Endometrial Cancer-Adjacent Tissues Express Higher Levels of Cancer-Promoting Genes than the Matched Tumors.

Molecular alterations in tumor-adjacent tissues have recently been recognized in some types of cancer. This phenomenon has not been studied in endometrial cancer. We aimed to analyze the expression of genes associated with cancer progression and metabolism in primary endometrial cancer samples and the matched tumor-adjacent tissues and in the samples of endometria from cancer-free patients with uterine leiomyomas. Paired samples of tumor-adjacent tissues and primary tumors from 49 patients with endometrial cancer (EC), samples of endometrium from 25 patients with leiomyomas of the uterus, and 4 endometrial cancer cell lines were examined by the RT-qPCR, for MYC, NR5A2, CXCR2, HMGA2, LIN28A, OCT4A, OCT4B, OCT4B1, TWIST1, STK11, SNAI1, and miR-205-5p expression. The expression levels of MYC, NR5A2, SNAI1, TWIST1, and STK11 were significantly higher in tumor-adjacent tissues than in the matched EC samples, and this difference was not influenced by the content of cancer cells in cancer-adjacent tissues. The expression of MYC, NR5A2, and SNAI1 was also higher in EC-adjacent tissues than in samples from cancer-free patients. In addition, the expression of MYC and CXCR2 in the tumor related to non-endometrioid adenocarcinoma and reduced the risk of recurrence, respectively, and higher NR5A2 expression in tumor-adjacent tissue increased the risk of death. In conclusion, tissues proximal to EC present higher levels of some cancer-promoting genes than the matched tumors. Malignant tumor-adjacent tissues carry a diagnostic potential and emerge as new promising target of anticancer therapy.

A Set of 17 microRNAs Common for Brain and Cerebrospinal Fluid Differentiates Primary Central Nervous System Lymphoma from Non-Malignant Brain Tumors.

The diagnosis of primary central nervous system (CNS) lymphoma, which is predominantly of the diffuse large B-cell lymphoma type (CNS DLBCL), is challenging. MicroRNAs (miRs) are gene expression-regulating non-coding RNAs that are potential biomarkers. We aimed to distinguish miR expression patterns differentiating CNS DLBCL and non-malignant CNS diseases with tumor presentation (n-ML). Next generation sequencing-based miR profiling of cerebrospinal fluids (CSFs) and brain tumors was performed. Sample source-specific (CSF vs. brain tumor) miR patterns were revealed. Even so, a set of 17 miRs differentiating CNS DLBCL from n-ML, no matter if assessed in CSF or in a tumor, was identified. Along with the results of pathway analyses, this suggests their pathogenic role in CNS DLBCL. A combination of just four of those miRs (miR-16-5p, miR-21-5p, miR-92a-3p, and miR-423-5p), assessed in CSFs, discriminated CNS DLBCL from n-ML samples with 100% specificity and 67.0% sensitivity. Analyses of paired CSF-tumor samples from patients with CNS DLBCL showed significantly lower CSF levels of miR-26a, and higher CSF levels of miR-15a-5p, miR-15b-5p, miR-19a-3p, miR-106b-3p, miR-221-3p, and miR-423-5p. Noteworthy, the same miRs belonged to the abovementioned set differentiating CNS DLBCL from non-malignant CNS diseases. Our results not only add to the basic knowledge, but also hold significant translational potential.

Tumor and Cerebrospinal Fluid microRNAs in Primary Central Nervous System Lymphomas.

