in colorectal adenocarcinoma

AIM： To evaluate the differences that exist bet- ween peripheral and mesenteric serum levels of carcinoembryonic antigen （CEA） and cytokeratins in patients with colorectal adenocarcinoma. METHODS： One hundred and thirty-eight patients with colorectal adenocarcinoma who underwent surgery at Hospital Sao Paulo （Discipline of Surgical Gastroenterology of UNIFESP-EPM） between December 1993 and March 2000 were retrospectively analyzed. Differences between CEA and cytokeratin （TPA-M） levels in peripheral blood （P） and in mesenteric blood （M） were studied. Associations were investigated between peripheral and mesenteric levels and the staging and histopathological variables （degree of cell differentiation, macroscopic appearance, tumor dimensions and presence of lymphatic and venous invasion）. RESULTS： Differences were observed in the numerical values of the marker levels： CEA （M） （39.10 mg/1 ± 121.19 mg/L） vs CEA （P） （38.5 mg/L ± 122.55 mg/L）, P 〈 0.05; TPA-M （M） （325.06 U/L ±527.29 U/L） vs TPA-M （P） （279.48 U/L ±455.81 U/L）, P 〈 0.01. The mesenteric CEA levels were higher in more advanced tumors （P 〈 0.01）, in vegetating lesions （34.44 mg/L ± 93.07 mg/L） （P 〈 0.01） and with venous invasion （48.41 mg/L ± 129.86 mg/L） （P 〈 0.05）. Peripheral CEA was higher with more advanced staging （P 〈 0.01）and in lesions with venous invasion （53.23 mg/L ± 258.57 mg/L） （P 〈 0.05）. The patients demonstrated increased mesenteric and peripheral TPA-M levels with more advanced tumors （P 〈 0.01 and P 〈 0.01） and in non-ulcerated lesions [530.45 U/L =1= 997.46 U/L （P 〈 0.05） and 457.95 U/L ± 811.36 U/L （P 〈 0.01）]. CONCLUSION： The mesenteric levels of the tumor markers CEA and cytokeratins were higher than the peripheral levels in these colorectal adenocarcinoma patients, Higher levels of these biologic tumor markers are associated with an advanced state of cancerous dissemination

Relationship between serum calcium and CA 19-9 levels in colorectal cancer

AIM:To examine the calcium metabolism of colorectal cancer (CRC)in patients with colorectal cancer and control patients. METHODS:Seventy newly diagnosed CRC patients were included.The healthy control group was age and gender matched(n=32).Particular attention was devoted to the relationship between serum calcium of patients,and levels of AFP,CEA,carbohydrate antigen 19-9(CA 19-9)(that could be considered as prognostic factors).Furthermore,the Ca-sensing receptor(CaSR)gene A986S polymorphism was investigated in these patients,as well as the relationship between different CaSR genotypes and the data stated above. RESULTS:A lower level of ionized calcium(also corrected for albumin)was found in the serum of CRC patients with normal 25(OH)vitamin D levels.The ionized calcium concentration was inversely correlated with the serum level of CA.19-9.There was no difference in the distribution of CaSR genotypes,between CRC patients and general population.The genotypes did not correlate with other data examined. CONCLUSION:Based on these results,lower levels of serum calcium might be a pathogenic and prognostic factor in colorectal cancer.

Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer

AIM: To investigate SBA2 expression in CRC cell lines and surgical specimens of CRC and autologous healthy mucosa. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used for relative quantification of SBA2 mRNA levels in 4 human CRC cell lines with different grades of differentiation and 30 clinical samples. Normalization of the results was achieved by simultaneous amplification of beta-actin as an internal control. RESULTS: In the exponential range of amplification, fairly good linearity demonstrated identical amplification efficiency for SBA2 and beta-actin (82%). Markedly lower levels of SBA2 mRNA were detectable in tumors, as compared with the coupled normal counterparts P【0.01). SBA2 expression was significantly (0.01】P 【 0.05) correlated with the grade of differentiation in CRC, with relatively higher levels in well-differentiated samples and lower in poorly-differentiated cases. Of the 9 cases with lymph nodes affected, 78% (7/9) had reduced SBA2 mRNA expression in contrast to 24% (5/21) in non-metastasis samples 0.01】P【0.05). CONCLUSION: SBA2 gene might be a promising novel biomarker of cell differentiation in colorectal cancer and its biological features need further studies.

C-reactive protein,procalcitonin,interleukin-6,vascular endothelial growth factor and oxidative metabolites in diagnosis of infection and staging in patients with gastric cancer

AIM:The current study was to determine the serum/pLasma levels of VEGF,IL-6,malondialdehyde (MDA),nitric oxide (NO),PCT and CRP in gastric carcinoma and correlation with the stages of the disease and accompanying infection. METHODS:We examined the levels of serum VEGF,IL-6, PCT,CRP and plasma MDA,NO in 42 preoperative gastric cancer patients and 23 healthy subjects.There were infection anamneses that had no definite origin in 19 cancer patients. RESULTS:The VEGF levels (mean±SD; pg/mL) were 478.05±178.29 and 473.85±131.24 in gastric cancer patients with and without infection,respectively,and these values were not significantly different (P>0.05).The levels of VEGF, CRP,PCT,It-6,MDA and NO in cancer patients were significantly higher than those in healthy controls and the levels of CRP,PCT,It-6,MDA and NO were statistically increased in infection group when compared with non- infection group (P<0.001). CONCLUSION:Although serum VEGF concentrations were increased in gastric cancer,this increase might not be related to infection.CRP,PCT,IL-6,MDA and NO have obvious drawbacks in the diagnosis of infections in cancer patients. These markers may not help to identify infections in the primary evaluation of cancer patients and hence to avoid unnecessary antibiotic treatments as well as hospitalization. According to the results of this study,IL-6,MDA,NO and especially VEGF can be used as useful parameters to diagnose and grade gastric cancer.

