Germline MUTYH mutations and high-grade gliomas: Novel evidence for a potential association.

There is growing body of evidence supporting the role of germline mutations in the pathogenesis of pediatric central nervous system (CNS) tumors, and the widespread use of next-generation sequencing (NGS) panels facilitates their detection. Variants of the MUTYH gene are increasingly recognized as suspected germline background of various extraintestinal malignancies, besides their well-characterized role in the polyposis syndrome associated with biallelic mutations. Using a multigene NGS panel (Illumina TruSight Oncology 500), we detected one H3 G34V- and one H3 K27M-mutant pediatric high-grade diffuse glioma, in association with c.1178G>A (p.G393D) and c.916C>T (p.R306C) MUTYH variants, respectively. Both MUTYH mutations were germline, heterozygous and inherited, according to the subsequent genetic testing of the patients and their first-degree relatives. In the H3 K27M-mutant glioma, amplifications affecting the 4q12 region were also detected, in association with KDR-PDGFRA, KIT-PDGFRA, and KDR-CHIC2 fusions, previously unreported in this entity. Among 47 other CNS tumors of various histological types tested with the same NGS panel in our institution, only one adult glioblastoma harbored MUTYH mutation. Together with a single previous report, our data raises the possibility of an association between germline MUTYH mutations and CNS malignancies, particularly in pediatric histone H3-mutant gliomas.

Prostate-specific membrane antigen expression in the vasculature of primary lung carcinomas associates with faster metastatic dissemination to the brain.

Glioblastomas and brain metastases (BM) of solid tumours are the most common central nervous system neoplasms associated with very unfavourable prognosis. In this study, we report the association of prostate-specific membrane antigen (PSMA) with various clinical parameters in a large cohort of primary and secondary brain tumours. A tissue microarray containing 371 cases of ascending grades of gliomas pertaining to astrocytic origin and samples of 52 cases of primary lung carcinomas with matching BM with follow-up time accounting to 10.4 years was evaluated for PSMA expression using immunohistochemistry. In addition, PSMA expression was studied in BM arising from melanomas and breast carcinomas. Neovascular expression of PSMA was evident alongside with high expression in the proliferating microvasculature of glioblastomas when compared to the tumour cell expression. This result correlated with the results obtained from the in silico (cancer genome databases) analyses. In gliomas, only the vascular expression of PSMA associated with poor overall survival but not the tumour cell expression. In the matched primary lung cancers and their BM (n = 52), vascular PSMA expression in primary tumours associated with significantly accelerated metastatic dissemination to the brain with a tendency towards poor overall survival. Taken together, we report that the vascular expression of PSMA in the primary and secondary brain tumours globally associates with the malignant progression and poor outcome of the patients.

Dissection of subclonal evolution by temporal mutation profiling in chronic lymphocytic leukemia patients treated with ibrutinib.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is inducing durable responses in chronic lymphocytic leukemia (CLL) patients with refractory/relapsed disease or with TP53 defect, with BTK and phospholipase C gamma 2 (PLCG2) mutations representing the predominant mechanisms conferring secondary ibrutinib resistance. To understand the landscape of genomic changes and the dynamics of subclonal architecture associated with ibrutinib treatment, an ultra-deep next-generation sequencing analysis of 30 recurrently mutated genes was performed on sequential samples of 20 patients, collected before and during single-agent ibrutinib treatment. Mutations in the SF3B1, MGAand BIRC3 genes were enriched during ibrutinib treatment, while aberrations in the BTK, PLCG2, RIPK1, NFKBIE and XPO1 genes were exclusively detected in posttreatment samples. Besides the canonical mutations, four novel BTK mutations and three previously unreported PLCG2 variants were identified. BTK and PLCG2 mutations were backtracked in five patients using digital droplet PCR and were detectable on average 10.5 months before clinical relapse. With a median follow-up time of 36.5 months, 7/9 patients harboring BTK mutations showed disease progression based on clinical and/or laboratory features. In conclusion, subclonal heterogeneity, dynamic clonal selection and various patterns of clonal variegation were identified with novel resistance-associated BTK mutations in individual patients treated with ibrutinib.

Decreased Expression of ZNF554 in Gliomas is Associated with the Activation of Tumor Pathways and Shorter Patient Survival.

Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein subfamily, is predominantly expressed in the brain and placenta in humans. Recently, we unveiled that ZNF554 regulates trophoblast invasion during placentation and its decreased expression leads to the early pathogenesis of preeclampsia. Since ZNF proteins are immensely implicated in the development of several tumors including malignant tumors of the brain, here we explored the pathological role of ZNF554 in gliomas. We examined the expression of ZNF554 at mRNA and protein levels in normal brain and gliomas, and then we searched for genome-wide transcriptomic changes in U87 glioblastoma cells transiently overexpressing ZNF554. Immunohistochemistry of brain tissues in our cohort (n = 62) and analysis of large TCGA RNA-Seq data (n = 687) of control, oligodendroglioma, and astrocytoma tissues both revealed decreased expression of ZNF554 towards higher glioma grades. Furthermore, low ZNF554 expression was associated with shorter survival of grade III and IV astrocytoma patients. Overexpression of ZNF554 in U87 cells resulted in differential expression, mostly downregulation of 899 genes. The "PI3K-Akt signaling pathway", known to be activated during glioma development, was the most impacted among 116 dysregulated pathways. Most affected pathways were cancer-related and/or immune-related. Congruently, cell proliferation was decreased and cell cycle was arrested in ZNF554-transfected glioma cells. These data collectively suggest that ZNF554 is a potential tumor suppressor and its decreased expression may lead to the loss of oncogene suppression, activation of tumor pathways, and shorter survival of patients with malignant glioma.

The genomic imprint of cancer therapies helps timing the formation of metastases.

A retrospective determination of the time of metastasis formation is essential for a better understanding of the evolution of oligometastatic cancer. This study was based on the hypothesis that genomic alterations induced by cancer therapies could be used to determine the temporal order of the treatment and the formation of metastases. We analysed the whole genome sequence of a primary tumour sample and three metastatic sites derived from autopsy samples from a young never-smoker lung adenocarcinoma patient with an activating EGFR mutation. Mutation detection methods were refined to accurately detect and distinguish clonal and subclonal mutations. In comparison to a panel of samples from untreated smoker or never-smoker patients, we showed that the mutagenic effect of cisplatin treatment could be specifically detected from the base substitution mutations. Metastases that arose before or after chemotherapeutic treatment could be distinguished based on the allele frequency of cisplatin-induced dinucleotide mutations. In addition, genomic rearrangements and late amplification of the EGFR gene likely induced by afatinib treatment following the acquisition of a T790M gefitinib resistance mutation provided further evidence to tie the time of metastasis formation to treatment history. The established analysis pipeline for the detection of treatment-derived mutations allows the drawing of tumour evolutionary paths based on genomic data, showing that metastases may be seeded well before they become detectable by clinical imaging.

Differentially Expressed miRNAs Influence Metabolic Processes in Pituitary Oncocytoma.

Spindle cell oncocytomas (SCO) of the pituitary are rare tumors accounting for 0.1-0.4% of all sellar tumors. Due to their rarity, little information is available regarding their pathogenesis. Our aim was to investigate miRNA expression profile of pituitary oncocytomas. Total RNA was extracted from 9 formalin-fixed paraffin embedded pituitary samples (4 primary, 3 recurrent oncocytomas and 2 normal tissues). Next-generation sequencing was performed for miRNA profiling. Transcriptome data of additional 6 samples' were obtained from NBCI GEO database for gene expression reanalysis and tissue-specific target prediction. Bioinformatical analysis, in vitro miRNA mimics transfection, luciferase reporter system and AlamarBlue assay were applied to characterize miRNA's function. 54 differentially expressed miRNAs and 485 genes in pituitary SCO vs. normal tissue and 8 miRNAs in recurrent vs. primary SCO were determined. Global miRNA downregulation and decreased level of DROSHA were detected in SCO samples vs. normal tissue. Transcriptome analysis revealed cell cycle alterations while miRNAs influenced mainly metabolic processes (tricarboxylic acid cycle-TCA, carbohydrate, lipid metabolism). Through miRNA-target interaction network the overexpressed Aconitase 2 potentially targeted by two downregulated miRNAs (miR-744-5p, miR-127-3p) was revealed. ACO2 and miR-744-5p interaction was validated by luciferase assay. MiR-127-3p and miR-744-5p significantly decreased cell proliferation in vitro. Our study firstly reported miRNA profile of pituitary oncocytoma. Our results suggest that tumor suppressor miRNAs may have an essential role in the pathogenesis of pituitary oncocytoma. Earlier reports showed downregulated TCA cycle in SCO which is extended by our results adding the role of miR-744-5p targeting ACO2.