Primary central nervous system lymphoma (PCNSL) is a rare, highly aggressive, extranodal form of non-Hodgkin lymphoma, predominantly diagnosed as primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL). Fast and precise diagnosis of PCNSL is critical yet challenging. microRNAs, important regulators in physiology and pathology are potential biomarkers. In 131 patients with CNS DLBCL and with non-malignant brain lesions (n-ML), miR-21, miR-19b and miR-92a, miR-155, miR-196b, miR-let-7b, miR-125b, and miR-9 were examined by RT-qPCR in brain biopsy samples (formalin-fixed paraffin-embedded tissues, FFPET; CNS DLBCL, n = 52; n-ML, n = 42) and cerebrospinal fluid samples (CSF; CNS DLBCL, n = 30; n-ML, n = 23) taken for routine diagnosis. FFPET samples were split into study and validation sets. Significantly higher CSF levels of miR-21, miR-19b, and miR-92a were identified in PCNSL but not in n-ML, and differentiated PCNSL from n-ML with 63.33% sensitivity and 80.77% specificity. In FFPETs, miR-155 and miR-196b were significantly overexpressed and miR-let-7b, miR-125b, and miR-9 were downregulated in PCNSL as compared to n-ML. Combined miR-155 and miR-let-7b expression levels in FFPETs discriminated PCNSL and n-ML with a 97% accuracy. In conclusion, tissue miR-155, miR-196b, miR-9, miR-125b, and miR-let-7b expression profiles differentiate PCNSL from n-ML. PCNSL CSFs and the relevant biopsy samples are characterized by specific, different microRNA profiles. A logistic regression model is proposed to discriminate between PCNSL and non-malignant brain lesions. None of the examined microRNAs influenced overall survival of PCNSL patients. Further ongoing developments involve next generation sequencing-based profiling of biopsy and CSF samples.

A comprehensive flow-cytometry-based immunophenotypic characterization of Burkitt-like lymphoma with 11q aberration.

We previously described a subset of MYC translocation-negative aggressive B-cell lymphomas resembling Burkitt lymphoma, characterized by proximal gains and distal losses in chromosome 11. In the 2016 WHO classification, these MYC-negative lymphomas were recognized as a new provisional entity, 'Burkitt-like lymphoma with 11q aberration'. Here we present an immunophenotype analysis of Burkitt-like lymphomas with 11q aberration. Cells were acquired by fine needle aspiration biopsy from 10 young adult patients, 80% of whom presented recurrence-free 5-year survival. Twenty-three MYC-positive Burkitt lymphomas, including three carrying both MYC rearrangement and 11q aberration, served as controls. By immunohistochemistry, all Burkitt-like lymphomas with 11q aberration were CD20+/CD10+/BCL6+/BCL2-/MUM1-/MYC+/EBV-, usually LMO2+/CD44-/CD43- and sometimes CD56+, and showed high proliferation rate. By flow cytometry, Burkitt-like lymphoma with 11q aberration immunophenotypically resembled MYC-positive Burkitt lymphoma, except for significantly (adjusted P<0.001) more frequent CD38higher expression in Burkitt lymphoma (91% MYC-positive Burkitt lymphoma vs 10% Burkitt-like lymphoma with 11q aberration), more frequently diminished CD45 expression in Burkitt lymphoma (74% vs 10%), an exclusive CD16/CD56 and highly restricted CD8 expression in Burkitt-like lymphoma with 11q aberration (60% vs 0% and 40% vs 4%, respectively). We showed high diagnostic accuracy and effectiveness of flow cytometry in Burkitt lymphoma. CD16/CD56 expression without CD38higher and the lack of CD16/CD56 with CD38higher expression proves to be a reliable, fast, and cost-effective method for diagnosing 11q aberration and MYC rearrangements in CD10(+) aggressive lymphomas, respectively. In addition, we confirmed a pattern of an inverted duplication with telomeric loss of 11q, as a recurrent 11q abnormality, but one case presented alternative changes, possibly resulting in an equivalent molecular effect. Our findings reveal similarities along with subtle but essential differences in the immunophenotype of Burkitt-like lymphoma with 11q aberration and MYC-positive Burkitt lymphoma, important for the differential diagnosis, but also for understanding the pathogenesis of Burkitt-like lymphoma with 11q aberration.

Changes in plasma miR-9, miR-16, miR-205 and miR-486 levels after non-small cell lung cancer resection.

