Genetic Profiling of Cell-Free DNA in Liquid Biopsies: A Complementary Tool for the Diagnosis of B-Cell Lymphomas and the Surveillance of Measurable Residual Disease.

PURPOSE: To assess the potential value of LiqBio as a complementary tool for diagnosis and surveillance of BCL. METHODS: This prospective multi-center study included 78 patients (25 follicular lymphomas (FL) and 53 large B-cell lymphomas (LBCL)). We performed next-generation sequencing (NGS) of cfDNA LiqBio and paired gDNA tissue biopsies at diagnosis and compared the mutational statuses. Also, through NGS of LiqBio, we identified MRD biomarkers and compared this novel LiqBio-MRD method with PET/CT in detecting MRD at follow-up. RESULTS: We identified mutations in 71% of LiqBio and 95% of tissue biopsies, and found a correlation between variant allele frequency of somatic mutations. Additionally, we identified mutations in 73% of LiqBio from patients with no available tissue samples or no mutations in them. Regarding the utility of LiqBio-MRD as a dynamic monitoring tool, when compared with the PET/CT method, a lower sensitivity was observed for LiqBio-MRD at 92.3% (vs. 100% for PET/CT), but a higher specificity of 91.3% (vs. 86.9% for PET/CT). CONCLUSION: Genetic profiling of tumor cfDNA in plasma LiqBio is a complementary tool for BCL diagnosis and MRD surveillance.

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

Craniopharyngiomas: A clinicopathological and molecular study of 52 cases - Experience in the Complejo Hospitalario de Toledo and Hospital Universitario 12 de Octubre (Madrid).

Craniopharyngiomas (CPs) are histologically benign tumors that are associated with high levels of morbidity. Two clinicopathological variants - adamantinomatous (ACP) and papillary (PCP) - have been described. They differ in their molecular features, whereby activating mutations in BRAF (V600E) and CTNNB1 genes characterize PCP and ACP, respectively. Recently, both variants have been shown to express elevated PD-L1 protein expression, but ACP also exhibited tumor cell-intrinsic PD-1 expression. In this study we analyze these molecular alterations in 52 cases with a long follow-up and examine their associations with immunohistochemical and clinical characteristics. ACPs comprise 73.1% of cases, while 21.2% are PCPs. Aberrant nuclear immunoreactivity for ß-catenin was observed in all ACPs. BRAF p.V600E mutations were observed in 90.9% of PCPs. Only one ACP case featured both alterations. Both types of CP exhibited strong nuclear staining for p63 with diffuse and basal distribution. ACP and PCP consistently expressed PD-L1, most in a substantial percentage of tumor cells, with a distinctive spatial distribution of expression in each subtype; only ACP demonstrated PD-1 expression. There was no evidence of differences in clinical prognosis between ACPs and PCPs. The identification of hallmark molecular signatures in the two CP variants is useful for sub-categorization in routine histopathology reporting. It is also pertinent to personalized therapy and for the development of improved non-invasive therapeutic strategies in this disease.

Frizzled 1 and Wnt1 as new potential therapeutic targets in the traumatically injured spinal cord.

Despite the experimental evidence pointing to a significant role of the Wnt family of proteins in physiological and pathological rodent spinal cord functioning, its potential relevance in the healthy and traumatically injured human spinal cord as well as its therapeutic potential in spinal cord injury (SCI) are still poorly understood. To get further insight into these interesting issues, we first demonstrated by quantitative Real-Time PCR and simple immunohistochemistry that detectable mRNA expression of most Wnt components, as well as protein expression of all known Wnt receptors, can be found in the healthy human spinal cord, supporting its potential involvement in human spinal cord physiology. Moreover, evaluation of Frizzled (Fz) 1 expression by double immunohistochemistry showed that its spatio-temporal and cellular expression pattern in the traumatically injured human spinal cord is equivalent to that observed in a clinically relevant model of rat SCI and suggests its potential involvement in SCI progression/outcome. Accordingly, we found that long-term lentiviral-mediated overexpression of the Fz1 ligand Wnt1 after rat SCI improves motor functional recovery, increases myelin preservation and neuronal survival, and reduces early astroglial reactivity and NG2+ cell accumulation, highlighting the therapeutic potential of Wnt1 in this neuropathological situation.

