Biotechnic & Histochemistry

Comparison of DNA damage and proliferative capacities in smear samples of HPV positive and negative patients by micronucleus counting and AgNOR staining

Human papillomavirus (HPV) is believed to cause cervical cancer. Thousands of women develop cancer and other diseases caused by HPV each year. HPV 16 and 18 types are found in approximately 70% of cervical cancers. Micronuclei are small chromosomal fragments that are considered indicators of DNA damage. AgNOR positive dots are useful for assessing proliferation. We investigated the relation between HPV-DNA, micronuclei and AgNOR in smear samples. Three groups were defined: HPV negative, 16/18 positive and other high-risk groups (31, 33, 35, 39, 45, 51, 52, 56, 58, 66 and 68) (HR). After typing, micronuclei were identified by Papanicolaou staining and AgNOR regions were detected by silver staining. Serum reactive protein (CRP) also was measured. We found that the average age of HPV negative patients was significantly greater than for the HPV positive groups. We also found that CRP levels were significantly higher in the HPV 16/18 positive group than HPV negative and other HPV group. We found that the number of micronuclei in the HPV 16/18 group was significantly greater than for the HPV negative group. Also, we found that AgNOR staining for the HPV 16/18 group was significantly greater than for the HPV negative group. We found that CRP level, cell proliferation and genome instability were increased in HPV positive patients. The AgNOR and micronucleus tests were useful for evaluating cell proliferation and DNA damage.

The use of immunohistochemical labeling to identify patterns of proliferation and protein expression in smooth muscle tumors of the uterus

Leiomyomas (fibroids) are the most common benign tumors of the uterus and are present in greater than half the female population over 50 years old in the United States. Leiomyosarcomas are the malignant variation of leiomyomas and, while far less common, have a high mortality rate. Differential protein expression between both benign and malignant tumors and normal tissue samples forms the basis of many treatment strategies. This study evaluated protein expression of several markers using immunohistochemistry (IHC) methods on 74 leiomyomas, 14 uterine leiomyosarcomas, and 26 normal uterine myometrial samples which had been formalin-fixed and paraffin-embedded. Markers included the Ki-67 proliferation marker, estrogen receptor (ER), progesterone receptor (PR), aldehyde dehydrogenase (ALDH), B-cell lymphoma 2 (BCL-2), and cytokeratin 8/18 (CK 8/18). The Ki-67 positivity was significantly higher in leiomyosarcomas when compared with benign uterine tissues and was also higher in leiomyomas than in normal uterine smooth muscle. ER and PR were highly expressed in benign tissues but exhibited reduced expression in malignant lesions. Both CK 8/18 and ALDH were expressed in a significantly higher proportion of normal myometrial tissues as compared with leiomyomas and leiomyosarcomas. Conversely, BCL-2 expression in normal tissues was lower than in both leiomyomas and leiomyosarcomas with leiomyosarcomas producing the highest expression. The Ki-67 value can reliably differentiate between benign and malignant smooth muscle uterine tissues. Because CK 8/18 and ALDH were more frequently or strongly expressed in normal myometrium vs. leiomyoma or leiomyosarcoma, pathological changes to the cells may be the cause for a reduction in protein production. Future investigations may determine that upregulation of either of these two markers in leiomyomas or leiomyosarcomas could lead to slower tumor growth.

Tumor apelin immunoreactivity is correlated with body mass index in ovarian high grade serous carcinoma

Ovarian cancer has a high mortality rate. Serous carcinoma is the most common subtype and can be detected by distant or lymph node metastasis in advanced stages. Apelin, an adipokine associated with obesity, and its receptor, APJ, participate in lymphatic invasion. Angiogenesis also can affect lymph node involvement in serous ovarian carcinomas. We investigated apelin/APJ receptor immunoreactivity in stages III and IV ovarian cancer with or without lymph node involvement and correlated the results with body mass index (BMI) to determine whether the potential relation of the two affects the outcome of the cancer. We investigated 30 patients diagnosed between 2014 and 2016 with high grade serous ovarian cancer. Tumor:stroma ratio, indirect immunoperoxidase method, H-score and MATLAB analysis were performed. In obese and pre-obese patients, tumor apelin immunoreactivity was stronger than for patients with normal BMI. Tumor:stroma ratio was correlated with survival and lymph node involvement. Strong apelin and moderate APJ immunoreactivity was detected in both lymph node negative and positive patients. BMI was related to both survival outcome and apelin immunoreactivity. BMI, adipokines such as apelin, and the stromal compartment play critical roles in advanced stage serous carcinomas.

