Chih Ping Han

Scoring mechanisms of p16INK4a immunohistochemistry based on either independent nucleic stain or mixed cytoplasmic with nucleic expression can significantly signal to distinguish between endocervical and endometrial adenocarcinomas in a tissue microarray study

BackgroundEndocervical adenocarcinomas (ECAs) and endometrial adenocarcinomas (EMAs) are malignancies that affect uterus; however, their biological behaviors are quite different. This distinction has clinical significance, because the appropriate therapy may depend on the site of tumor origin. The purpose of this study is to evaluate 3 different scoring mechanisms of p16INK4a immunohistochemical (IHC) staining in distinguishing between primary ECAs and EMAs.MethodsA tissue microarray (TMA) was constructed using formalin-fixed, paraffin-embedded tissue from hysterectomy specimens, including 14 ECAs and 24 EMAs. Tissue array sections were immunostained with a commercially available antibody of p16INK4a. Avidin-biotin complex (ABC) method was used for antigens visualization. The staining intensity and area extent of the IHC reactions was evaluated using the semi-quantitative scoring system. The 3 scoring methods were defined on the bases of the following: (1) independent cytoplasmic staining alone (Method C), (2) independent nucleic staining alone (Method N), and (3) mean of the sum of cytoplasmic score plus nucleic score (Method Mean of C plus N).ResultsOf the 3 scoring mechanisms for p16INK4a expression, Method N and Method Mean of C plus N showed significant (p-values < 0.05), but Method C showed non-significant (p = 0.245) frequency differences between ECAs and EMAs. In addition, Method Mean of C plus N had the highest overall accuracy rate (81.6%) for diagnostic distinction among these 3 scoring methods.ConclusionAccording to the data characteristics and test effectiveness in this study, Method N and Method Mean of C plus N can significantly signal to distinguish between ECAs and EMAs; while Method C cannot do. Method Mean of C plus N is the most promising and favorable means among the three scoring mechanisms.

Scoring of p16INK4a immunohistochemistry based on independent nuclear staining alone can sufficiently distinguish between endocervical and endometrial adenocarcinomas in a tissue microarray study

Endocervical adenocarcinomas and endometrial adenocarcinomas are malignancies that affect uterus; however, their biological behaviors are quite different. This distinction has clinical significance, because the appropriate therapy may depend on the site of tumor origin. The purpose of this study is to evaluate four different scoring methods of p16INK4a immunohistochemical staining in distinguishing between primary endocervical adenocarcinomas and endometrial adenocarcinomas from limited sizes of tissue specimens. A tissue microarray was constructed using formalin-fixed, paraffin-embedded tissue from hysterectomy specimens, including 14 endocervical adenocarcinomas and 21 endometrial adenocarcinomas. Tissue array sections were immunostained with a commercially available antibody of p16INK4a. Avidin–biotin complex method was used for antigens visualization. The staining intensity and area extent of the immunohistochemistry was evaluated using the semiquantitative scoring system. Of the four scoring methods for p16INK4a expression, Method Nucleus, Method Dominant Cytoplasm or Nucleus, and Method Mean of Cytoplasm plus Nucleus showed significant (P values <0.05), but Method Cytoplasm did not show significant (P=0.432), frequency distinction between endocervical adenocarcinomas and endometrial adenocarcinomas. In addition, Method Mean of Cytoplasm plus Nucleus had the highest overall accuracy rate (80%) for diagnostic distinction among these four score-counting methods. According to the data in this tissue microarray study, Method Nucleus is the most convenient and efficient method to distinguish between endocervical adenocarcinomas and endometrial adenocarcinomas. Although Method Dominant Cytoplasm or Nucleus as well as Method Mean of Cytoplasm plus Nucleus also revealed statistically significant results, they are relatively more inconvenient due to complicated score calculating means on the basis of mixed cytoplasmic and nuclear p16INK4a expressions. Method Cytoplasm is of no use in the diagnostic distinction between endocervical adenocarcinomas and endometrial adenocarcinomas.