Spatio-temporal and Cellular Expression Patterns of PTK7 in the Healthy and Traumatically Injured Rat and Human Spinal Cord.

Despite the emerging role of protein tyrosine kinase 7 (PTK7) as a Wnt co-receptor and the relevant functions of the Wnt family of proteins in spinal cord injury (SCI), the potential involvement of PTK7 in SCI is currently unknown. As a first essential step to shed light on this issue, we evaluated the spatio-temporal and cellular expression patterns of PTK7 in healthy and traumatically injured rat and human spinal cords. In the uninjured rats, PTK7 expression was observed in the ependymal epithelium, endothelial cells, meningeal fibronectin-expressing cells, and specific axonal tracts, but not in microglia, astrocytes, neurons, oligodendrocytes, or NG2+ cells. After rat SCI, the mRNA expression of PTK7 was significantly increased, while its spatio-temporal and cellular protein expression patterns also suffered evident changes in the injured region. Briefly, the expression of PTK7 in the affected areas was observed in axons, reactive astrocytes, NG2+ and fibronectin-expressing cells, and in a subpopulation of reactive microglia/macrophages and blood vessels. Finally, in both healthy and traumatically injured human spinal cords, PTK7 expression pattern was similar to that observed in the rat, although some specific differences were found. In conclusion, we demonstrate for the first time that PTK7 is constitutively expressed in the healthy adult rat and human spinal cord and that its expression pattern clearly varied after rat and human SCI which, to our knowledge, constitutes the first experimental evidence pointing to the potential involvement of this co-receptor in physiological and pathological spinal cord functioning.

Cells in the adult human spinal cord ependymal region do not proliferate after injury.

In vertebrates that regenerate the injured spinal cord, cells at the ependymal region proliferate and coordinate the formation of bridges between the lesion stumps. In mammals, these cells also proliferate profusely around the central canal after spinal cord injury, although their actual contribution to repair is controversial. In humans, however, the central canal disappears from early childhood in the majority of individuals, being replaced by astrocyte gliosis, ependymocyte clusters, and perivascular pseudo-rosettes. In this human ependymal remnant, cells do not proliferate under normal conditions, but it is not known if they do after a lesion. Here, we studied the human ependymal remnant after traumatic spinal cord injury using samples from 21 individuals with survival times ranging from days to months post-injury. With three different monoclonal antibodies raised against two different proliferation markers (Ki67 and MCM2), we found that the ependymal remnant in adult humans does not proliferate after injury at any time or distance from the lesion. Our results seriously challenge the view of the spinal cord ependymal region as a neurogenic niche in adult humans and suggest that it would not be involved in cell replacement after a lesion. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Leishmania Uses Mincle to Target an Inhibitory ITAM Signaling Pathway in Dendritic Cells that Dampens Adaptive Immunity to Infection.

C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection. Mincle deficiency enhanced adaptive immunity against the parasite, correlating with increased activation, migration, and priming by Mincle-deficient dendritic cells (DCs). Leishmania triggered a Mincle-dependent inhibitory axis characterized by SHP1 coupling to the FcRγ chain. Selective loss of SHP1 in CD11c+ cells phenocopies enhanced adaptive immunity to Leishmania. In conclusion, Leishmania shifts Mincle to an inhibitory ITAM (ITAMi) configuration that impairs DC activation. Thus, ITAMi can be exploited for immune evasion by a pathogen and may represent a paradigm for ITAM-coupled receptors sensing self and non-self.

Glomerular C3d as a novel prognostic marker for renal vasculitis.