Influence of mutagenic versus non-mutagenic pre-operative chemotherapy on the immune infiltration of residual breast cancer.

Background: Chemotherapeutic agents are often mutagenic. Induction of mutation associated neo-epitopes is one of the mechanisms by which chemotherapy is thought to increase the number of tumor-infiltrating lymphocytes. It is not known, however, whether treatment with various chemotherapeutic agents with different mutagenic capacity induce a significantly different number of stromal tumor-infiltrating lymphocytes (StrTIL) in residual cancer.Methods: One hundred and twenty breast carcinoma cases with residual disease that were treated with one of three types of pre-operative chemotherapy regimens were selected for the study. The percentage of StrTIL was evaluated in pretreatment core biopsies (pre-StrTIL) and post-treatment surgical tumor samples (post-StrTIL). TIL changes (ΔStrTIL) were calculated from the difference between post-StrTIL and pre-StrTIL.Results: When analyzing the pre-StrTIL and post-StrTIL among the three treatment groups, we detected significant StrTIL increase independently of the treatment applied. Based on distant metastases-free survival analysis, both post-StrTIL and ΔStrTIL was found to be independent prognostic factor in HR negative cases. Conclusions: Significant increase of StrTIL in the residual disease was observed in patients treated with the highly (platinum), moderately (cyclophosphamide) and marginally mutagenic chemotherapeutic agents (taxane, anthracycline). Increase in StrTIL in residual cancer compared to pretreatment tumor tissue is associated with improved distant metastasis-free survival in cases with HR negative breast carcinoma.

Tumor necrosis correlates with PD-L1 and PD-1 expression in lung adenocarcinoma.

Background: Predictive biomarkers for immunotherapy in lung cancer are intensively investigated; however, correlations between PD-L1/PD-1 expressions and clinical features or histopathological tumor characteristics determined on hematoxylin and eosin stained sections have not extensively been studied. Material and methods: We determined PD-L1 expression of tumor cells (TC) and immune cells (IC), and PD-1 expression of IC by immunohistochemistry in 268 lung adenocarcinoma (LADC) patients, and correlated the data with smoking, COPD, tumor grade, necrosis, lepidic growth pattern, vascular invasion, density of stromal IC, and EGFR/KRAS status of the tumors. Results: There was a positive correlation between PD-L1 expression of TC and IC, as well as PD-L1 and PD-1 expression of IC. Tumor necrosis was associated with higher PD-L1 expression of TC and PD-1 expression of IC. A negative correlation was observed between lepidic growth pattern and PD-L1 expression of TC and PD-L1/PD-1 expression of IC. EGFR mutation seemed to negatively correlate with PD-1 expression of IC, but this tendency could not be verified when applying corrections for multiple comparisons. No significant effect of the KRAS mutation on any of the studied variables could be established. Conclusion: Here we first demonstrate that the presence of necrosis correlates with higher PD-L1 expression of TC and PD-1 expression of IC in LADC. Further studies are required to determine the predictive value of this observation in LADC patients receiving immunotherapy.

EZH2 is upregulated in the proliferation centers of CLL/SLL lymph nodes.

Lymph node involvement of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) is characterised by the diffuse infiltration of small neoplastic lymphocytes, which is accompanied by the presence of proliferation centres (PCs) comprising prolymphocytes and paraimmunoblasts. There is increasing evidence of accumulation of various molecular alterations in the tumour cells of PCs, which may explain why extended PCs are related to a less favourable prognosis. To further characterize PCs, we compared the expression level of EZH2 protein, the overexpression of which has recently been recognized as poor prognostic factor in CLL/SLL, in the PCs and the intervening small cell areas in lymph nodes of 15 patients with CLL/SLL. We also investigated the mutational profile of EZH2 and the expression of its upstream regulators c-Myc, E2F1, pRB and miR-26a. Our results showed a significantly increased expression of EZH2 in the PCs. No EZH2 mutations were detected, however, overexpression of c-Myc, E2F1 and pRb proteins as well as reduced expression of the tumor suppressor miR-26a were demonstrated in the PCs. In summary our findings indicate that EZH2 pathway is significantly upregulated in the PCs of CLL/SLL lymph nodes, providing further evidence for the distinguished biological features of the PCs.

