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The detection of mutations of isocitrate dehydrogenase 1 and 2 (IDH 1/2) as tumor driver genes in chondromas and chondrosarcomas more than ten years ago was a first major step for better understanding the molecular carcinogenenesis of these rare mesenchymal tumors. Within the TCA cycle, wild-typ IDH1/2 catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). IDH mutations catalyze the production of a non-physiological metabolite, D-2hydroxyglutarate (D-2HG) from α-KG. D-2HG can inhibit the class of α-KG-dependent enzymes by binding competitively to its receptor. Important enzyme families, such as the Ten-Eleven Translocation (TET) family of 5-methylcytosine hydroxylases and the Jumonji family of histone lysine demethylases are α-KG dependent. Many of the TET and Jumonji family-dependent enzymes regulate epigenetic factors, such as DNA methylation, histone modification, and nucleosome remodeling, underscoring the central role of the epigenome in cancer development. When D-2HG acts with these enzymes instead α-KG their functions will be in disarray with heavily hypermethylated DNA and dysregulations in histone metylation. NcRNAs have increasingly been described as a cornerstone of cancer development. Therefore this review describes exemplarily the oncogenic functions of miRNAs in chondrosarcoma in more detail. Particularly in chondrosarcomas additional carcinogenic features are aquired by interactions of ncRNAs with α-KG-dependent epigenetic regulators. Distinct ncRNAs, miRNAs and lncRNAs alike, are involved in deregulating important cellular signalling pathways and thus contributing further to malignant transformation and development of malignant cellular traits in these rare mesenchymal tumors. This review specially empasizes the complex interactions between the world of ncRNAs and genetics and epigenetics.

Purpose In a post hoc analysis of the MAGIC trial, patients with curatively resected gastric cancer (GC) and mismatch repair (MMR) deficiency (MMRd) had better median overall survival (OS) when treated with surgery alone but worse median OS when treated with additional chemotherapy. Further data are required to corroborate these findings. Methods Between April 2013 and December 2018, 458 patients with curatively resected GC, including cancers of the esophagogastric junction Siewert type II and III, were identified in the German centers of the staR consortium. Tumor sections were assessed for expression of MLH1, MSH2, MSH6 and PMS2 by immunohistochemistry. The association between MMR status and survival was assessed. Similar studies published up to January 2021 were then identified in a MEDLINE search for a meta-analysis. Results MMR-status and survival data were available for 223 patients (median age 66 years, 62.8% male), 23 patients were MMRd (10.3%). After matching for baseline clinical characteristics, median OS was not reached in any subgroup. Compared to perioperative chemotherapy, patients receiving surgery alone with MMRd and MMRp had a HR of 0.67 (95% CI 0.13–3.37, P = 0.63) and 1.44 (95% CI 0.66–3.13, P = 0.36), respectively. The meta-analysis included pooled data from 385 patients. Compared to perioperative chemotherapy, patients receiving surgery alone with MMRd had an improved OS with a HR of 0.36 (95% CI 0.14–0.91, P = 0.03), whereas those with MMRp had a HR of 1.18 (95% CI 0.89–1.58, P = 0.26). Conclusion Our data support a positive prognostic effect for MMRd in GC patients treated with surgery only and a differentially negative prognostic effect in patients treated with perioperative chemotherapy. MMR status determined by preoperative biopsies may be used as a predictive biomarker to select patients for perioperative chemotherapy in curatively resectable GC.

Brain metastases frequently occur during the course of disease in patients suffering from lung cancer. Occasionally, neurological symptoms caused by brain metastases (BM) might represent the first sign of systemic tumor disease (so called precocious metastases), leading to the detection of the primary lung tumor. The biological basis of precocious BM is largely unknown, and treatment options are not well established for this subgroup of patients. Therefore, we retrospectively analyzed 33 patients (24 non-small cell lung cancer (NSCLC)), 9 small cell lung cancer (SCLC)) presenting with precocious BM focusing on molecular alterations potentially relevant for the tumor's biology and treatment. We found five FGFR1 amplifications (4 adenocarcinoma, 1 SCLC) among 31 analyzed patients (16.1%), eight MET amplifications among 30 analyzed tumors (7 NSCLC, 1 SCLC; 26.7%), three EGFR mutations within 33 patients (all adenocarcinomas, 9.1%), and five KRAS mutations among 32 patients (all adenocarcinomas; 15.6%). No ALK, ROS1 or RET gene rearrangements were detected. Our findings suggest that patients with precocious BM of lung cancer harbor EGFR mutations, MET amplifications or FGFR1 amplifications as potential targeted treatment options.