Pauci-immune necrotizing crescentic glomerulonephritis is the histologic substrate of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Several studies in animal models have demonstrated the crucial role of complement activation in the pathogenesis of ANCA-associated vasculitis, but only small series have analyzed the prognostic implications of complement glomerular deposits. This study aimed to assess the clinical and prognostic implications of C3d- and C4d-positive glomerular staining in renal vasculitis. Eighty-five patients with a diagnosis of pauci-immune necrotizing crescentic glomerulonephritis were included in the study. C3d and C4d were analyzed by immunohistochemical staining using a polyclonal antibody. The primary predictors were glomerular C3d- and C4d-positive staining. The primary end point was the cumulative percentage of patients who developed end-stage renal disease. Glomerular staining for C3d and C4d was observed in 42 (49.4%) of 85 biopsies and 38 (44.7%) of 85 biopsies, respectively. C3d-positive staining was associated with the severity of renal impairment and with a lower response rate to treatment (P=.003 and P=.04, respectively). Renal survival at 2 and 5 years was 60.9% and 51.8% in C3d-positive patients compared with 87.7% and 78.9% in C3d-negative patients (P=.04). C4d-positive staining did not show any impact in renal outcome. When adjusted by renal function and other histologic parameters, C3d staining remained as an independent predictor for renal survival (hazard ratio, 2.5; 95% confidence interval, 1.1-5.7; P=.03). Therefore, this study demonstrates that C3d-positive glomerular staining is an independent risk factor for the development of end-stage renal disease in ANCA-associated renal vasculitis.

Gene expression changes associated with erlotinib response in glioma cell lines.

Erlotinib (ERL), a tyrosine kinase inhibitor that acts on the epidermal growth factor receptor (EGFR), is used as a second line treatment for glioma therapy, with controversial findings regarding its response. Here, we analysed the gene expression profiles of a series of human glioma cell lines with differing sensitivities to ERL to identify the gene expression changes associated with ERL response. The varying responses to ERL were associated with different expression levels of specific genes (HRAS, CTFG, ERCC5 and HDAC3) and genes associated with specific pathways (apoptosis and cell death). PI3K pathway genes were primarily affected by ERL, as we found that PIK3R3 was repressed by ERL treatment in sensitive glioma cell lines. The cell cycle and ubiquitin pathways were also affected by EGFR inhibition, as GAS5, PLK1 and BIRC5 were the most significantly affected genes. In this study we have identified several genes such as PIK3R3 and GAS5, that can be targeted in order to enhance the response to ERL therapy.

Cancer-associated fibroblast and M2 macrophage markers together predict outcome in colorectal cancer patients.

Tumor epithelial cells within a tumor coexist with a complex microenvironment in which a variety of interactions between its various components determine the behavior of the primary tumors. Cancer-associated fibroblasts (CAF) and M2 macrophages, characterized by high expression of different markers, including α-SMA, FSP1 and FAP, or CD163 and DCSIGN, respectively, are involved in the malignancy of different tumors. In the present study, expression of the above markers in CAF and M2 macrophages was analyzed using RT-PCR and immunohistochemistry in the normal mucosa and tumor tissue from a cohort of 289 colorectal cancer patients. Expression of CAF and M2 markers is associated with the clinical outcome of colorectal cancer patients. Moreover, the combination of CAF and M2 markers identifies three groups of patients with clear differences in the progression of the disease. This combined variable could be a decisive factor in the survival of advanced-stage patients. Taken together, these analyses demonstrate the prognostic involvement of interrelationships between DCSIGN, CD163, α-SMA, FSP1 and FAP markers in the survival of colon cancer patients.

Genetic alterations associated with progression and recurrence in meningiomas.

Meningiomas are the most common primary brain tumors; they arise from the coverings of the brain. Although meningiomas are generally benign, some are more clinically aggressive, as reflected by their histopathological features or by their unexpected recurrence. We hypothesized that recurrent histologically benign meningiomas might have genetic features in common with those showing a more aggressive histology. By comparing gene expression profiles associated with meningioma progression and recurrence in 128 tumor samples (i.e. 83 benign World Health Organization [WHO] Grade I, 37 atypical WHO Grade II, and 8 anaplastic WHO Grade III) from 121 patients, we identified a 49-gene signature of meningioma aggressivity. This signature classified the tumors into 2 groups showing different clinical and pathological behaviors. The signature was composed of genes involved in the cell cycle (TMEM30B, CKS2, and UCHL1) and other pathways previously described as being altered in meningiomas, that is, WNT (SFRP1 and SFRP4) and transforming growth factor-ß pathways (LTBP2 and LMO4). Overall, gene downregulation was observed in advanced and recurrent samples versus benign and original ones. We propose that this gene repression may be caused by gene promoter hypermethylation, as in the case of UCHL1 and SFRP1, suggesting that this epigenetic event, together with loss of specific chromosomal regions, may play an important role in meningioma progression and recurrence.