PURPOSE: The majority of non-small cell lung cancer (NSCLC) patients presents with an advanced-stage disease and, consequently, exhibits a poor overall survival rate. We aimed to assess changes in plasma miR-9, miR-16, miR-205 and miR-486 levels and their potential as biomarkers for the diagnosis and monitoring of NSCLC patients. METHODS: Plasma was collected from 50 healthy donors and from NSCLC patients before surgery (n = 61), 1 month after surgery (n = 37) and 1 year after surgery (n = 14). microRNA levels were quantified using qRT-PCR. RESULTS: We found in NSCLC patients before treatment, both with squamous cell carcinoma (SQCC) and adenocarcinoma (ADC), significantly higher plasma miR-16 and miR-486 levels than in healthy individuals. Pre-treatment miR-205 concentrations were found to be significantly higher in SQCC than in ADC patients, and only SQCC patients presented significantly higher circulating miR-205 levels than healthy donors. SQCC plasma miR-9 levels were not different from normal control levels, but in ADC they were found to be significantly decreased. A combination of plasma miR-16, miR-205 and miR-486 measurements was found to discriminate NSCLC patients from healthy persons, with a specificity of 95% and a sensitivity of 80%. Following tumor resection, we found that the miR-9 and miR-205 levels significantly decreased, even below the normal level, whereas the increased miR-486 level persisted up to one year after surgery, and the miR-16 level decreased to normal. After tumor resection, none of the miR levels tested was found to relate to recurrence. CONCLUSIONS: Our data indicate that miR-9, miR-16, miR-205 and miR-486 may serve as NSCLC biomarkers. The observed cancer-related pre- and post-operative changes in their plasma levels may not only reflect the presence of a primary cancer, but also of a systemic response to cancer.

miR-7 expression in serous ovarian carcinomas.

BACKGROUND/AIM: miR-7 has recently been linked to cancer. Some miR-7 targets, including B-cell lymphoma 2 (BCL2) and epidermal growth factor receptor (EGFR), are involved in ovarian cancer (OC) pathogenesis. The majority of OCs display TP53 mutations, which are critically important for OC development. We aimed to study the expression level of miR-7 and of two of its postulated target genes, BCL2 and EGFR, in serous ovarian carcinomas of different TP53 status and tumour grade. MATERIALS AND METHODS: Gene and miR expression was assessed by real-time reverse transcription polymerase chain reaction in 45 clinical samples of low- (G1+G2) and high- (G3) grade primary serous OC with wild-type (wt) or mutated TP53, as well as in three OC cell lines, each representing a different TP53 status. The results obtained in patients with OC were analysed against their disease-free survival (DFS). RESULTS: In high-grade OC with TP53 mutations, the level of miR-7 expression significantly exceeded (by several fold) that in wtTP53 cancer (p<0.01). Within the wtTP53 tumour series, the level of miR-7 expression was significantly higher (by over 10-fold) in high-grade than in low-grade OC (p<0.01). miR-7 expression was not found to influence DFS. The differences in miR-7 expression depending on TP53 status found in clinical OC samples were not observed in OC cell lines. miR-7 overexpression correlated with diminished BCL2 expression, but there was no relationship between miR-7 and EGFR expression, neither in tumour samples nor in the cell lines. CONCLUSION: There is a link between miR-7 expression and TP53 status and tumour grade in serous OC. Molecular mechanisms of these relationships need to be elucidated. Of the two postulated miR-7 target genes we examined, BCL2, but not EGFR, seems to be a possible miR-7 target in OC.

miR expression in MYC-negative DLBCL/BL with partial trisomy 11 is similar to classical Burkitt lymphoma and different from diffuse large B-cell lymphoma.

Fast and reliable differential diagnosis of Burkitt lymphoma (BL) vs. diffuse large B cell lymphoma (DLBCL) is of major importance for therapeutic decisions and patient outcome. Aggressive B cell non-Hodgkin lymphomas (B-NHLs) that do not belong to the abovementioned entities were categorized by the current WHO lymphoma classification as "B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL" (DLBCL/BL). We have recently described a DLBCL/BL subgroup with recurrent chromosome 11q aberrations, resembling BL (B-NHLs[11q]). Here, we analyzed 102 prospectively collected fine needle aspirates from patients with aggressive B-NHLs in order to investigate the potential of microRNA (miR)-155, its precursor BIC, as well as miR-21 and miR-26a to differentiate BL from DLBCL, and from DLBCL/BL that include B-NHLs[11q]. Both BL and DLBCL/BL cases, including B-NHLs[11q], demonstrated significantly lower expression levels of miR-155/BIC, miR-21, and miR-26a compared to primary DLBCL. In conclusion, the miRs expression in B-NHLs[11q] provides a new suggestion, in addition to pathomorphological and clinical similarities between classical, i.e., MYC translocation-positive BL, and B-NHLs[11q], to recognize the B-NHLs[11q] subgroup of DLBCL/BL category as a MYC translocation-negative variant of BL in most cases, and points to the potential utility of miR-155/BIC/miR-21/miR-26a for the differential diagnosis of a heterogeneous category of DLBCL/BL.

