Radek Trojanec

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are a promising prognostic biomarker of gliomas. The purpose of our study was to examine the clinical prognostic properties of IDH1/2 mutations in a glioma patient cohort from the Czech Republic using an improved platform for simple and reliable IDH genotyping.We retrospectively analyzed a group of 145 glioma patients by testing for the three most frequent IDH mutations, IDH1 R132H, IDH1 R132C, and IDH2 R172K, through the competitive amplification of differentially melting amplicons (CADMA) polymerase chain reaction (PCR). O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, copy number of EGFR, p53, RB1, MDM2, CDKN2A genes, and deletions in 1p, 19q and 10p chromosomal regions were also analyzed and correlated with clinical characteristics.Of 145 gliomas, 36 harbored IDH1 R132H mutation and 1 IDH1 R132C mutation. We did not detect any IDH2 R172K mutation. IDH1 mutations were positively associated with MGMT methylation (OR 3.08, 95% CI 1.387-7.282; p = 0.007), 1p/19q co-loss (OR 8.85, 95% CI 2.367-42.786; p = 0.002) and negatively associated with epidermal growth factor receptor amplification (OR 0.12, 95% CI 0.019-0.437; p = 0.006) and 10p loss (OR 0.09, 95% CI 0.005-0.436; p = 0.019). The overall survival of IDH-mutant was 25 months, but only 9 months in IDH-wild type gliomas (p = 0.035); at the same time, survival associated with methylated vs. unmethylated MGMT promoter did not significantly differ (p = 0.166).Despite IDH1 mutations being closely associated with MGMT methylation in glioma patients, IDH1 mutations in glioblastoma patients are stronger marker of overall survival than MGMT methylation and should be the marker of choice, especially when using genotyping by CADMA PCR.Key words: isocitrate dehydrogenase - polymerase chain reaction - glioma - glioblastoma.

To date, no comprehensive prognostic or predictive marker profiling analysis has been performed in association with the age of patients with breast cancer. In the present study, 632 breast cancer tissue samples were analyzed for expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), B-cell lymphoma (Bcl)-2 protein, HER2 gene amplification, proliferation [as evaluated by proliferating cell nuclear antigen (PCNA) and Ki-67 index], tumor grade, histological type and molecular subtype. The data revealed correlations with the age of patients. A statistically significant positive correlation was identified between patient age and expression of ER (P<0.0001). There was no significant association between patient age and PR, HER2 protein expression, HER2 gene amplification or PCNA. A significant negative correlation between age and Ki-67 expression (P<0.0001) as well as grade of tumor (P=0.007) was identified. The spectrum of molecular subtypes differed according to age (P=0.0003). The highest incidence of aggressive triple-negative and HER2-positive breast cancer was present in patients aged between 20 and 39 years. Luminal A subtype was the most frequent cancer subtype in patients from age 40 onwards, where proliferation activity declined with age and expression of hormone receptors increased along with Bcl-2 expression. Aggressive forms of breast cancer were more common in younger patients. Prognostic and predictive markers have a complex age-specific distribution. The findings of less aggressive luminal A and B subtypes in older patients, and the positive correlation with ER, PR and Bcl-2 expression reveal the potential efficacy of Bcl-2 as a marker of hormone responsiveness in these patients.

Gliosarcoma is a rare, malignant CNS tumor with a very poor prognosis. Gliosarcoma is a variant of glioblastoma multiforme, which is characterized by the presence of both glial and mesenchymal components. The treatment strategy for gliosarcomas has not yet been determined clearly. This case report presents a 23-year-old female patient who complained of increasing headaches, nausea and vomiting, and slight motor weakness in her left arm. An MRI scan of the brain showed a tumor filling the anterior part of the right lateral ventricle and extending into the right frontal lobe. Tumor extirpation was performed. Histology revealed gliosarcoma. Subsequently, the patient received concomitant chemoradiotherapy with temozolomide in the Stupp regimen. Following the fourth cycle of maintenance temozolomide chemotherapy, at eight months after diagnosis, an MRI scan detected progression of the tumor residue. The patient underwent another surgery and then received 10 cycles of second-line chemotherapy in the ICE (ifosfamide, carboplatin, and etoposide) regimen. She completed oncological therapy with minimal toxicity and follow-up MRI scans showed virtually no residual tumor. Another follow-up MRI scan, performed 28 months after diagnosis, demonstrated progression of the tumor residue again. A third tumor resection was performed 29 months after initial diagnosis. Histology again confirmed gliosarcoma. An early postoperative MRI scan showed subtotal resection with a tumor residue in eloquent areas and also suspected implantation metastasis in the spinal canal at the C2 level. From the neurological perspective, the patient was fully self-sufficient, and had only a very mild motor deficit in her left arm. Currently, at 31 months after initial diagnosis, the patient is in a stable condition and fully self-sufficient. Our case report shows that long-term survival can be achieved in a gliosarcoma patient exhibiting all the unfavorable features in clinical-pathological terms. The minimal recommended treatment is maximal resection followed by adjuvant radiotherapy. Our patient also underwent chemoradiotherapy with temozolomide in the Stupp regimen. Recurrence at eight months after diagnosis was managed by a repeat operation and high-dose combination chemotherapy, which kept the disease in remission for 20 months after the initial relapse. The lack of unequivocal rules for chemotherapy provides an opportunity to test less common treatment regimens.Key words: gliosarcoma - surgery - chemotherapy - radiotherapy - survivalThis study was supported in part by the grant No. NT13581-4/2012(86-91) of the Internal Grant Agency of the Czech Ministry of Health.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 26. 3. 2016Accepted: 27. 4. 2016.