Nodal marginal zone lymphoma: gene expression and miRNA profiling identify diagnostic markers and potential therapeutic targets.

Nodal marginal zone lymphoma (NMZL) is a small B-cell neoplasm whose molecular pathogenesis is still essentially unknown and whose differentiation from other small B-cell lymphomas is hampered by the lack of specific markers. We have analyzed gene expression, miRNA profile, and copy number data from 15 NMZL cases. For comparison, 16 follicular lymphomas (FLs), 9 extranodal marginal zone lymphomas, and 8 reactive lymph nodes and B-cell subtypes were included. The results were validated by quantitative RT-PCR in an independent series, including 61 paraffin-embedded NMZLs. NMZL signature showed an enriched expression of gene sets identifying interleukins, integrins, CD40, PI3K, NF-κB, and TGF-ß, and included genes expressed by normal marginal zone cells and memory B cells. The most highly overexpressed genes were SYK, TACI, CD74, CD82, and CDC42EP5. Genes linked to G(2)/M and germinal center were down-regulated. Comparison of the gene expression profiles of NMZL and FL showed enriched expression of CHIT1, TGFB1, and TACI in NMZL, and BCL6, LMO2, and CD10 in FL. NMZL displayed increased expression of miR-221, miR-223, and let-7f, whereas FL strongly expressed miR-494. Our study identifies new candidate diagnostic molecules for NMZL and reveals survival pathways activated in NMZL.

Prognostic impact of ΔTAp73 isoform levels and their target genes in colon cancer patients.

PURPOSE: Cumulative data support the role of ΔTAp73 variants in tumorigenic processes such as drug resistance. We evaluate the impact of TP73 isoforms and their putative target genes ABCB1, HMGB1, and CASP1 on the survival of colon cancer patients and the correlation between their expressions. EXPERIMENTAL DESIGN: We determined in 77 colon cancer patients the expression of ΔEx2p73, ΔEx2/3p73, ΔNp73, TAp73, ABCB1, HMGB1, and CASP1 by quantitative real-time reverse transcriptase-PCR. Tumor characteristics, disease-free survival, and overall survival (OS) were examined in each patient. Functional experiments were carried out to check whether ectopic expression of ΔNp73 modifies the proliferation, drug resistance, migration, and invasion properties of colon tumor cells and the expression of ABCB1, HMGB1, and CASP1. RESULTS: Positive correlations were observed between the expression levels of ΔTAp73 variants and HMGB1. Furthermore, a trend was observed for ABCB1. Overexpression of ΔEx2/3p73 and ΔNp73 isoforms was significantly associated with advanced stages (P = 0.04 and P = 0.03, respectively) and predicted shortened OS (P = 0.04 and P = 0.05, respectively). High levels of ABCB1 and HMGB1 were associated with shorter OS (P = 0.04 and P = 0.05, respectively). Multivariate analysis showed that, in addition to the tumor stage, ABCB1 and HMGB1 had independent relationships with OS (P = 0.008). Ectopic expression of ΔNp73 was associated with an increase in proliferation and drug resistance. CONCLUSIONS: The positive correlation between ΔTAp73 variants and HMGB1 and ABCB1 expression supports them as TP73 targets. The fact that upregulation of ΔTAp73 isoforms was associated with shortened OS, increase in proliferation, and drug resistance confirms their oncogenic role and plausible value as prognostic markers. ABCB1 and HMGB1, putative ΔTAp73 target genes, strongly predict OS in an independent manner, making clear the importance of studying downstream TP73 targets that could predict the outcome of colon cancer patients better than ΔTAp73 variants themselves do.

Stimulation of ALK by the growth factor midkine renders glioma cells resistant to autophagy-mediated cell death.

Δ9-tetrahydrocannabinol (THC), the main active component of marijuana, promotes cancer cell death via autophagy stimulation. We find that activation of the tyrosine kinase receptor ALK by its ligand midkine interferes with the signaling mechanism by which THC promotes autophagy-mediated glioma cell death.

Differential expression profiling analyses identifies downregulation of 1p, 6q, and 14q genes and overexpression of 6p histone cluster 1 genes as markers of recurrence in meningiomas.