Immortalization of T-cells is accompanied by gradual changes in CpG methylation resulting in a profile resembling a subset of T-cell leukemias.

We have previously described gene expression changes during spontaneous immortalization of T-cells, thereby identifying cellular processes important for cell growth crisis escape and unlimited proliferation. Here, we analyze the same model to investigate the role of genome-wide methylation in the immortalization process at different time points pre-crisis and post-crisis using high-resolution arrays. We show that over time in culture there is an overall accumulation of methylation alterations, with preferential increased methylation close to transcription start sites (TSSs), islands, and shore regions. Methylation and gene expression alterations did not correlate for the majority of genes, but for the fraction that correlated, gain of methylation close to TSS was associated with decreased gene expression. Interestingly, the pattern of CpG site methylation observed in immortal T-cell cultures was similar to clinical T-cell acute lymphoblastic leukemia (T-ALL) samples classified as CpG island methylator phenotype positive. These sites were highly overrepresented by polycomb target genes and involved in developmental, cell adhesion, and cell signaling processes. The presence of non-random methylation events in in vitro immortalized T-cell cultures and diagnostic T-ALL samples indicates altered methylation of CpG sites with a possible role in malignant hematopoiesis.

Telomerase upregulation is a postcrisis event during senescence bypass and immortalization of two Nijmegen breakage syndrome T cell cultures.

Our knowledge on immortalization and telomere biology is mainly based on genetically manipulated cells analyzed before and many population doublings post growth crisis. The general view is that growth crisis is telomere length (TL) dependent and that escape from crisis is coupled to increased expression of the telomerase reverse transcriptase (hTERT) gene, telomerase activity upregulation and TL stabilization. Here we have analyzed the process of spontaneous immortalization of human T cells, regarding pathways involved in senescence and telomerase regulation. Two Nijmegen breakage syndrome (NBS) T cell cultures (S3R and S4) showed gradual telomere attrition until a period of growth crisis followed by the outgrowth of immortalized cells. Whole genome expression analysis indicated differences between pre-, early post- and late postcrisis cells. Early postcrisis cells demonstrated a logarithmic growth curve, very short telomeres and, notably, no increase in hTERT or telomerase activity despite downregulation of several negative hTERT regulators (e.g. FOS, JUN D, SMAD3, RUNX2, TNF-a and TGFb-R2). Thereafter, cMYC mRNA increased in parallel with increased hTERT expression, telomerase activity and elongation of short telomeres, indicating a step-wise activation of hTERT transcription involving reduction of negative regulators followed by activation of positive regulator(s). Gene expression analysis indicated that cells escaped growth crisis by deregulated DNA damage response and senescence controlling genes, including downregulation of ATM, CDKN1B (p27), CDKN2D (p19) and ASF1A and upregulation of CDK4, TWIST1, TP73L (p63) and SYK. Telomerase upregulation was thus found to be uncoupled to escape of growth crisis but rather a later event in the immortalization process of NBS T cell cultures.

Molecular signature of cell cycle exit induced in human T lymphoblasts by IL-2 withdrawal.