Gastric cancer (GC) is one of the most common and lethal gastrointestinal malignancies worldwide. Many studies have shown that development of GC and other malignancies is mainly driven by alterations of cellular signaling pathways. MicroRNAs (miRNAs) are small noncoding molecules that function as tumor-suppressors or oncogenes, playing an essential role in a variety of fundamental biological processes. In order to understand the functional relevance of miRNA dysregulation, studies analyzing their target genes are of major importance. Here, we chose to analyze two miRNAs, miR-20b and miR-451a, shown to be deregulated in many different malignancies, including GC. Deregulated expression of miR-20b and miR-451a was determined in GC cell lines and the INS-GAS mouse model. Using Western Blot and luciferase reporter assay we determined that miR-20b directly regulates expression of PTEN and TXNIP, and miR-451a: CAV1 and TSC1. Loss-of-function experiments revealed that down-regulation of miR-20b and up-regulation of miR-451a expression exhibits an anti-tumor effect in vitro (miR-20b: reduced viability, colony formation, increased apoptosis rate, and miR-451a: reduced colony forming ability). To summarize, the present study identified that expression of miR-20b and miR-451a are deregulated in vitro and in vivo and have a tumor suppressive role in GC through regulation of the PI3K/AKT/mTOR signaling pathway.

External quality assessment (EQA) is an essential part of performance monitoring for molecular laboratories. At the moment, a national law regulates participation in EQA schemes for clinical biology and pathology in Belgium. This study aimed (1) to get insights on how laboratories organize their EQA participation, (2) to poll satisfaction with the current situation (selection of EQA programs in advance by a governmental body), (3) to provide guidance for choosing the most relevant EQA provider and (4) to propose a new model for national performance monitoring. A survey was sent to Belgian laboratories performing molecular tests in the field of microbiology, hematology and pathology with (1) general questions on how they select an EQA provider and (2) their satisfaction of each provider. In total, 25 molecular laboratories [microbiology (N = 13), hematology (N = 8) and pathology (N = 4)] from 14 different hospitals completed the survey regarding their EQA organization. All three laboratory groups indicated to prefer EQA schemes using real patient materials as well as those with varying targets and concentrations. For molecular microbiology and hematology, schemes with a syndromic approach are sought. Since annual participation in EQA becomes burdensome in most laboratories, this paper also offers a risk-based strategy for determining the participation frequency. Based on the needs of Belgian laboratories, three proposals were made: (1) for the proper selection of an EQA scheme, (2) for determining the minimal participation frequency and (3) for the national organization of EQA schemes.

Background: Sorafenib has shown efficacy in patients with imatinib-, sunitinib-, and regorafenib-resistant gastrointestinal stromal tumors (GISTs). No biomarker is currently available for predicting response to sorafenib in patients with GIST. Methods: We herein report 3 patients with imatinib-, sunitinib-, and regorafenib-resistant metastasized GISTs, who were treated with sorafenib. Besides receptor tyrosine kinase KIT and platelet-derived growth factor receptor α, also BRAF was tested for mutations. Results: Sorafenib therapy induced a long-term disease control in 2 out of 3 patients over a period of 49 and 19 months, respectively. Sorafenib-responsive GISTs were BRAF wild-type, whereas the sorafenib-resistant GIST carried a BRAF V600E mutation. Conclusion: We confirm sorafenib as an effective therapeutic option in patients with imatinib-, sunitinib-, and regorafenib-resistant GISTs. Larger studies are required to corroborate whether BRAF mutation may predict sorafenib resistance in GISTs.