Nuclear Receptor Interaction Protein (NRIP) expression assay using human tissue microarray and immunohistochemistry technology confirming nuclear localization

BackgroundA novel human nuclear receptor interaction protein (NRIP) has recently been discovered by Chen SL et al, which may play a role in enhancing the transcriptional activity of steroid nuclear receptors in prostate (LNCaP) and cervical (C33A) cancer cell lines. However, knowledge about the biological functions and clinical implications of NRIP, is still incomplete. Our aim was to determine the distribution of NRIP expression and to delineate the cell types that express NRIP in various malignant tumors and healthy non-pathological tissues. This information will significantly affect the exploration of its physiological roles in healthy and tumor cells.MethodsBy using tissue microarray (TMA) technology and an anti-NRIP monoclonal antibody immunohistochemical (IHC) survey, NRIP expression was examined in 48 types of tumors and in a control group of 48 matched or unmatched healthy non-neoplastic tissues.ResultsOur survey results showed that ten cases were revealed to express the NRIP in six malignancies (esophageal, colon, breast, ovarian, skin, and pancreatic cancers), but not all of these specific tumor types consistently showed positive NRIP expression. Moreover, malignant tumors of the stomach, prostate, liver, lung, kidney, uterine cervix, urinary bladder, lymph node, testis, and tongue revealed no NRIP expression. Among the control group of 48 matched and unmatched non-neoplastic tissues, all of them demonstrated IHC scores less than the cut-off threshold of 3. In addition, ten cores out of thirty-six carcinomatous tissues revealed positive NRIP expression, which indicated that NRIP expression increases significantly in carcinoma tissue cores, comparing to the matched controlled healthy tissues.ConclusionThis is the first study to use a human TMA and IHC to validate the nuclear localization for this newly identified NRIP expression. In considering the use of NRIP as a potential diagnostic tool for human malignancies survey, it is important to note that NRIP expression carries a sensitivity of only 23%, but has a specificity of 100%. There is also a significant difference in positive NRIP expression between primary carcinomatous tissues and matched controlled healthy tissues. Although further large-scale studies will merit to be conducted to evaluate its role as a potential adjunct for cancer diagnosis, data from this study provides valuable references for the future investigation of the biological functions of NRIP in humans.

Adding the p16INK4a marker to the traditional 3-marker (ER/Vim/CEA) panel engenders no supplemental benefit in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study

Endocervical adenocarcinomas (ECAs) and endometrial adenocarcinomas (EMAs) are malignancies that affect the uterus; however, their biologic behaviors are quite different. This distinction has clinical significance, because the appropriate therapy may depend on the site of tumor origin. In this study, we not only compare the individual expression status of 4 immunomarkers [estrogen receptor (ER), vimentin (Vim), carcinoembryonic antigen (CEA), and p16INK4a], but also evaluate whether p16INK4a adds value to the ER/Vim/CEA panel characteristics and performance in distinguishing between primary ECA and EMA. A tissue microarray (TMA) was constructed using paraffin-embedded, formalin-fixed tissues from 38 hysterectomy specimens, including 14 ECAs and 24 EMAs. Tissue microarray sections were immunostained with 4 antibodies, by the avidin-biotin complex method for antigen visualization. The staining intensity and area extent of the immunohistochemical reactions were evaluated using the semiquantitative scoring system. The 3 markers (ER, Vim, CEA) and their respective panel expressions showed statistically significant (P<0.05) frequency differences in ECA and EMA tumors. The p16INK4a marker also revealed a significant frequency difference (P<0.05) between ECA and EMA, but did not demonstrate any supplementary benefit to the traditional 3-marker panel. In conclusion, when histomorphologic and clinical doubt exist as to the primary site of origin, we suggest that the conventional 3-marker (ER/Vim/CEA) panel is appropriate. Ancillary p16INK4a-marker testing does not add value to the 3-marker panel in distinguishing between primary ECA and EMA.

