Detection of oncogenic mutations in cervical carcinoma using method High Resolution Melting (HRM).

Oncogenic mutations in proto-oncogenes and tumor suppressor genes represent one of key events in cancerogenesis. In this study, we analysed mutation status in PIK3CA, KRAS and EGFR proto-oncogenes and TP53 tumor suppressor gene in a cohort of twenty-four patients diagnosed with squamous cell carcinoma or adenocarcinoma using the screening method "High Resolution Melting" (HRM). Positive findings were confirmed and identified by Sanger sequencing. Totally, we detected DNA sequence changes in targeted regions in seven patients (7/24, 29.2%). In PIK3CA gene, we found six sequence changes in four patients (4/24, 16.7%) and four of them were confirmed as oncogenic mutations. In KRAS gene, we detected sequence changes in four patients (4/24, 16.7%). Conversely, we identified pathogenic or potentially pathogenic sequence changes neither in EGFR nor TP53 genes. Our results suggest that sequence changes are specific neither for a certain histological subtype, clinical stage nor lymph node involvement and they appear independently on the presence of HPV (human papillomavirus) infection since early clinical stages. We observed the correlation between the presence of DNA sequence changes and hTERC gene amplification, but we did not find a significant relationship between the identified DNA sequence changes and detected copy-number alterations using the technique of array-CGH (array-based comparative genomic hybridization). Regardless our results confirmed an important role of oncogenic mutations in PIK3CA and KRAS genes in the neoplastic transformation process in the cervical carcinoma pathogenesis. Their identification in the early clinical stages should encourage further studies to better understand these mutations and exploit them for more detailed diagnostics.

Molecular cytogenetic analyses of hTERC (3q26) and MYC (8q24) genes amplifications in correlation with oncogenic human papillomavirus infection in Czech patients with cervical intraepithelial neoplasia and cervical carcinomas.

UNLABELLED: It is known that cervical cancer develop from precancerous intraepithelial neoplasia (CIN) which is characterized by series of genetic abnormalities. The progression of CIN to cervical carcinoma has been associated especially with the genomic integration of oncogenic human papilloma virus (HPV) and gain of the human telomerase RNA gene hTERC (3q26) and MYC (8q24). In this study, cytology specimens of cervical intraepithelial neoplasia and cervical carcinoma from 74 Czech women were analyzed using the triple-color Cervical FISH Probe Kit designed for identification of HPV infected cells and copy number aberration of the hTERC and MYC genes. HPV-positivity exhibited 70% of patients with premalignant lesions (CIN I - CIN III, carcinoma in situ), chromosomal changes were found in 53.3% of cases - MYC amplification had 33.3% of women with CIN I - CIN III and 50% with carcinoma in situ. Amplification of hTERC was detected in 16.7% of patient with CIN I, in 50% with CIN II, in 58.3% with CIN III and in 66.7% with carcinoma in situ. Based on HPV-positivity and the occurrence of chromosomal aberrations, patients were divided into high-, intermediate- and low-risk group. Among women with cervical carcinomas, HPV infection was detected in 90.1% of specimens and chromosomal aberrations were found in 87.5% of samples. Amplification of MYC gene was detected in 25% and hTERC gene in 62.5% of patients. According to the histopathological grade of tumors, MYC gene amplification occurred more frequently in specimens of spinocellular carcinoma than adenocarcinoma (p=0.029). We found no association between the frequency of cytogenetic lesions and the incidence of lymphangiogenesis or lymph node metastases in cervical carcinoma patients. Simultaneous hTERC and MYC genes amplification was significantly more frequent in samples of cervical carcinomas than in premalignant lesions (p=0.008).In a cohort of 26 patients with cervical carcinoma we used oligo-based GGH+SNP microarray technique for the high resolution mapping of copy number changes of hTERC and MYC genes. We found that recurrent gain of genetic material in chromosome 3q26 area carrying hTERC gene of size 43.6 Mb between 3q25.1-3qter and duplication of 3q were the most common genomic identifications of amplified gene. In MYC locus array-CGH profiling identified duplication of 8q and trisomy 8 as frequent genomic changes.Our work confirmed that in cervical carcinoma gains of hTERC and MYC genes are specific genomic changes associated with developing of malignant phenotype. We also showed that in premalignant stages HPV-FISH assay can be used as an effective diagnostic procedure to identify patients carrying highly risking HPV infection and chromosomal aberrations associated with this malignancy. KEYWORDS: cervical cancer, cervical dysplasia, HPV infection, hTERC amplification, MYC amplification, FISH, array-CGH.