// Ugne Gyvyte 1, * , Simonas Juzenas 1, * , Violeta Salteniene 1 , Juozas Kupcinskas 1, 2 , Lina Poskiene 3 , Laimutis Kucinskas 1 , Sonata Jarmalaite 4, 5 , Kristina Stuopelyte 4, 5 , Ruta Steponaitiene 1 , Georg Hemmrich-Stanisak 6 , Matthias Hubenthal 6 , Alexander Link 7 , Sabine Franke 8 , Andre Franke 6 , Dalia Pangonyte 3 , Vaiva Lesauskaite 9 , Limas Kupcinskas 1, 2, # , Jurgita Skieceviciene 1, # 1 Institute for Digestive Research, Academy of Medicine, Lithuanian University of Health Sciences, Kaunas, Lithuania 2 Department of Gastroenterology, Academy of Medicine, Lithuanian University of Health Sciences, Kaunas, Lithuania 3 Department of Pathological Anatomy, Academy of Medicine, Lithuanian University of Health Sciences, Kaunas, Lithuania 4 Division of Human Genome Research Centre, Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania 5 National Cancer Institute, Vilnius, Lithuania 6 Institute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, Kiel, Germany 7 Department of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke University Hospital Magdeburg, Magdeburg, Germany 8 Institute of Pathology, Otto-von-Guericke University, Magdeburg, Germany 9 Institute of Cardiology, Academy of Medicine, Lithuanian University of Health Sciences, Kaunas, Lithuania * These authors contributed equally to this work # These authors jointly supervised this work Correspondence to: Jurgita Skieceviciene, email: jurgita.skieceviciene@lsmuni.lt Keywords: GIST, microRNA, microRNA profiling, small RNA-seq Received: May 24, 2016 Accepted: March 12, 2017 Published: March 29, 2017 ABSTRACT Deregulation of miRNAs has been observed virtually in all major types of cancer, whereas the miRNA signature in GIST is not well characterized yet. In this study the first high-throughput miRNA profiling of 15 paired GIST and adjacent normal tissue samples was performed using small RNA-seq approach and differentially expressed miRNAs as well as isomiRNAs were defined. Highly significantly deregulated miRNAs were selected for validation by Taq-Man low-density array in replication group of 40 paired samples. Validated miRNAs were further subjected to enrichment analysis, which revealed significantly enriched KEGG pathways in the main GIST associated pathways. Further, we used an integrated analysis of miRNA-mRNA correlations for KIT and PDGFRA target genes and found a significant correlation between all of the enriched miRNAs and their target gene KIT . Results of the phenotype analysis showed miR-509-3p to be up-regulated in epithelioid and mixed cell types compared to spindle type, whereas miR-215-5p showed negative correlation with risk grade of GIST. These data reveal a detailed miRNA profile of GIST and highlight new candidates that may be important in the development of malignant disease.

Cathepsin X (CTSX, also called cathepsin Z/P) is a cysteine protease that still plays an unknown role in human cancer. It has been shown to bind cell surface heparin sulphate proteoglycans and integrins, indicating possible functions of CTSX in cellular adhesion, phagocytosis, and immune response. Our previous studies have shown an association between Helicobacter pylori (H. pylori) infection, a strong up-regulation of CTSX, and development of gastric cancer. In this study, yeast two-hybrid analysis revealed that RPLP0, a ribosomal protein P0, interacts with the human CTSX protein in gastric cancer. The CTSX/RPLP0 interaction was confirmed by co-immunoprecipitation assays. In addition, co-localization studies in cancer cell line N87 and gastric cancer tissue samples were performed. Laserscan microscopy revealed a shuttling of RPLP0 (and CTSX) from cytoplasm to the nucleus after CTSX knockdown. Down-regulation of RPLP0 resulted in G1 arrest of gastric cancer cells, whereas knockdown of CTSX led to G1 arrest and apoptosis after 48 h. Knockdown of both proteins caused increased apoptosis. RPLP0 deficiency could suppress cell growth and cell cycle progression by down-regulating CDK2. It was further demonstrated that RPLP0 affected p21 expression, but did not change the expression of Cyclin E. Down-regulation of both proteins at least through CDK2 suggests an anti-apoptotic effect on gastric cancer cells and opens up new possibilities for apoptotic immune modulation and gastric cancer therapy.