Use of asprosin and subfatin for differential diagnosis of serous ovarian tumors

Asprosin (ASP) and subfatin are hormones that regulate glucose metabolism. The role of ASP and subfatin in serous ovarian tumors has not been investigated. We investigated the expression of subfatin and asprosin in 30 serous benign, 30 serous borderline, 30 malignant and 30 control ovarian tissues. We investigated ASP and subfatin immunoreactivity and quantification was achieved using an ELISA method. ASP and subfatin were localized in the epithelial parts of normal ovarian tissues; however, in cancer tissues, immunoreactivity was detected in the parenchymal areas. Biochemical analysis of ovarian tissues revealed significantly decreased ASP and subfatin compared to the control. We propose that ASP and subfatin are promising candidates for biomarkers to distinguish serous benign, serous borderline and malignant ovarian cancers.

Micronucleus assay for predicting side effects of radiotherapy for cervical cancer

Radiotherapy (RT) is an important treatment for cervical cancer. The quality of life of patients undergoing RT may be compromised during and following treatment by nausea, diarrhea, vomiting, burns, erythema and fistula. Cytokinesis-block micronucleus (CBMN) assays may be useful for predicting adverse effects of RT for cancer. The CBMN test is easy to perform and is reproducible for screening subjects exposed to ionizing radiation. We investigated the use of the frequency of micronuclei (MN) from peripheral blood samples, irradiated in vitro, as a possible biomarker to predict the side effects of RT in patients with cervical cancer. We used 10 patients with cervical cancer receiving RT and chemotherapy. We found a strong relation between the frequency of MN and the appearance of acute side effects of RT for cervical cancer. We suggest that the methodology presented here may be useful for predicting side effects of RT for patients affected by cervical cancer and who have undergone chemotherapy.

1p36 is a chromosomal site of genomic instability in cervical intraepithelial neoplasia

We investigated the association between progressive stages of cervical neoplasia and DNA damage in 1p36 DNA sequences of chromosome 1 in cervical epithelium using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). We used a hospital based unmatched case control study of 29 women that were grouped according to disease stage and selected according to histological diagnosis: 10 with low grade squamous intraepithelial lesions (LG-SILs), 10 with high grade SILs (HG-SILs) and nine with no cervical lesions; the 1pter sequence was used as internal control. We found a significant increase in the number of patients with HG-SIL compared to patients with LG-SILs or with no cervical lesions. 1p36 Genomic instability was validated by DBD-FISH using neutral comets. Genetic instability at specific gene loci, such as 1p36, might be characteristic of cervical cancer progression. DBD-FISH appears to be a useful approach for detecting and comparing damage to specific chromosomal regions related to the progression of cervical cancer.

Laminin receptor 1 expression in premalignant and malignant squamous lesions of the cervix

Laminin receptor 1 (LAMR) may have a role in the progression of premalignant squamous epithelial lesions to cervical cancer. Therefore, we aimed to investigate the expression of laminin receptor 1 (LAMR) in normal, premalignant, and malignant tissues of the uterine cervix. Paraffin blocks of 129 specimens with the diagnoses of normal cervical tissue (n = 33), cervical intraepithelial neoplasia (CIN) 1 (n = 30), CIN 2 (n = 14), CIN 3 (n = 28), and squamous cell carcinoma (n = 24) were immunohistochemically stained with LAMR antibody and its expression percentage, pattern, and intensity in these tissues were assessed. Compared to the other groups, the nonstaining with LAMR was highest in low grade squamous intraepithelial lesion (LSIL) (p < 0.0001). LAMR expression, which was positive in less than 50% of cells with weak staining, increased significantly between normal cervical epithelium and high-grade squamous intraepithelial lesion (HSIL) or invasive carcinoma, as well as between LSIL and HSIL (p < 0.0001). Between LSIL and invasive carcinoma, a significant increment was also observed for weak staining in less than 50% of cells (p < 0.001). LAMR expression, which was positive in more than 50% of cells with strong staining, was significantly higher in normal cervical tissue compared to the other groups (p < 0.0001). Disease progression related gradual increment of LAMR expression from normal cervical epithelium or LSIL towards HSIL or cervical cancer reveals that LAMR may play an important role in the transition from premalignant to malignant state in cervical lesions.

Antiproliferative and apoptosis inducing effects of the methanolic extract of Centaurea hermannii in human cervical cancer cell line

We investigated the antiproliferative and apoptosis inducing effects of a methanolic extract of