[Aspects of nutrition therapy of an infant with central nervous system tumour]. / A táplálásterápia alkalmazásának szempontjai egy központi idegrendszeri daganatos csecsemõ ellátása során.

The atypical teratoid/rhabdoid tumour (ATRT) is a rare type of central nervous system tumour appearing usually under 2 years of age. The survival of patients is insufficient despite the combined treatment (neurosurgical removal, intensive chemo- and radiotherapy). ATRT recurs one year after completion of treatment in 60% of cases. Maintaining appropriate nutritional status during treatment is of great importance in this young age group. Nutritional treatment of patients with ATRT is especially difficult due to young age and possible neurological sequelae. A successful case of a three-month-old female infant is presented, with special emphasis on the importance of feeding therapy.

Catalog of genetic progression of human cancers: non-Hodgkin lymphoma.

The recent application of next-generation sequencing technologies lead to significant improvements in our understanding of genetic underpinnings of non-Hodgkin lymphomas with identification of an unexpectedly high number of novel mutation targets across the different B-cell lymphoma entities. These recently discovered molecular lesions are expected to have a major impact on development of novel biomarkers and targeted therapies as well as patient stratification based on the underlying genetic profile. This review will cover the major discoveries in B-cell lymphomas using next-generation sequencing technologies over the last few years, highlighting alterations associated with relapse and progression of these diseases.

Low CD23 expression correlates with high CD38 expression and the presence of trisomy 12 in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by a neoplastic B-cell population coexpressing CD5 and CD23; however, the expression of CD23 is variable. In human, two isotypes of CD23 have been identified and related to different functions. The aim of our study was to investigate the relative expression of the two CD23 isotypes in CLL and find possible correlation with other prognostic factors. The expression of CD23 isotypes was analyzed in 54 cases of CLL by polymerase chain reaction (PCR) and quantitative real-time PCR. The immunophenotype of CLL cells was characterized by flow cytometry. We demonstrated a higher CD23a than CD23b expression of CLL cells. Our results also revealed two subsets of CLL cases with a distinct CD23 isotype expression pattern. Thirty-two percent of the cases (group CLL1) showed both low mRNA level of CD23 isotypes and high protein levels of CD20 and CD38 in contrast to group CLL2 with high CD23 mRNA levels. By correlating these results to the presence of prognostic factors determined by fluorescence in situ hybridization, we found that the majority of the cases of group CLL1 (14/17) carried trisomy 12. In summary, our results confirm a high CD23a/CD23b ratio of the CLL cells and demonstrate that in a subset of CLL cases, low CD23 expression together with high CD20 and CD38 expressions may serve as a surrogate for trisomy 12. Copyright © 2015 John Wiley & Sons, Ltd.

[The genetic landscape of chronic lymphocytic leukemia]. / A krónikus limfocitás leukémia genetikai háttere az újgenerációs szekvenálás korszakában.

Chronic lymphocytic leukemia (CLL) is the most frequent mature B-cell non-Hodgkin's lymphoma in the Western countries. The recent next-generation sequencing (NGS) studies lead to an exponential increase in our knowledge of the pathogenesis and progression of CLL. Whole genome and exome sequencing studies revealed a remarkable inter- and intra-patient genetic heterogeneity with a significant therapy-induced clonal evolution in the majority of the patients. Driver mutations were identified in components of various signalling pathways and cellular processes with notable prognostic and therapeutic relevance. Interestingly, these studies revealed only a few genes mutated in at least 15-20% of the patients with a larger number of genes mutated in a smaller proportion of patients. This improved understanding of the genomic landscape of CLL has opened new avenues for a more precise patient stratification and rational application of novel, more effective targeted therapies.

[The role of the pathologist in the diagnosis and therapy planning of central nervous system tumors. Prognostic and predictive markers]. / A patológus szerepe a központi idegrendszer tumorainak diagnosztikájában, a terápiatervezésben. Prognosztikus és prediktív markerek vizsgálata.

Despite advances in imaging methods, the standard of diagnosis and treatment of the tumours of the nervous system remains the histological report issued by a neuropathologist. For reliable, definitive diagnosis, close collaboration with other medical professions is essential, correlation of histological findings with clinical and imaging results is necessary. Neuropathology became a subspecialty because of the specific knowledge and experience it requires. In more complex cases consultation with neuropathologists is important to ensure adequate diagnosis and subsequent treatment. In both establishing the diagnosis and treatment planning, the molecular testing of brain tumors becomes more and more important. These tests are reliably available only in larger centers. Out of the molecular markers, in current practice the investigation of codeletion at 1p/19q, IDH mutations, ß-katenin nuclear positivity and MGMT methylation gained acceptance. Besides these tests already in practice, a vast array of potential diagnostic and prognostic markers are being investigated, which in the future may assist in delivering better and more individualized therapy.