BACKGROUND: The molecular mechanisms of cell cycle exit are poorly understood. Studies on lymphocytes at cell cycle exit after growth factor deprivation have predominantly focused on the initiation of apoptosis. We aimed to study gene expression profile of primary and immortalised IL-2-dependent human T cells forced to exit the cell cycle by growth factor withdrawal, before apoptosis could be evidenced. RESULTS: By the Affymetrix microarrays HG-U133 2.0 Plus, 53 genes were distinguished as differentially expressed before and soon after IL-2 deprivation. Among those, PIM1, BCL2, IL-8, HBEGF, DUSP6, OSM, CISH, SOCS2, SOCS3, LIF and IL13 were down-regulated and RPS24, SQSTM1, TMEM1, LRRC8D, ECOP, YY1AP1, C1orf63, ASAH1, SLC25A46 and MIA3 were up-regulated. Genes linked to transcription, cell cycle, cell growth, proliferation and differentiation, cell adhesion, and immune functions were found to be overrepresented within the set of the differentially expressed genes. CONCLUSION: Cell cycle exit of the growth factor-deprived T lymphocytes is characterised by a signature of differentially expressed genes. A coordinate repression of a set of genes known to be induced during T cell activation is observed. However, growth arrest following exit from the cell cycle is actively controlled by several up-regulated genes that enforce the non-dividing state. The identification of genes involved in cell cycle exit and quiescence provides new hints for further studies on the molecular mechanisms regulating the non-dividing state of a cell, the mechanisms closely related to cancer development and to many biological processes.

Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia.

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

Spontaneously immortalized T lymphocytes from Nijmegen Breakage Syndrome patients display phenotypes typical for lymphoma cells.

We found that the peripheral T lymphocytes from four of eight patients with the lymphoma predisposing Nijmegen Breakage Syndrome (NBS) acquired an unlimited growth potential following in vitro mitogen stimulation and subsequent interleukin-2-dependent propagation. The immortal T cell lines revealed morphological and other features typical for anaplastic large cell lymphoma (ALCL). In addition, multiple copies of ALK, but with no ALK gene rearrangements were found in a subpopulation of cells of one of the immortalized lines. These cell lines may be useful for the in vitro elucidation of mechanisms involved in the development of ALCL.

Spontaneously immortalized human T lymphocytes develop gain of chromosomal region 2p13-24 as an early and common genetic event.

To gain further insight into the molecular events responsible for the extended life span and immortalization of human lymphoid cells, we analyzed a series of spontaneously immortalized, IL2-dependent human T-cell lines using molecular cytogenetic techniques. Two of the cell lines were derived from normal spleen and three from patients with Nijmegen breakage syndrome (NBS), a recessive disorder characterized by a high incidence of lymphoid malignancies. Here we show that spontaneous immortalization of the five T-cell lines was associated with the acquisition of copy number gains involving chromosomal region 2p13-24 as common early alterations. In addition, we found an amplification of 8q21-24 after prolonged propagations in all three NBS-derived cell lines as well as early development of near-tetraploidy in two of these lines. Gains involving the short arm of chromosome 2 recently were found in several lymphoid malignancies. Therefore, the cell lines described here can be used for identification and characterization of genes involved in the pathogenesis of lymphoid neoplasms and would also provide a useful tool for better understanding the mechanisms responsible for cell immortalization.

Telomere maintenance and cell cycle regulation in spontaneously immortalized T-cell lines from Nijmegen breakage syndrome patients.

Nijmegen breakage syndrome (NBS) is a rare genetic instability syndrome associated with a high incidence of lymphoid malignancies. The NBS1 protein has been implicated in telomere biology suggesting that cells from NBS patients might have deficient telomere maintenance capacity. In this study we characterized spontaneously immortalized T-cell lines derived from three NBS patients regarding growth characteristics, telomere biology, expression of cell-cycle regulators, and response to DNA damage to understand the role of NBS1 in the immortalization process. In all the NBS T-cell lines the acquisition of an immortal phenotype was associated with telomere length stabilization, high telomerase activity, and increased mRNA expression of the catalytic subunit of telomerase (hTERT), together with c-myc up-regulation. Our findings provide evidence that telomere length maintenance was intact in the T lymphocytes in the absence of a full-length NBS protein, presumably due to the presence of an alternatively transcribed NBS protein of 70 kDa. Normal protein expression patterns for pRb and p53 in all the immortal lines coincided with altered expression of some cell-cycle proteins as well as with an impaired G1/S arrest after gamma irradiation, despite a seemingly normal p53/p21 pathway. The here described, spontaneously immortalized NBS derived T-cell lines can be useful in future analysis of the biologic effects in the NBS.