Significant association of genetic polymorphism of human nonmetastatic clone 23 type 1 gene with an increased risk of endometrial cancer

OBJECTIVE To investigate the association of single nucleotide polymorphisms (SNPs) of nonmetastatic clone 23 type 1 (nm23-H1) gene with endometrial cancer and their implication in clinicopathologic characteristics of women in Taiwan. METHODS Three hundred and fifty-nine blood samples were collected from 268 healthy women and 91 patients with endometrial cancer to analyze SNPs rs16949649 and rs2302254 of nm23-H1 promoter using real time polymerase chain reaction and genotyping. The association of genotype and allele differences of nm23-H1 SNPs with endometrial cancer and their implication in some clinicopathologic variables were analyzed using Pearsons Chi-square or Fisher exact tests. RESULTS Women with heterozygous genotypes TC in rs16949649 or CT in rs2302254 exhibited higher risk to develop endometrial cancer as compared to those with their wild-type or homozygous genotypes (odds ratio 3.30 and 1.86; 1.84 and 1.90 for respective SNP). Individuals with CC genotype were at less risk (OR: 0.08; P=0.037) to have non-endometrioid type as compared to those with TT genotype in rs16949649. However, a trend of increased risk (OR: 26.67; P=0.01) of advanced stage endometrial cancer (stage III-IV) was observed in patients with TT genotype as compared to those with CC genotype in rs2302254. CONCLUSIONS Heterozygous genotypes TC in rs16949649 and CT in rs2302254 of nm23-H1 promoter are potential susceptibility factors for endometrial cancer in Taiwan women. Once having the endometrial cancer, Taiwan women with variant homozygote CC in rs1694964 were at less risk to have non-endometrioid type, while women with variant homozygote TT in rs2302254 tended to have advanced stage cancer.

Clinical implications of aldo-keto reductase family 1 member C3 and its relationship with lipocalin 2 in cancer of the uterine cervix.

OBJECTIVE Over-expression of the aldo-keto reductase family 1 member C3 (AKR1C3) has been demonstrated in many human cancers. Lipocalin 2 (LCN2) is reported to inhibit cervical cancer metastasis but little is known regarding its relationship with AKR1C3 in the development and progression of uterine cervical cancer. This study aimed to investigate the involvement of AKR1C3 and its relationship with LCN2 in cervical cancer. METHODS The roles of AKR1C3 and LCN2 were investigated using the lentivirus shRNA system in SiHa and Caski cervical cancer cells. LCN2 and matrix metalloproteinase-2 (MMP-2) promoters were constructed to demonstrate transcriptional regulation by shAKR1C3 and shLCN2, respectively. The influences of metastatic phenotypes were analyzed by wound healing, Boyden chamber, and immunofluorescence assays. The activity of MMP-2 was determined by zymography assay. The impacts of AKR1C3 and LCN2 on patient prognosis were evaluated using tissue microarrays by Cox regression and Kaplan-Meier models. RESULTS Silencing of the AKR1C3 gene increased the expression of LCN2 and decreased the migratory and invasive abilities and changed the cytoskeleton of cervical cancer cells. When AKR1C3 was over-expressed, it decreased LCN2 promoter activity and LCN2 expression and increased cell migration. The mRNA level and enzyme activity of MMP-2 increased in silenced LCN2 cells. Positive AKR1C3 and negative LCN2 were correlated with higher recurrence and poorer survival of cervical cancer patients. CONCLUSIONS Silencing of AKR1C3 increases LCN2 expression and inhibits metastasis in cervical cancer. Both AKR1C3 and LCN2 serve as molecular targets for cancer therapy to improve the clinical outcome of cervical cancer patients.