The majority of meningiomas are probably benign but a number of tumors display considerable histological and/or clinical aggressivity, sometimes with unexpectedly high recurrence rates after radical removal. Understanding the potential behavior of these tumors in individual patients is critical for rational therapeutic decision-making. This study aimed to identify gene expression profiles and candidate markers associated with original and recurrent meningiomas. Unsupervised hierarchical clustering of the samples confirmed 2 main groups of meningiomas with distinct clinical behaviors. The gene expression profiling study identified genes and pathways potentially associated with meningioma recurrence, revealing an overall lower level of gene expression. The differential gene expression profiling analyses of original and recurrent meningiomas identified 425 known genes and expressed sequence tags related to meningioma recurrence, with SFRP1 (8p12), TMEM30B (14q23), and CTGF (6q23) showing the most disparate expression. Most of the differentially expressed genes were located at 1p, 6q, and 14q and were underexpressed in recurrences. Loss of such chromosomal regions has previously been associated with a higher risk of meningioma recurrence or malignant progression. Thus, at these locations, we propose the existence of novel candidate genes that could be involved in meningioma recurrence. In addition, the overexpression of genes of histone cluster 1 (6p) in recurrent meningiomas is reported here for the first time. Finally, the altered genes related to meningioma recurrence are involved in pathways such as Notch, TGFß, and Wnt, as described previously, and in other pathways such as cell cycle, oxidative phosphorylation, PPAR, and PDGF, not related before to meningioma recurrence.

The MHC-related protein 1 (MR1) is expressed by a subpopulation of CD38+, IgA+ cells in the human intestinal mucosa.

MHC-related 1 (MR1) molecule is a non-classical member of the MHC class I family of proteins. The sequence homology between classical MHC class I molecules and MR1 is very high, although the MR1 gene is not polymorphic and is highly conserved between species. MR1 is the restriction molecule of a sub-population of T lymphocytes, which are CD4-,CD8- and display conserved TCR alpha chain. The function of these cells is currently unknown, but they are believed to have regulatory properties similar to those of the CD1d restricted NKT cells. The MR1 gene is ubiquitously transcribed; however it is unknown what types of cells express the MR1 protein "in vivo". In the present work we analyzed the expression of the MR1 protein using specific antisera and monoclonal antibodies in different human cell lines, in primary cells and in mucosal tissues. We found some lymphoid cell lines that express MR1 on the cell surface but at levels much lower than the MR1 transfected cell lines. In addition, we observed that expression of MR1 in the mucosa is restricted to a subpopulation of plasma cells or plasmablasts, CD38+ or CD138+ and IgA+, located in the human intestinal mucosa. This suggests a function for MR1 in the development of IgA producing plasma cells.

Novel genomic alterations and mechanisms associated with tumor progression in oligodendroglioma and mixed oligoastrocytoma.

Combined 1p/19q deletions are very prevalent in oligodendrogliomas (OGs) and, to a lesser extent, in oligoastrocytomas (OAs). These losses are associated with responsiveness to therapy. Using array-based comparative genomic hybridization, we screened for recurrent genomic alterations in OG and oligoastrocytoma subtypes on chromosome 19. Concomitant 1p/19q loss was detected in most of the tumors with allelic loss, but array-based comparative genomic hybridization revealed some tumors to have unrelated 1p/19q arm losses, suggesting alternative mechanisms of loss to that related to the reported t(1;19) translocation. Analyses of 1p/19q loss by fluorescence in situ hybridization and loss of heterozygosity assays and correlations of genomic data with the Ki-67 proliferation marker were also performed. Four 1q (or 19p) and 2 1p (or 19q) fluorescence in situ hybridization probe signals together with homozygosity of the 1p/19q microsatellites suggested a hypothetical mechanism of genome duplication consecutive to the loss of the derivative chromosome der(1p;19q) from the t(1;19)(1q;19p) translocation. This genome duplication was frequent in high-grade OGs and was strongly correlated with Ki-67 expression; thus, it could be related to tumor progression. Finally, in addition to the frequent 1p/19q loss, we report a novel 17q amplified region in OGs with BIRC5 as one of the possible candidate target genes of the amplicon.