Levels of v5 and v6 CD44 splice variants in serum of patients with colorectal cancer are not correlated with pT stage,histopathological grade of malignancy and clinical features

AIM:This study was designed to compare the levels of v5 and v6 splice variants of CD44 evaluated using EITSA test in the serum of patients with colorectal cancer in different stages of progression of the disease estimated in pT stage according to WHO score,histopathological grade of malignancy and some clinicopathological features. METHODS:The serum obtained from 114 persons with colorectal adenocarcinomas was examined using ELISA method,pT stage and grade of malignancy of the tumour were examined in formalin fixed and paraffin embedded materials obtained during operation. RESULTS:Only the level of CD44 v5 in the serum of patients before operation with G2 pT4 tumour was lower than that in other probes and the difference was statistically significant. We did not find any other correlations between the level of v5 and v6 CD44 variants and other evaluated parameters. CONCLUSION:The level of CD44 v5 and v6 estimated by ELISA test in the serum can not be used as a prognostic factor in colorectal cancer.

Role of serum total sialic acid in differentiating cholangiocarcinoma from hepatocellular carcinoma

AIM:This study was designed to evaluate the clinical application of serum total sialic acid (TSA) in the diagnosis of cholangiocarcinoma (CCA).METHODS: Serum TSA was determined by periodateresorcinol microassay in 69 patients with CCA, 59 patients with hepatocellular carcinoma (HCC), 37 patients with cirrhosis, 61 patients with chronic hepatitis and 50 healthy blood donors.RESULTS: The mean serum TSA concentration in CCA (2.41±0.70 mmol/L) was significantly higher than those of HCC, cirrhosis, chronic hepatitis and healthy blood donors (1.41±0.37 mmol/L, 1.13±0.31 mmol/L, 1.16±0.26 mmol/L, and 1.10±0.14 mmol/L, respectively; P＜0.001). Based on ROC curve analysis, a cut-off point of 1.75 mmol/L discriminated between CCA and HCC with a sensitivity,specificity and accuracy of 82.6%, 83.1%, and 82.8%,respectively.CONCLUSION: Based on our results, serum TSA would be a useful marker for the differential diagnosis of CCA from HCC.

Rising plasma nociceptin level during development of HCC:A case report

AIM:Although liver cirrhosis is a predisposing factor for hepatocellular carcinoma (HCC),relatively few reports are available on HCC in primary biliary cirrhosis.High plasma nociceptin (N/OFQ) level has been shown in Wilson disease and in patients with acute and chronic pain. METHODS:We report a follow-up case of HCC,which developed in a patient with primary biliary cirrhosis.The tumor appeared 18 years after the diagnosis of PBC and led to death within two years.Alfa fetoprotein and serum nociceptin levels were monitored before and during the development of HCC. Nociceptin content was also measured in the tumor tissue. RESULTS:The importance and the curiosity of the presented case was the novel finding of the progressive elevation of plasma nociceptin level up to 17-fold (172 pg/mL) above the baseline (9.2±1.8 pg/mL),parallel with the elevation of alpha fetoprotein (from 13 ng/mL up to 3 480 ng/mL) during tumor development.Nociceptin content was more than 15-fold higher in the neoplastic tissue (0.16 pg/mg) than that in the tumor- free liver tissue samples (0.01 pg/mg) taken during the autopsy. CONCLUSION:Results are in concordance with our previous observation that a very high plasma nociceptin level may be considered as an indicator for hepatocellular carcinoma.

Retrospective analysis of 67 consecutive cases of pure ovarian immature teratoma

OBJECTIVE: To investigate the development regularity, treatment methods and prognosis of ovary immature teratoma (POIT). METHODS: Sixty-seven patients with POIT, admitted from 1958 to 1998, were retrospectively analyzed. There were 31 patients with stage I, 4 with stage II, 2 with stage III and 1 with stage IV lesions. Twenty-seven patients had recurrences and 2 had distant metastases. Unilateral adnexectomy was performed for stage I lesions. From the 1980s, this was followed by four-cycles of combination chemotherapy (VAC, PVB or BEP x 3 cycles) as post-operative adjuvant therapy. Combined chemotherapy and multiple operations were performed for advanced and recurrent lesions. RESULTS: The overall survival rate was 75% (50/67). However, there was a remarkable difference in the results from the various periods. From 1958 to 1983, the 5-year survival rate was 40% (6/15), and it was raised to 79% (26/33) from 1984 to 1993. In the period 1994 to 1998, 95% (18/19)of patients were rescued. Thirty-five patients who had early lesions (stage I and II) had a 5-year survival rate of 91.4% (32/35). Thirty-two patients with recurrent or advanced lesions had a 5-years survival rate of 56% (18/32). There were 8 patients with grade III tumors and their 5-year survival rate was only 25% (2/8). The chief prognostic factors for this disease are clinical stage, pathological grade and adequate treatment. CONCLUSION: POIT is a potentially curable disease in today's practice. It is characterized by the fact that recurrent tumors may be converted back to mature ones as time goes on. With chemotherapy, these is a good opportunity to rescue those patients with recurrent tumors. At present, treatment of POIT gives the most satisfactory results among all malignant ovarian germ cell tumor types. Tests of serum specific tumor markers (CA19-9, AFP, CA125, CEA) performed preoperatively or before chemotherapy and during follow-up have been found helpful in the evaluation of prognosis.