The lysosomal cysteine carboxypeptidase cathepsin X (CTSX), localized predominantly in immune cells, has been associated with the development and progression of cancer. To determine its specific role in colorectal carcinoma (CRC), we analyzed CTSX expression in non-malignant mucosa and carcinoma of 177 patients as well as in 111 adenomas and related it with clinicopathological parameters. Further, the role of CTSX in the adhesion and invasion of the colon carcinoma cell lines HT-29 and HCT116 was investigated in an in vitro culture cell system with fibroblasts and monocytes, reflecting the situation at the tumor invasion front. Epithelial CTSX expression significantly increased from normal mucosa to adenoma and carcinoma, with highest expression levels in high grade intraepithelial neoplasia and in early tumor stages. Loss of CTSX occurred with tumor progression, and correlated with advanced local invasion, lymph node and distal metastasis, lymphatic vessel and vein invasion, tumor cell budding and poorer overall survival of patients with CRC. The subcellular distribution of CTSX changed from vesicular paranuclear expression in the tumor center to submembranous expression in cells of the invasion front. Peritumoral macrophages showed highest expression of CTSX. In vitro assays identified CTSX as relevant factor for cell–cell adhesion and tumor cell anchorage to fibroblasts and basal membrane components, whereas inhibition of CTSX caused increased invasiveness of colon carcinoma cells in mono- and co-culture. In conclusion, CTSX is involved in early tumorigenesis and in the stabilization of tumor cell formation in CRC. The results suggest that loss of CTSX may be needed for tumor cell detachment, local invasion and tumor progression. In addition, CTSX in tumor-associated macrophages indicates a role for CTSX in the anti-tumor immune response.

Background:Chemoresistance is a main obstacle to effective esophageal cancer (EC) therapy. We hypothesize that altered expression of microRNAs (miRNAs) play a role in EC cancer progression and resistance to 5-fluorouracil (5-FU) based chemotherapeutic strategies.Methods:Four pairs of esophageal adenocarcinoma (EAC) cell lines and corresponding 5-FU resistant variants were established. The expression levels of miRNAs previously shown to be involved in the general regulation of stem cell pathways were analyzed by qRT-PCR. The effects of selected miRNAs on proliferation, apoptosis, and chemosensitivity were evaluated both in vitro and in vivo. We identified a particular miRNA and analyzed its putative target genes in 14 pairs of human EC tumor specimens with surrounding normal tissue by qRT-PCR as well as Wnt pathway associated genes by immunohistochemistry in another 45 EAC tumor samples.Results:MiR-221 was overexpressed in 5-FU resistant EC cell lines as well as in human EAC tissue. DKK2 was identified as a target gene for miR-221. Knockdown of miR-221 in 5-FU resistant cells resulted in reduced cell proliferation, increased apoptosis, restored chemosensitivity, and led to inactivation of the Wnt/-catenin pathway mediated by alteration in DKK2 expression. Moreover, miR-221 reduction resulted in alteration of EMT-associated genes such as E-cadherin and vimentin as well as significantly slower xenograft tumor growth in nude mice. RT2 profiler analysis identified a substantial dysregulation of 4 Wnt/-catenin signaling and chemoresistance target genes as a result of miR-221 modulation: CDH1, CD44, MYC, and ABCG2.Conclusion:MiR-221 controls 5-FU resistance of EC partly via modulation of Wnt/-catenin-EMT pathways by direct targeting of DKK2 expression. MiR-221 may serve as a prognostic marker and therapeutic target for patients with 5-FU resistant EAC.