Patients with GBM continue to have a dismal prognosis, with a median survival of about 12 months. This prospective population-based study is focused on the relation among the selected gene aberrations and overall survival of primary Glioblastoma Multiforme patients only with resection. We collected clinical data of 140 patients treated in our hospital from Jully 2006 to June 2014. All tumour samples were submitted to histologic analysis and were investigated for the aberrations of TP53, EGFR1, PTEN, MDM2, RB1, CCND1, BCR, 9p21 (CDKN2A, p16), 10p11, 19q13, 1p36, IDH1 mutation, and MGMT promoter methylation. The younger age, Karnofsky score an chemoradiotherapy (14.6 vs 5.3 months in radiotherapy alone) at diagnosis was a positive and smoking was a negative prognostic factor. Temporal lobe tumour origin was associated with a shorter period of Performance free status in the group with chemoradiotherapy. Relation with OS according to univariate Cox regression model: p53 high copy number (HCN), CCND1 HCN, 10p11HCN and partly MGMT promoter methylation were linked to prolonged OS. Cox proportional regression models for survival revealed that TP53 HCN was associated with a prolonged OS of all patients and the chemoradiotherapy group. CCND1 HCN, 10p11HCN and partly MGMT promoter methylation significantly extended OS in the group with chemoradiotherapy. The effect of these gene changes on OS was reduced in the group of all patients. Author made the efforts to gain clinical and genetic factors, which easily usable in the clinical practice. Contrary literature data, there were confirmed TP53, CCND1 as predictive and prognostic factors.

Gliomas are the most common, malignant, and highly infiltrating diffuse brain tumors and their prognosis still remains unfavorable despite aggressive therapy. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are probably crucial in gliomagenesis, but are also associated with better prognosis and have predictive potential for therapy. Here, we investigated association of IDH mutations with other molecular genetic markers and clinical characteristics of glioma patient cohort from the Czech Republic. A group of 153 glioma patients was retrospectively analyzed for the presence of three most frequent IDH mutations - IDH1 R132H, IDH1 R132C, and IDH2 R172K by competitive amplification of differentially melting amplicons (CADMA) PCR. Copy number of EGFR, p53, RB1, MDM2, CDKN2A genes and 1p, 19q and 10p chromosomal regions were investigated by fluorescent in situ hybridization. MGMT promoter methylation status was determined by bisulfite conversion followed by methylation-specific PCR. Results of molecular genetic analysis were correlated with clinical characteristics. Thirty six IDH1 R132H, one IDH1 R132C and no IDH2 R172K mutations were found. In the group of 52 gliomas, the CADMA PCR results were confirmed by PCR followed by restriction analysis. In the group of all gliomas, the MGMT methylation was found in 50% (76/151), EGFR amplification in 25% (32/127), CDKN2A loss in 30% (38/127), 1p loss in 30% (39/129), RB1 loss in 25% (32/128), p53 loss in 16% (19/121), 10p loss in 20% (24/122), 19q loss in 17% (22/127), 1p/19q co-loss in 9% (12/127), MDM2 gain in 24% (30/125). IDH1 mutations were positively associated with MGMT methylation (OR = 3.38, 95%CI = 1.532, 7.96, P = 0.003), 1p/19q co-loss (OR = 7.2, 95%CI = 2.092, 28.839, P = 0.002) and negatively associated with EGFR amplification (OR = 0.14, 95%CI = 0.021, 0.497, P = 0.009) and 10p loss (OR = 0.09, 95%CI = 0.005, 0.463, P = 0.022). Overall survival of IDH1 mutated glioblastomas (both primary and secondary) were almost 3 times longer than these without IDH1 mutations (P = 0.035), unlike MGMT methylated vs. unmethylated glioblastomas (P = 0.166). In conclusion, we retrospectively detected IDH mutations in a group of the Czech glioma patients by CADMA PCR method and investigated association between IDH status and other analyzed molecular genetics and clinical characteristics. Our findings point out that in glioblastomas, IDH1 mutations are probably stronger prognostic marker than MGMT methylation. We suggested IDH1/2 mutations analysis as a prime procedure in glioma sample genetic examination. This work was supported by grants from the Ministry of Health (NT 13581) and Technology Agency (TE02000058) of Czech Republic. The infrastructural part of this project (Institute of Molecular and Translational Medicine) was supported by the National Program of Sustainability (NPU LO1304).

