Vedrana Milinkovic

Identification of genes associated with non-small-cell lung cancer promotion and progression

Lung cancer is the most common cause of neoplasia-related death worldwide. One of the crucial early events in carcinogenesis is the induction of genomic instability and mutator phenotype. We investigated genomic instability in 30 patients with non-small-cell lung cancer (NSCLC) by comparing DNA fingerprints of paired tumor and normal tissues using arbitrarily primed polymerase chain reaction (AP-PCR). Selected 21 DNA bands with altered mobility were isolated from polyacrylamide gels, cloned and sequenced. Obtained sequences were submitted to homology search in GenBank database which revealed the following genes: TSPAN14, CDH12, RDH10, CYP4Z1, KIR, E2F4, PHACTR3, PHF20, PRAME family member and SLC2A13. Following the identification of these genes we examined their relation to the clinicopathological parameters and survival of the patients. Our study revealed that genetic alterations of TSPAN14, SLC2A13 and PHF20 appeared prevalently in tumors of grade 1, stage I suggesting that structural changes of these genes could play a role in NSCLC promotion. Contrary to this CYP4Z1, KIR and RDH10 were prevalently mutated in tumors of grade 3, stage III suggesting that they could play a role in NSCLC progression. E2F4, PHACTR3, PRAME family member and CDH12 most probably play important role in NSCLC geneses. In conclusion, our study revealed altered genes previously not described in regard to this type of cancer.

Identification of Novel Genetic Alterations in Samples of Malignant Glioma Patients

Glioblastoma is the most frequent and malignant human brain tumor. High level of genomic instability detected in glioma cells implies that numerous genetic alterations accumulate during glioma pathogenesis. We investigated alterations in AP-PCR DNA profiles of 30 glioma patients, and detected specific changes in 11 genes not previously associated with this disease: LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2, KCNG2, PDE4D, KIR3DL3, and INPP5A. Further correlations revealed that 8 genes might play important role in pathogenesis of glial tumors, while changes in GP2, KCNG2 and KIR3DL3 should be considered as passenger mutations, consequence of high level of genomic instability. Identified genes have a significant role in signal transduction or cell adhesion, which are important processes for cancer development and progression. According to our results, LHFPL3 might be characteristic of primary glioblastoma, SGCG, HTR4, ITGB1, CPS1, PROS1 and INPP5A were detected predominantly in anaplastic astrocytoma, suggesting their role in progression of secondary glioblastoma, while alterations of PDE4D seem to have important role in development of both glioblastoma subtypes. Some of the identified genes showed significant association with p53, p16, and EGFR, but there was no significant correlation between loss of PTEN and any of identified genes. In conclusion our study revealed genetic alterations that were not previously associated with glioma pathogenesis and could be potentially used as molecular markers of different glioblastoma subtypes.

Amplification of Cycline D1, C-MYC And EGFR Oncogenes in Tumour Samples of Breast Cancer Patients / AMPLIFIKACIJA CIKLIN D1, C-MYC AND EGFR ONKOGENA U TUMORSKIM UZORCIMA PACIJENTKINJA OBOLELIH OD KANCERA DOJKE

Summary Background: Breast cancer is the most common form of cancer in women. It arises from multiple genetic changes in oncogenes and tumor suppressor genes. Among so far studied oncogenes relatively few, including epdermal growth factor receptor (EGFR), cyclinD1 (CCND1)and cmyc, have been found to play an important role in progression of this type of human malignancy. The aim of this study was to examine the prognostic potential of CCND1, c-myc and EGFR amplification and their possible cooperation in breast carcinogenesis. Methods: Copy number analyses of CCND1 and c-myc genes were done by TaqMan based quantitative real time PCR. Am pli fication status of EGFR was determined by differential PCR. Results: Amplification of CCND1, c-myc and EGFR onco- gene has been found in 20.4%, 26.5% and 26.5% of breast cancer cases, respectively. Analysis showed that amplification of CCND1 oncogene was significantly associated with the stage II of disease while amplification of EGFR gene was sig- nificantly associated with overexpression of HER-2/neu. Tu- mour stage and expression of HER-2/neu appeared to be significant predictors of patients outcome. Stage I patients lived significantly longer then stage III patients (p=0.04) while patients with FiER-2/neu overexpression had worse prognoses and lived significantly shorter (p=0.001). Finally, survival of patients who underwent hormone therapy only was significantly longer (p=0.001) then survival of the rest of patients. Conclusions: Amplification of CCND1 or EGFR oncogene is associated with the progression of breast cancer and bad prognosis. No co-ordination in amplification of CCND1, c- myc and EGFR oncogenes were established in this cohort of breast cancer patients. Kratak sadržaj Uvod: Kancer dojke je najčešči tip maligniteta koji se javlja kod žena. Tumori dojke nastaju kao rezultat akumulacije genetičkih promena kako u onkogenima tako i u tumor supresorskim genima. Medu mnogim onkogenima čija je uloga u genezi tumora dojke ispitivana do danas, samo se neki smatraju značajnim za razviče ovih karcinoma. U tu se grupu svakako ubrajaju receptor za epidermalni factor rasta (EGFR), c-myc i ciklinDI (CCND7). Cilj rada je bio utvrditi prognostickí značaj amplifikacije CCND1, c-myc i EGFR onkogena u razvicu tumora dojke kao i eventualne medusob- ne koalteracije ovih gena. Metode: Amplifikacioni status CCND1 i c-myc gena odreden je kvantitativnim PCR-om u reálnom vremenu, a amplifikacioni status EGFR onkogena je definisan diferencijalnim PCR-om. Rezultati: Amplifikacija CCND1 gena detektovana je kod 20,4%, a c-myc i EGFR onkogena kod 26,5% ispitanih uzo- raka. Analize su pokazale da je amplifikacija CCND1 onko- gena statistički značajno povezana sa stadijumom II tumora dojke kao i da amplifikacija EGFR-a značajno korelira sa povečanom ekspresijom HER2/neu. Analize kliničkih i histo- patoloških parametara su jasno pokazale da stadijum tumo- ra i nivo ekspresije HER2/neu gena predstavljaju značajne pokazatelje daljeg toka bolesti, odnosno sudbine pacijenta. Utvrdeno je da pacijentkinje sa tumorima dojke stadijuma I žive značajno duže od onih sa tumorom stadijuma III (p= 0,04) kao i da pacijentkinje sa HER2/neu pozitivnim statu- som imaju goru prognózu i žive značajno krače (p=0,001). Na kraju, študija je pokazala da pacijentkinje podvrgnute samo hormonskoj terapiji imaju najbolju prognózu i žive značajno duže od ostalih (p=0,001). Zaključak: Amplifikacija CCND1 i EGFR onkogena je po- vezana sa losom prognozom i progresijom karcinoma dojke. U ispitivanom tumorskom uzorku nisu detektovane nikakve koalteracije CCND1, c-myc i EGFR onkogena.