[Preinvasive lesions in gynecology -  vulva]. / Prekancerózy v gynekologii -  vulva.

For preinvasive lesions of vulva, a common term VIN -  vulval intraepithelial neoplasia is used. VIN is a histological dia-gnosis based on abnormal squamous epithelial proliferation. There are two types of VIN apart from their association with human papillomavirus (HPV). Undifferentiated (usual) vulval intraepithelial neoplasia is commonly associated with carcinogenic genotypes of HVP, whereas differentiated vulval intraepithelial neoplasia is not associated with high-risk genotypes of HPV. The article presents an overview of VIN occurence and epidemiology, its classification system and dia-gnostics. In conclusion, VIN therapeutical possibilities are presented. It can be treated with surgical therapy (local excision, partial vulvectomy, vulvectomy, laser vaporization) or medical therapy (imiquimod).

[Preinvasive lesions in gynaecology -  vagina]. / Prekancerózy v gynekologii -  pochva.

Preinvasive lesions of the vagina are relatively rare, clearly defined afflictions, originating most of all in association with the highrisk human papillomavirus infection (HR HPV). The most frequent appearance is in coincidence with preinvasive lesions of uterine cervix and in vaginal cuff after hysterectomy. Preinvasive vaginal lesions are divided into squamous lesions (vaginal intraepithelial neoplasia) and nonsquamous lesions. Vaginal adenosis belongs to nonsquamous vaginal preinvasive lesions and is a precursor lesion of vaginal clear cell carcinoma. Surgical and nonsurgical techniques including laser destructive methods and local administration of 5- fluorouracile are used in the treatment of preinvasive lesions of vagina.

[Preinvasive lesions in gynaecology - uterine cervix]. / Prekancerózy v gynekologii - delozní hrdlo.

Preinvasive lesion of the uterine cervix can give rise to cervical cancer. High-risk human papillomaviruses with high oncogenic potential are considered to be the main etiopathological factors with interaction of other risk factors (recurrent inflammation of the cervix, injury of the cervix, immunosuppressive conditions, sexual promiscuity, etc.). Early dia-gnosis of these changes at regular gynecological examinations and adequate treatment can prevent of malignant transformation. Organized cervical screening and implementation of nationwide vaccination against human papillomavirus promises to reduce the incidence of cervical cancer.

[Preinvasive lesions in gynaecology -  endometrium]. / Prekancerózy v gynekologii -  endometrium.

Preinvasive lesions of the endometrium are histopathological and molecular alterations related to high-risk of uterine carcinoma development. Incidence has increasing tendency. Atypical endometrial hyperplasia is the preinvasive lesion of type I endometrial carcinoma, developing under a hyperestrogenic background. Carcinoma in situ of the endometrium is considered to be precursor lesion of type II endometrial carcinoma, first of all uterine serous carcinoma. Hysterectomy and bilateral oophorectomy is the main therapeutical modality in both preinvasive lesions of the endometrium. The hormonal therapy with progestogens is the possibility of fertility sparing approach in treatment of histological findings of atypical endometrial hyperplasia in young women. Even though preinvasive lesions of the endometrium are clearly defined, the possibilities of endometrial cancer screening are markedly limited.

[Preinvasive lesions in gynaecology -  ovary]. / Prekancerózy v gynekologii -  ovarium.

