CHEK2 1100delC and Del5395bp mutations in BRCA-negative individuals from Serbian hereditary breast and ovarian cancer families.

PURPOSE: Hereditary breast/ovarian cancer (HBOC) occurs in families with several members affected. Most HBOC is caused by mutations in high penetrance BRCA genes accounting for about 5% of all breast cancers. There is a large number of genes with moderate or low penetrance, contributing to non-BRCA aggregation of breast and ovarian cancers. In the present study, we evaluated the presence and frequency of 1100delC and Del5395 mutations in CHEK2 gene in Serbian BRCA-negative HBOC cases. METHODS: We analyzed 57 BRCA-negative subjects from high risk breast/ovarian cancer families from Serbia for CHEK2 1100delC and Del5395 mutations. We used two multiplex allele specific PCR in order to detect them. All suspected positive cases were compared with controls and confirmed by DNA sequencing. RESULTS: 1100delC was not detected in the tested group. However, we detected one Del5395 mutation in a female diagnosed with breast cancer at the age of 32 and with apparent family history of breast cancer (her mother and sister were diagnosed with breast cancer at 42 and 39 years of age, respectively). The frequency of Del5395 mutation in our tested group was 1.7% (1/57). CONCLUSIONS: 1100delC variant in CHEK2 gene was not present in the tested subjects from HBOC families in Serbia. However, the finding of Del5395 mutation does not allow us to discard a possible involvement of this gene in breast cancer susceptibility in Serbian population. It would be of great interest to assess the distribution of this large deletion in other countries from the Balkan region in order to assess its geographical distribution and possible founder effect.

KRAS and BRAF mutations in Serbian patients with colorectal cancer.

PURPOSE: Mutations of KRAS and BRAF genes represent molecular biomarkers of response to targeted therapy in patients with metastatic colorectal cancer (mCRC). Since these mutations have been shown to exert different biological effects and impacts on patients' outcome, there is a need to determine reliably the frequency and types of KRAS mutations for diagnostic and individual therapeutic purposes. Despite having a wild type (wt) KRAS, some patients fail to respond to treatment. BRAF V600E mutation is an additional molecular determinant of response to the same therapy. In this study we described the KRAS and the BRAF V600E mutation spectra and frequencies in a group of Serbian mCRC specimens. METHODS: KRAS mutations were determined with DxS TheraScreen® K-RAS Mutation Kit and KRAS StripAssay(™), and for the BRAF V600E mutation we applied High Resolution Melting (HRM) analysis. RESULTS: KRAS mutations were present in 34.7% of 190 analyzed samples. The 7 most frequent mutation types observed were: G12D 43.9%, G12V 21.2%, G12A 10.6%, G12C 7.6%, G12S 4.5%, G12R 1.5%, G13D 10.6%. Among the wt KRAS patients, 17.8% carried the BRAF V600E mutation. CONCLUSIONS: We have shown that the spectrum and frequency distribution of the identified KRAS and BRAF mutations in Serbian study population are in good accordance with literature data. We believe that our results are significant concerning aspects related to tumor molecular biology as well as to patient selection in the diagnostic settings.

Therapeutic cancer vaccines in cervical cancer: phase I study of Lovaxin-C.

Producing effective therapeutic vaccines has proved much more difficult and challenging than developing cancer preventive vaccines. Despite huge research in the area of cancer immunology, FDA/EMEA have not approved any type of cancer treatment vaccine so far. More than 99% of cervical cancers have detectable amounts of human papillomavirus (HPV) DNA. Integration of high-risk HPV into the host cell genome is followed by continual expression of HPV E6 and E7 oncoproteins, making them excellent targets for developing vaccines which could be used in high grade precancerous (CIN) lesions or invasive cancer or in the prevention of cancer recurrence. Therapeutic cervical cancer vaccines have been extensively studied. Strategies used were vaccination with HPV peptides or proteins, alone or in pulsed dendritic cells, DNA vaccines, virus-like particles or viral and bacterial vectors. Lovaxin-C is a recombinant live-attenuated Listeria monocytogenes (Lm) that secretes the antigen HPV-16 E7 fused to a non-hemolytic listeriolysin O protein. In a phase I study Lovaxin-C was administered to advanced cervical cancer patients refractory to existing therapies. The dose-limiting toxicity was hypotension and flue-like syndrome. There were no serious adverse events. Specific T-cell response was detected as well as clinical response to Lovaxin-C. Several other therapeutic HPV vaccines are in clinical development and in most of the studies specific immunological and clinical responses were seen. Efficacious therapeutic vaccine for the treatment of cervical cancer should be expected in the near future.