Concurrent alteration of p16 and PTEN tumor suppressor genes could be considered as potential molecular marker for specific subgroups of NSCLC patients

p16 and PTEN are tumor suppressors that are commonly inactivated in human cancers. Loss of each of these molecules is widely studied in lung cancer, including non-small cell lung carcinoma (NSCLC), its most common clinical form. However, the importance of their mutual alterations for NSCLC pathogenesis has been barely examined so far. In this study we tested hypothesis that aberrant p16 might cooperate with inactive PTEN during pathogenesis of NSCLC, particularly in promoting tumor aggressiveness and invasiveness. Initially, we screened NSCLC tumor samples from patients for the presence of the most common genetic and epigenetic alterations of p16 and further correlated them with previously detected aberrations in PTEN gene. Statistical analyses showed that aberrant p16 directly correlated with altered PTEN. Such significant correlation was also observed in groups of patients with high genomic instability, with squamocellular histological subtype, with disease grade 2 and with lymph node invasion. Finally, survival analyses revealed dramatic decrease in survival rate of patients with mutual alterations of p16 and PTEN, but without prognostic significance. Overall results implicate cooperation between aberrant p16 and PTEN in pathogenesis of NSCLC and suggest that their combination might be considered as potential molecular marker for specific subgroups of NSCLC patients.

The impact of TP53 and RAS mutations on cerebellar glioblastomas.

Cerebellar glioblastoma (cGBM) is a rare, inadequately characterized disease, without detailed information on its molecular basis. This is the first report analyzing both TP53 and RAS alterations in cGBM. TP53 mutations were detected in more than half of the samples from our cohort, mainly in hotspot codons. There were no activating mutations in hotspot codons 12/13 and 61 of KRAS and HRAS genes in cGBM samples but we detected alterations in other parts of exons 2 and 3 of these genes, including premature induction of STOP codon. This mutation was present in 3 out of 5 patients. High incidence of RAS mutations, as well as significantly longer survival of cGBM patients compared to those with supratentorial GBM suggest that cGBM may have different mechanisms of occurrence. Our results suggest that inactivation of TP53 and RAS may play an important role in the progression of cerebellar GBM.

Prolonged survival after neoadjuvant chemotherapy related with specific molecular alterations in the patients with nonsmall-cell lung carcinoma.

Lung cancer is the most common cause of neoplasia-related death worldwide. Accounting for approximately 80% of all lung carcinomas, the non-small cell lung carcinoma (NSCLC) is the most common clinical form with its two predominant histological types, adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Although surgical resection is the most favorable treatment for patients with NSCLC, relapse is still high, so neoadjuvant chemotherapy (NAC) is an accepted treatment modality. In this study we examined whether some of the key molecules associated with the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have predictive and prognostic value for the NAC application. To that end we examined the expression status of PTEN, pAKT, pERK and loss of heterozygosity (LOH) of PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC. LOH PTEN and low pERK expression is shown to be correlated with the longest survival of patients with SCC and ADC, respectively, who received NAC. These results point that the application of NAC is beneficial in the NSCLC patients with specific molecular alterations which could further help to improve constant search for the druggable molecular targets used in personalized therapy.