In comparison to malignant tumors of vulva, vagina, cervix and uterine corpus, clear morphologic and molecular genetic features of precursor lesions of ovarian carcinoma have not been defined yet. We can see an effort to describe preinvasive lesions to allow dia-gnostics and treatment prior to development of invasive ovarian cancer. This tendency is magnified by the fact that ovarian carcinomas have the highest mortality from all gynecological malignancies. Currently, reports confirming different morphology, pathogenesis and molecular alterations in heterogeneous group of ovarian carcinomas have been described. There is a tendency to divide epithelial malignant tumors into two groups. Low grade ovarian serous carcinoma, low grade endometrioid, clear cell, mucinous ovarian cancers and Brenner tumors of ovary are categorized as type I ovarian tumors. Highgrade serous carcinoma, undifferentiated carcinomas and malignant mixed mesodermal tumors of the ovary (MMMTs) belong to type II tumors. A potential precursor lesion of highgrade serous ovarian cancer has been defined -  serous tubal intraepithelial carcinoma.

[Prognostic factors for cervical cancer]. / Prognostické faktory karcinomu delozního hrdla.

Standardized gynecological oncological therapeutical guidelines are based on ordinary predictive factors, such as depth of stromal invasion, histopathological type of tumor, lymphovascular space invasion, lymph node metastases. Unfortunately, the power of these prognostic factors is not able to determine the safety of this procedure in the relation to disease recurrence in a group of patients who are indicated for conservative operations. This is the appropriate area for new, especially biomolecular prognostic factors (proteins: p63, TAp63, p16, p21, p27, COX-2, genes: hTERC, MYCC). Moreover, comprehensive evaluation of cervical cancer prognostic factors and assessment of new biomarkers of cancer can ease prediction of risk of spread outside primary localization and determine probability of disease recurrence. This information can help to individualize surgical, radiotherapeutic and chemotherapeutic treatment.

[Sentinel lymph node identification using hysteroscopy in patients with endometrial cancer]. / Detekce sentinelové uzliny u pacientek s karcinomem endometria s vyuzitím hysteroskopie.

BACKGROUNDS: Endometrial carcinoma is the most frequent gynecologic malignancy. The incidence is 30 : 100,000 with an increasing tendency. The main therapeutic modality remains radical surgery. The purpose of the study is to evaluate the feasibility of sentinel lymph node (SLN) detection in endometrial cancer using hysteroscopic administration of radiocolloid and the combination of preoperative lymphoscintigraphy with intraoperative gamma-detection probe examination. PATIENTS AND METHODS: From May 2006 to January 2009, 99mTc-labelled nanocolloid (100 MBq) was administered preoperatively in 21 patients with endometrial cancer. On the day of surgery, radiocolloid together with blue dye was injected via 20-gauge needle under the endometrium using hysteroscopy. Lymphoscintigraphy was performed in all patients after 60 minutes. Therapeutic surgery followed the tracer administration 2 hours later in extensity of abdominal hysterectomy, bilateral salpingo-oophorectomy, peritoneal wash, pelvic lymphadenectomy and in patients with positive high-risk prognostic factors of paraaortic lymphadenectomy. SLN was located by use of gamma-detecting probe intraoperatively. RESULTS: At least one SLN was detected in 17 of 21 (81%) patients included in the study. The mean number of detected SLNs was 2 (range 1-5). 8 of 17 (47%) patients had radioactive nodes only in the paraaortic area. Synchronic appearance of SLNs in the pelvic and paraaortic areas was detected in 1 of 17 (6%) cases. Overall, in 4 of 9 (44%) cases of sentinel lymph node localization in the paraaortic area the SLNs were detected at the level above the inferior mesenteric artery. The metastatic involvement of 3 sentinel lymph nodes was detected in one patient (3 lymph nodes with micrometastases). All the remaining lymph nodes not labelled as SLNs were histologically negative in this case. The sensitivity and specificity for SLN metastases detection was 100%. CONCLUSION: SLN detection in endometrial cancer appears to be a promising method with the potential to reduce unnecessary surgery radicality and to clarify staging. The utilization of hysteroscopic application of radiocolloid respects the anatomical consequences and natural lymphatic drainage of the endometrium. The combination of pre-operative lymphoscintigraphy and intra-operative detection using a handheld gamma probe can be beneficial.