Polymorphisms of Human Nonmetastatic Clone 23 Type 1 Gene and Neoplastic Lesions of Uterine Cervix

Hypothesis: Single-nucleotide polymorphisms (SNP) in promoter of human nonmetastatic clone 23 type 1 (nm23-H1) may affect their binding with transcription factors and affect promoter activity as well as gene transcription. Therefore, we investigated the impact of the nm23-H1 gene polymorphisms on the neoplastic lesions of uterine cervix in mid-Taiwan women (women who live in the central area of Taiwan). We expected that women with different genotypes in nm23-H1 polymorphisms, such as rs34214448, rs16949649, or rs2302254, may have different incidences of cervical neoplasia. Materials and Methods: In total, 366 blood samples were collected from 244 healthy women and 122 patients with cervical neoplasia to analyze 3 nm23-H1 gene single-nucleotide polymorphisms (rs34214448, rs16949649, and rs2302254). Results: The heterozygous genotypes, TG in rs34214448 or TC in rs16949649, were differentially distributed between patients with cervical neoplasia and normal women (Hommel adjusted P = .0440 and .0435, respectively) as compared to their homozygotes. Moreover, compared to those with wild-type homozygotes and heterozygotes, women with variant homozygotes TT in rs34214448 or CC in rs16949649 exert different distributions between patients with cervical neoplasia and normal women (P = .058 and .058). Interestingly, we found the genotype distribution of rs34214448 has significant association with that of rs16949649 with high consistency. Conclusions: Mid-Taiwan women with the polymorphic heterozygotes TG in rs34214448 or TC in rs16949649 of human nonmetastatic clone 23 type 1 promoter have the tendency to develop cervical neoplasia while compared to their homozygous counterparts. However, women with variant homozygotes TT in rs34214448 or CC in rs16949649 exhibit less tendency as compared to those with wild-type homozygotes and heterozygotes.

Significant Relation of Tissue Inhibitor of Matrix Metalloproteinase-2 and Its Combination With Matrix Metalloproteinase-2 to Survival of Patients With Cancer of Uterine Cervix

Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) has high affinity for matrix metalloproteinase-2 (MMP-2). Few studies simultaneously investigate their implication in prognosis of patients with cervical cancer. We used reverse transcription-polymerase chain reaction and immunohistochemical method for cervical tissues and microarrays to investigate the association among TIMP-2, MMP-2, clinicopathological parameters, and prognosis of patients with cancer. Our results showed that cancer tissues exhibited less TIMP-2 expression and patients with pelvic lymph node metastasis had less TIMP-2 expression. Positive TIMP-2 constellated with negative MMP-2 indicated lower recurrence probability and better overall survival. The protective effect of TIMP-2 expression may overcome the adverse effect of MMP-2 expression in terms of disease-free interval and overall survival while neither TIMP-2 nor MMP-2 alone can be used to predict outcome. We suggest that following patients other than those with positive TIMP-2 and negative MMP-2 expression more closely and intensely may improve their prognosis.

Assessment of common bile duct using laparoscopic ultrasound during laparoscopic cholecystectomy.

Background Several reports have shown that laparoscopic ultrasound (LUS) examination of the bile duct is promising and suggest it as a primary method for bile duct imaging during laparoscopic cholecystectomy (LC). This study was designed to evaluate the feasibility of LUS during LC, and the occurrence rate of common bile duct (CBD) stones during LC. Materials and Methods One hundred and fifteen consecutive patients with gallstones were enrolled into this study. LUS was used to assess the CBD routinely during LC, which was successfully performed in 112 cases. Choledocholithiasis was rated before LC as being of low, intermediate, or high probability on the basis of clinical, laboratory, and/or imaging findings (Cotton criteria). Duct calculi were defined as echogenic material within the CBD, which cast discrete acoustic shadows. Sludge was defined as mobile or floating low-amplitude echogenic material without discrete acoustic shadowing. Results The CBD could be evaluated in 112 of 115 LC (97.4%) patients (72 females and 40 males). The mean age was 54±16 years old. The occurrence rate of CBD stones in the low-risk group was 7%, that in the intermediate group was 36.4%, and the high-risk group was 78.9%. The overall incidence of CBD stones was 25.0%. Conclusions With increasing experience, LUS can become the routine method for evaluating the bile duct during LC. A more aggressive preoperative evaluation of CBD is mandated in the intermediate and high-risk groups of patients suspected of having CBD stones.