Purpose The introduction of new therapeutic strategies has resulted in increased complete remission rates in B-cell chronic lymphocytic leukemia (CLL). Preliminary data have suggested that the absence of minimal residual disease (MRD) is an endpoint of therapy that, if achieved, translates into an improved survival. We developed and validated a novel, combined method to assess MRD in CLL using fluorescence-activating cell sorting (FACS) and interphase fluorescence in situ hybridization (FISH) for the detection of numeric chromosomal aberrations. Materials and Methods Cell sorting was performed on a FACS-Aria based on the CD19+CD5+ co-expression. Interphase FISH analysis was applied to purified cells with commercially available probes (Vysis/Abbott). Spiking experiments of CLL cells harboring a numeric chromosomal abnormality into normal blood with dilutions of 10−3 to 10−6 were performed; FISH detection of the specific chromosomal aberration in CD19+CD5+ purified cells allowed discrimination of CLL cells from normal precursor B-cells. Results Positive results were demonstrated in all dilutions up to 10−5 or even 10−6. This approach for the assessment of MRD in CLL reaches sensitivity at least as high as and even higher than other methods, such as 4-color flow cytometry or quantitative allele-specific polymerase chain reaction. Conclusion It can be used for ≥ 80% of patients with CLL, including all patients with CLL with poor prognosis, as assessed by the presence of the deletion 11q (ataxia telangiectasia-mutated) or the deletion 17p (p53). Furthermore, it allows easy standardization among laboratories that apply FACS because it is based on 2-color labeling only and on FISH assays using commercially available probes.

Several lines of evidences suggest that T cell/histiocyte-rich B-cell lymphoma (T/HRBCL) represents an aggressive variant of the clinically indolent entity nodular lymphocyte predominance Hodgkin’s lymphoma (LPHL). Still, this view has not yet been supported by firm genetic evidence. In this study, we analyzed 17 T/HRBCL cases using comparative genomic hybridization (CGH) combined with microdissection of single CD20+ neoplastic cells and DNA amplification by degenerate oligonucleotide primed-polymerase chain reaction, an approach we previously used in LPHL. Genomic imbalances were detected in all cases (in total, 80 changes). The most common imbalances included gain of Xq, 4q13q28, Xp21p11, and 18q21, and loss of 17p. Of note, a partial gain of 4q, a rare change in lymphoma, is also among the genomic imbalances most frequently encountered in LPHL. On the other hand, the CGH profiles of T/HRBCL and LPHL showed several distinct features, in particular with respect to the number of genomic imbalances (average of 4.7 in T/HRBCL versus 10.8 in LPHL) and their distribution (usually 1 to 5 in T/HRBCL versus 6 to 22 in LPHL). Altogether, our CGH findings of shared as well as distinctive cytogenetic features in both diseases suggest that T/HRBCL constitutes a separate lymphoma entity, possibly originating from the same precursor cell as LPHL.

Single-cell polymerase chain reaction (PCR) has been used as a tool to demonstrate clonality and B-cell origin of Reed-Sternberg (RS) cells in Hodgkin disease (HD). An analogous approach was used to investigate genomic imbalances in a (cyto)genetically poorly characterized subentity: lymphocyte predominance Hodgkin disease (LPHD). Nineteen cases of LPHD were selected for a comparative genomic hybridization (CGH) study. CGH was performed with degenerate oligonucleotide primed–PCR (DOP-PCR)–amplified DNA from 4-5 microdissected CD20+ malignant cells. All analyzed cases revealed a high number of genomic imbalances (average 10.8 per case), involving all chromosomes but the excluded 19, 22, and Y, indicating a high complexity of LPHD. The majority of detected aberrations were recurrent. Gain of 1, 2q, 3, 4q, 5q, 6, 8q, 11q, 12q, and X, and loss of chromosome 17 were identified in 36.8% to 68.4% of the analyzed cases. Some of them have also been found in non-Hodgkin lymphoma (NHL), and possibly represent secondary changes associated with disease progression. Gain of 2q, 4q, 5q, 6, 11q, however, are much more rarely observed in NHL and could be more specifically associated with LPHD. Particularly interesting is a frequent overrepresentation of chromosome arm 6q, a region usually deleted in NHL. Rearrangement of theBCL6 gene (3q27) demonstrated by cytogenetics and fluorescence in situ hybridization in 2 cases in this study suggests its contribution in pathogenesis of LPHD. In conclusion, the data show a consistent occurrence of genomic alterations in LPHD and highlight genomic regions that might be relevant for development and/or progression of this lymphoma entity.