[Childhood brain tumors: diagnosis and therapy - comprehensive genomic profiling]. / Gyermekkori agydaganatok: diagnózis és terápia ­ komprehenzív genomikai profilozás.

With the advancement of molecular oncology, numerous new opportunities are available for the effective and efficient treatment of patients diagnosed with childhood brain tumors. This includes gene panel analysis aiding personalized treatment used in clinical trials, and the application of targeted therapy independent of tissue type (tumor agnostic therapy). Most personalized therapies inhibit certain kinases. In our review, we present the modern pathological diagnosis of childhood brain tumors, as well as the complex intracellular regulation of signal transduction pathways important from the point of view of clinical practice, and we describe their further targets defined on the basis of pharmacological characteristics of the pathway, based on international and our own results. Despite common mutations affecting kinases, personalized therapy is not available in many types of tumors. Through the example of childhood brain tumors, we demonstrate the expected future therapeutic significance of tyrosine kinases.

Novel, clinically relevant genomic patterns identified by comprehensive genomic profiling in ATRX-deficient IDH-wildtype adult high-grade gliomas.

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the TERT gene promoter (pTERT) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative mechanisms of telomere lengthening. In this study, we performed the first complex genomic analysis specifically concentrating on the latter subgroup. Comprehensive genomic profiling of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas revealed that ATRX and pTERT mutations are mutually exclusive. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. RAS-MAPK pathway alterations, including NF1 mutations, were more characteristic in the ATRX-deficient group. Variants of genes related to homologous recombination repair showed different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these contained a novel fusion involving the NTRK2 and LRRFIP2 genes, while the other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Genetic characteristics of ATRX-deficient IDH-wildtype adult high-grade gliomas suggest that these tumors are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA repair inhibitors.

PD-1 and PD-L1 expression in rare lung tumors.

Background: Our knowledge is still limited about the characteristics and treatment of rare lung tumors. The aim of our study was to determine programmed cell death ligand-1 (PD-L1) and programmed cell death-1 (PD-1) expression in rare pulmonary tumors to assess the potential role of immunotherapy. Methods: 66 pathologically confirmed rare lung tumors including 26 mucoepidermoid carcinomas (MECs), 27 adenoid cystic carcinomas (ACCs), and 13 tracheobronchial papillomas (TBPs) were collected retrospectively. Immunohistochemical (IHC) staining was performed on formalin fixed paraffin embedded (FFPE) tumor tissues, and PD-L1 expression on tumor cells (TCs) and immune cells (ICs), and PD-1 expression on ICs were determined. The cut off value for positive immunostaining was set at 1% for all markers. Results: PD-L1 expression on TCs was observed in two cases of MEC (7.7%), one case of ACC (3.7%), and was absent in TBP samples. PD-L1 expression on ICs could be demonstrated in nine cases of MEC (34.6%), four cases of ACC (14.8%), and was absent in TBPs. All PD-L1 TC positive tumors were also PD-L1 IC positive. Higher expression level than 5% of PD-L1 TC and/or IC was observed only in one ACC and in two MEC patients. Among them, strong PD-L1 immunopositivity of >50% on TCs and of >10% on ICs could be demonstrated in one MEC sample. PD-L1 expression of ≥1% on ICs was significantly more common in MEC, than in TBP (p < 0.001). In MEC ≥1% PD-L1 TC or IC expressions were significantly more common in patients aged 55 or older, than in younger patients (p = 0.046, and p = 0.01, respectively). PD-1 expression on ICs was found in five cases of MEC (19.2%), four cases of ACC (14.8%), and in two cases of TBP (15.4%). Only one MEC case showed a higher than 5% expression level of PD-1 on ICs. Conclusion: This retrospective study comprehensively demonstrated the rare expression of PD-L1 and PD-1 in pulmonary MEC, ACC, and TBP. However, we found very strong PD-L1 immunopositivity on both TCs and ICs in one MEC sample, which warrants further investigations in a larger cohort.