TP53 mutations in breast cancer: association with ductal histology and early relapse of disease.

PURPOSE: This study aimed to investigate the incidence of core domain TP53 mutations in Serbian breast cancer patients in view of their possible correlation with prognostic parameters, tumor characteristics and clinical disease course. METHODS: 145 breast cancer patients were included. Data on clinical disease course were available for 100 patients including 30 node-negative and 70 node-positive patients. After surgery, node-positive patients underwent adjuvant chemotherapy, mostly CMF. TP53 mutations were detected by PCR-SSCP. RESULTS: 31 mutations were found in 27/145 patients including 4/59 node-negative patients and 23/83 node-positive patients (4 double mutations). 26/31 TP53 mutations were found in patients with invasive ductal carcinoma and only 2 in patients with invasive lobular carcinoma. The presence of TP53 mutations was correlated with clinical disease course in premenopausal node-positive patients (n=70). 11/20 patients with TP53 mutations relapsed. Within the first 24 months of follow-up, significantly shorter disease-free intervals were observed in TP53-mutated patients. CONCLUSIONS: TP53 mutations correlated only with nodal status and ductal histology. The significance of the predominant distribution of TP53 mutations in tumors with a ductal histology for the aggressive behavior of these tumors has yet to be proved, since the favorable biological features of tumors with a lobular histology do not result in a better prognosis. Early relapse in mutated-TP53 carriers may support data on its predictive value with respect to adjuvant CMF.

p53 gene alterations and human papillomavirus type 16 infection in early stages of cervical carcinoma in Serbia.

PURPOSE: The incidence rate (age-standardized) of cervical carcinoma in Serbia is the highest in Europe. p53 is mainly inactivated at protein level in carcinomas associated with human papillomavirus (HPV) infection, such as cervical carcinomas. These tumors show low rate of p53 mutations. It is not clear if p53 mutations confer additional impact on disease prognosis. The role of polymorphic variant at codon 72 of p53 gene on patient's prognosis is controversial. The aim of this study was to determine the frequency of p53 mutations and to assess polymorphic variants of codon 72 among cervical carcinoma patients. PATIENTS AND METHODS: 53 patients, mainly FIGO stage I (n=50), with squamous cell carcinoma (n=49) were included. 30/32 (94%) patients who received adjuvant radiotherapy were followed-up (median 15 months, range 4-39). DNA was isolated by the salting out method from tumor tissue (n=53) and blood (42/53). p53 mutations were detected by PCR-SSCP (polymerase chain reaction - single-stranded conformational polymorphism) electrophoresis. Codon 72 polymorphism was assessed by the restriction fragment-length polymorphism method. RESULTS: Six p53 mutations were detected in 5/53 (9%) patients with FIGO stage I squamous cell carcinoma (one patient had double mutations). 25/42 (60%) patients exhibited Arg/Arg genotype. HPV16 type was detected in 29/51 (57%) cervical carcinoma samples. Relapse of disease occurred in only 2 patients- both with Arg/Arg genotype and HPV16 positive. One of them exhibited p53 mutation. CONCLUSION: Our results showed low incidence of p53 mutations and prevalence of Arg/Arg genotype polymorphic variant of codon 72 of p53 gene in early stages of cervical carcinoma.