Steroid hormones induce transcription from the mouse mammary tumor virus (MMTV) promoter by complex mechanisms requiring binding of the hormone receptors to the hormone responsive element (HRE) of the long terminal repeat region. Here we show that the MMTV promoter contains two degenerated octamer motifs immediately upstream of the TATA box that together bind OTF-1 (Oct-1, NFIII) with an affinity similar to the octamer consensus. In transfection experiments, mutation of these octamer motifs interferes with the hormonal response of the MMTV promoter. In vitro, these mutations do not influence basal transcription but completely abolish the stimulatory effect of purified progesterone receptor. Progesterone receptor and glucocorticoid receptor bound to the HRE facilitate binding of OTF-1 to the two octamer motifs. Thus, OTF-1 is a natural mediator of hormonal induction of the MMTV promoter and acts through cooperation with the hormone receptors for binding to DNA.

Multiple myeloma (MM) is a clonal plasmocyte disorder often preceded by a premalignant condition, monoclonal gammopathy of undetermined significance (MGUS). Chromosomal abnormalities in MM are complex, of which some are at present considered the most important prognostic indicators in MM. Their role in the pathogenesis of MM and in the transformation from MGUS towards MM is however not fully understood, at least partly due to the low-level bone marrow infiltration hampering cytogenetic analysis. In this study, a unique combination of techniques was used to allow whole genome screening for numerical chromosomal abnormalities of immunophenotypically pure, aberrant plasmocyte populations selected from the bone marrow by flow cytometry. Fourteen bone marrow samples from patients with either MM (n= 11) (first diagnosis or relapsing) or MGUS (n=3), with plasmocyte percentages as low as 1 % (range 1 – 57 %), were analyzed. Aberrant/monoclonal plasmocyte populations were identified based on the expression of CD56 and/or light chain restriction within a CD138+/CD38++ gate and sorted using a FACSAria® (BD, US). Whole genomic DNA was extracted from the CD56+or−/κ or λ+ plasmocyte populations, amplified with degenerate oligonucleotide primer-PCR and used for comparative genomic hybridization (CGH). CGH results were confirmed by interphase fluorescent in situ hybridization. All cases (MM and MGUS) showed the presence of multiple chromosomal abnormalities with a minimum of 5 each. All chromosomes were at least one time involved. Relapsing MM showed a mean number of chromosomal changes of 10 compared to 7 and 6 in newly diagnosed MM and MGUS cases respectively, suggesting karyotypic instability during the course of the disease. The presence of chromosomal aberrations known to be frequently occurring in MM, was confirmed such as gains of 1q (4/11, 36%), 7q (5/11, 45%) and 5q11-q32 (7/11, 64%) and losses of 13q (6/11, 55 %) and 16q (4/11, 36%). This study also showed a high incidence of chromosomal abnormalities not previously or only rarely described in MM, such as gains of 4q11-q22 (9/11, 82 %) and 8q21-q23 (5/11, 45%) and loss of 20q (5/11, 45 %). Some abnormalities were detected in both MM and MGUS, such as gain of 5q and loss of 16q, and may be considered very early, primary aberrations. Gain of 4q11-q22 was detected in almost all MM cases but in none of the MGUS cases, suggesting this abnormality might be involved in disease progression. These results indicate a higher complexity and diversity of chromosomal abnormalities in both MM and MGUS, than has already been described. CGH on flow sorted, immunophenotypically pure, aberrant plasmocytes allows an adequate genetic analysis of MGUS, which should ultimately result in the identification of genetic changes involved in the transformation of MGUS towards MM.

This paper summarizes the minimal workout of chronic lymphoproliferative disorders in a routine laboratory of haematology as recommended by a team of experienced laboratory supervisors in Belgium, taking into account the specific organisation of healthcare in Belgium, the innovations in the field of molecular analyses and related reimbursement. The starting point was essentially based upon clinical and/or haematological indications and it is emphasized that conclusions should be drawn in close dialogue with the clinician and experts in cytogenetics and histopathology. Reports made in the laboratory should be based upon an integration of cytomorphological, immunophenotypical and molecular data. These guidelines are not intended to be used as universal' diagnostic pathways', but should be useful in developing local diagnostic pathways. It is well understood that this consensus, being valid anno 2009, may rapidly change with new technologies being introduced and new targets discovered.