Importance of Copy Number Alterations of FGFR1 and C-MYC Genes in Triple Negative Breast Cancer

Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical course and is unresponsive to anti-HER2 and endocrine therapy. TNBC is difficult to treat and is often lethal. Given the need to find new targets for therapy we explored clinicopathological significance of copy number gain of FGFR1 and c-MYC. Our aim was to determine the impact of FGFR1 and c-MYC copy number gain on clinical course and outcome of TNBC. Methods: FGFR1 and c-MYC gene copy number alterations were evaluated in 78 archive TNBC samples using TaqMan based quantitative real time PCR assays. Results: 50% of samples had increased c-MYC copy number. c-MYC copy number gain was associated with TNBC in contrast to ER positive cancers. Our results showed significant correlation between c-MYC copy number gain and high grade of TNBCs. This suggests that c-MYC copy number could be an useful prognostic marker for TNBC patients. c-MYC copy number gain was associated with high pTNM stage as well as lobular and medullary tumor subtypes. 43% of samples had increased FGFR1 copy number. No correlations between FGFR1 copy number gain and clinicopathological variables were observed. Conclusions: We identified c-MYC copy number gain as a prognostic marker for TNBC. Our results indicate that cKratak sadr`aj

Abstract B42: Molecular and cytogenetic characterization of novel multidrug-resistant human tumor cell lines

Multidrug resistance (MDR) is the major cause of failure to a successful cancer treatment. Cancer cells manifesting MDR phenotype are cross-resistant to many structurally and functionally unrelated drugs. The purpose of this study was to establish in vitro models, i.e., resistant cell lines, to further investigate the mechanisms of MDR. We used the human colon cancer cell line DLD1 and the glioma cell line U87 to establish paclitaxel-resistant cell lines, DLD1 -TxR and U87-TxR, by continuous exposure to stepwise increasing concentrations of the drug. The newly established, DLD1 -TxR and U87-TxR cell lines showed extensive resistance to paclitaxel (301-fold and 103-fold respectively). They also demonstrated cross-resistance to other anticancer agents such as doxorubicin, epirubicin, vinblastin, and etoposide. Cytogenetic data based on inverted-DAPI Banding, demonstrated that both resistant cell lines retained several karyotypic characteristics of their sensitive counterparts and also acquired novel structural or numerical chromosomal aberrations that may be related to resistance to chemotherapy. Interestingly, while the dividing parental U87 cells were mainly near-tetraploid, chemoresistance in the U87-TxR cell line was accompanied by one level ploidy reduction as verified by the increased prevalence of near-diploid cell populations. In addition, the DLD1 -TxR cells showed an increase tendency to loose the Y chromosome. Gene expression analyses revealed a 4-fold increase in the level of mdr1 mRNA in DLD1 -TxR cells. U87-TxR cells demonstrated prominent mdr1 activity since the expression in parental cells was undetectable. In contrast, the level of mrp1 mRNA was 40% decreased in both resistant cell lines. The expression of other MDR-related genes, topo IIα, and lrp, was unaffected by paclitaxel resistance. Flow cytometry analyses showed that the accumulation of rhodamine in resistant cells DLD1 -TxR and U87-TxR was significantly lower than in their sensitive counterparts, 17-fold and 8-fold, respectively. These results indicate that P-gp, encoded by mdr1 gene, is significantly overexpressed in both resistant cell lines and may account for paclitaxel resistance. In conclusion, the two novel resistant cell lines, DLD1 -TxR and U87-TxR may serve as appropriate models for the study of the mechanisms of paclitaxel resistance offering novel insights into potential targets for overcoming MDR that would improve the response to chemotherapy and patient outcome. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B42.

Abstract B2: Alterations of p53, PTEN, and EGFR genes in glioblastoma multiforme

Glioblastomas are the most frequent and the most aggressive human brain tumors. Glioblastoma multiforme is one of the most intensively investigated human malignancy but the molecular mechanisms associated with the evolution of this type of tumor are still poorly understood. The aim of this study was to investigate alterations in EGFR oncogene and PTEN and p53 tumor supressor genes in 35 glioma specimen and to evaluate their role in glioma pathogenesis. DNA from tumor and blood of 35 patients with glioma was isolated and used to perform analysis of p53 mutational status using single-strand conformational polymorphism analysis (PCR- SSCP) and sequencing, evaluate loss of heterozigosity (LOH) in p53 and PTEN by fragment analysis, and to asseess EGFR gene amplification by differential PCR. Obtained results were further correlated with clinicopathological parameters. Loss of heterozygosity of PTEN was the most frequent alteration present in 62.86% of samples, followed by EGFR amplification detected in 40% of cases, while p53 gene was mutated in 28.57% of cases and inactivated by LOH in 20% of cases. All three genes were altered in 11.43% of samples, while 45.71 % of samples had only one of these three genes aberrant. We also identified 5 novel mutations in p53 that were not associated with brain tumors before. Correlation analysis indicated that p53 alterations were significantly associated with younger age ( Citation Information: Clin Cancer Res 2010;16(14 Suppl):B2.