Glioblastoma multiforme (GBM) grade IV (WHO) represent 50–60% of diagnosed adult primary CNS tumours. Patients have unfavorable prognosis, with a median survival of about 12 months. GBM is a highly invasive, amenable to a complete resection what is the main cause of recurrence or disease progression. The treatment is quite limited and only few molecular or genetic predictive / prognostic markers were described. The aim of this study is to verify the importance of the selected molecular and genetic markers for the disease prediction or as the possible therapeutic targets. Formalin-fixed, paraffin-embedded (FFPE) samples from 149 glioma patients treated in the University Hospital Olomouc, Czech Republic, have been collected prospectively and systematically since 2006. All patients were discussed in a comprehensive oncology team and underwent early post-surgical MRI (up to 72 hours) to determine resection radicality. Whenever possible, patients were recommended to the Stupp therapy protocol. FISH analysis was performed on FFPE tissues for detection of genes and chromosomal region status (1p, 9p, 19q, EGFR, MDM2, PTEN, TP53, CCND1, C-MYC and RB1, including enumeration of chromosomes on which the genes are localized (chromosome 7, 10, 11, 12, 13, 17 and 22). Using real-time methylation specific PCR, MGMT methylation status was also described. Association of clinical parameters and molecular markers were analyzed by Pearson chi-square test and Fisher test, to analyze differences in survival groups was used log-rank test. It was analyzed overall survival (OS) and graphically depicted by the Kaplan-Meier curves. Effect of different markers was analyzed by the Cox model, even with adjustment, the main clinical factors, e.g. age at diagnosis, performance status or type of treatment. We found statistically significant correlation. For example, MGMT methylation status and the copy numbers of the CCND1, TP53 and RB1 genes seems to be important for predicting response in patients undergoing radiotherapy or chemo-radiotherapy. On other hand, high amplification of EGFR gene (despite is often found in GBM) does not seem to have any prognostic or predictive impact, although increased copy of chromosome 7, where EGFR is localized, seems to be marker of lower hazard ratio. Some other genes are contemporary analyzed and the complete results will be presented. Despite intensive research, radiotherapy and Temodal remains practically as the only treatment for GBM gr.IV. Our study is trying to find other markers for better prediction of tumor prognosis or for biological treatment of the individual patient. The project was supported by Support of Sustainability of the Institute of Molecular and Translational Medicine NPU LO1304; Center of Competence for Molecular Diagnostics and Personalized Medicine TE02000058 and IGA MZČR NT13581 project.

Eligibility to anti‐HER2 therapy for breast tumors strictly depends on demonstrating HER2 overexpression (by immunohistochemistry) or HER2 gene amplification by in situ hybridization (ISH), usually defined by the ratio of HER2 gene to chromosome 17 centromere (CEP17) copies. However, the CEP17 copy number increase (CNI) has been proven responsible for misleading HER2 FISH results and recent small cohort studies suggest that chromosome 17 polysomy is actually very rare. Here we investigated by FISH the frequency of true chromosome 17 polysomy in a consecutive cohort of 5,477 invasive breast cancer patients. We evaluated and selected the LSI 17p11.2 probe for chromosome 17 enumeration on a training cohort of 67 breast cancer samples (CEP17 ≥ 2.5). LSI 17p11.2 was used in the 297/5,477 patients from the validation cohort displaying CEP17 CNI (CEP17 ≥ 3.0). Using HER2/17p11.2 scoring criteria, 37.3%/1.5% patients initially classified as equivocal/non‐amplified were reclassified as amplified. For a more accurate assessment of chromosome 17 and ploidy in the samples, we tested six markers located on chromosome 17 and centromeric regions of chromosome 8 (CEP8) and 11 (CEP11) in 67 patients with CEP17 and LSI 17p11.2 CNI. True polysomy (hyperdiploidy) according to these markers was found in 0.48% of cases (24/5,020). CEP8 and CEP11 CNI (≥3.0) was more frequent in the hyperdiploid than CEP17 non‐polysomic group (55.6% vs. 6.1% and 25% vs. 2.3%, respectively). Our results suggest that chromosome 17 polysomy is a rare event found in <1% breast cancer cases and that polysomy of other chromosomes frequently occurs with chromosome 17 polysomy.