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Background: Ultraconserved regions or elements (UCEs) are greater than 100-base pairs in length and are perfectly (100%) conserved across large evolutionary distances in the genomes of at least 3 of 5 placental mammals: human, cow, dog, rat, and mouse. Most UCEs are located in non-coding regions of the genome; however, some UCEs overlap with coding exons. In addition, some UCEs have been found to be transcriptionally active as long non-coding RNAs and are involved in a variety of cellular processes such as cellular proliferation. Although a few UCE sequence variations were reported to be associated with human diseases, the molecular functions of somatic mutations in UCEs remain largely unexplained in human cancers. In this study, we characterized the distribution of somatic UCE mutations throughout a spectrum of cancers and investigated the biological functions of cancer associated UCEs. Methods: We examined somatic UCE mutations in 2,449 cases of 22 cancer types using the PCAWG and ICGC platforms. To validate these mutation patterns, UCE sequencing was performed on the Illumina NovaSeq 6000 platform for MD Anderson patient cohorts. A custom AsCpf1 guide library was built to identify potential regulatory UCE functions in colorectal cancer models, and 3 guides were designed to target 2,247 UCEs. The proliferation ratio of knockout UCEs was analyzed. We then generated stable mutated UCE clones using the CRISPR AsCpf1 technology in colorectal cancer cells. We conducted RNA sequencing (RNA-seq) of mutated UCE cells. The expression of target proteins and genes were analyzed by qRT-PCR and western blotting. To address the effects of the mutated UCE_11311 tumor growth, DLD1 cells were injected in athymic nude mice. Results: We analyzed the WGS data on 2,449 cases of 22 cancer types and identified 24,039 somatic mutations in 10,090 (73.46%) UCEs. These were mostly located in non-coding DNAs, mainly in introns.Based on the RNA-seq data, one of the strongest impacts of mutated UCEs was on ARID1B. We confirmed that specific mutated UCE decreased ARID1B mRNA and protein levels. Our in vivo results demonstrated that UCE mutations enhanced the tumorigenicity of DLD1 xenograft tumors. As a summary, UCE_11311 has a transcriptional enhancer activity on the ARID1B gene and UCE_11311 mutations are actively participating in the tumorigenesis at least in part through regulating ARID1B expression. Discussion: We identified hundreds of unexplored tumorigenic UCEs that need to be further characterized functionally and clinically, and proved one of them to be a transcriptional enhancer of the tumor suppressor ARID1B. Collectively, these data support the concept that certain somatic UCE mutations are frequent and functional in cancer evolution, acting as driver mutations that can be used as new therapeutic targets; other somatic UCE mutations are cancer specific or patient specific, are markers of aggressiveness, and can be used to personalized therapy. Citation Format: Recep Bayraktar, Yitao Tang, Mihnea P. Dragomir, Linda Fabris, Giulio F. Draetta, Han Liang, George A. Calin. The mutational landscape of ultraconserved elements in human cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5750.

The ability of a receptor to preferentially activate only a subset of available downstream signal cascades is termed biased signaling. Although comprehensively recognized for the G protein–coupled receptors (GPCR), this process is scarcely explored downstream of receptor tyrosine kinases (RTK), including the cancer-relevant insulin-like growth factor-1 receptor (IGF1R). Successful IGF1R targeting requires receptor downregulation, yet therapy-mediated removal from the cell surface activates cancer-protective β-arrestin–biased signaling (β-arr-BS). As these overlapping processes are initiated by the β-arr/IGF1R interaction and controlled by GPCR-kinases (GRK), we explored GRKs as potential anticancer therapeutic targets to disconnect IGF1R downregulation and β-arr-BS. Transgenic modulation demonstrated that GRK2 inhibition or GRK6 overexpression enhanced degradation of IGF1R, but both scenarios sustained IGF1–induced β-arr-BS. Pharmacologic inhibition of GRK2 by the clinically approved antidepressant, serotonin reuptake inhibitor paroxetine (PX), recapitulated the effects of GRK2 silencing with dose- and time-dependent IGF1R downregulation without associated β-arr-BS. In vivo, PX treatment caused substantial downregulation of IGF1R, suppressing the growth of Ewing's sarcoma xenografts. Functional studies reveal that PX exploits the antagonism between β-arrestin isoforms; in low ligand conditions, PX favored β-arrestin1/Mdm2-mediated ubiquitination/degradation of IGF1R, a scenario usually exclusive to ligand abundancy, making PX more effective than antibody-mediated IGF1R downregulation. This study provides the rationale, molecular mechanism, and validation of a clinically feasible concept for “system bias” targeting of the IGF1R to uncouple downregulation from signaling. Demonstrating system bias as an effective anticancer approach, our study reveals a novel strategy for the rational design or repurposing of therapeutics to selectively cross-target the IGF1R or other RTK. Significance: This work provides insight into the molecular and biological roles of biased signaling downstream RTK and provides a novel “system bias” strategy to increase the efficacy of anti–IGF1R-targeted therapy in cancer.

Primary myelofibrosis (PMF) is a pluripotent hematopoietic stem cell-derived malignancy, included in the heterogeneous group of myeloproliferative neoplasms (MPNs). PMF diagnosis is based on a composite assessment of clinical and laboratory data. The three major diagnostic criteria are: screening for driver mutations, exclusion of other conditions that can cause myelofibrosis, and bone marrow biopsy displaying megakaryocyte changes and fibrosis. PMF treatment options are only partially disease-modifying and consist mainly of symptom control. Recently, a new targeted therapy was introduced for PMF patients, JAK-STAT inhibitors (i.e. ruxolitinib). However, specific subgroups of patients do not benefit from the JAK-STAT inhibitors: (1) those who are carrying JAK2 mutations, but ruxolitinib does not reduce the spleen size; (2) triple negative patients (no JAK2, CALR, or MPL mutations); and (3) those who discontinue JAK-STAT therapy because of side effects. These subgroups are in need of new therapeutic approaches. Mature microRNAs (miRNAs) range from 16 to 28 nucleotides (nt) in length and regulate specific messenger RNAs at the post-transcriptional level. Numerous in vitro and in vivo studies have reported specific miRNAs, as well as complex miRNA networks, to be dysregulated in PMF. Several of these miRNAs were shown to be implicated in essential events of PMF pathophysiology: increase of bone marrow fibrosis, progression to acute myeloid leukemia, resistance to JAK-STAT inhibitors, and activation of differentiation of hematopoietic stem/progenitor cells into megakaryocytes. Hence, we propose miRNAs as a potential minimally invasive diagnostic tool for PMF and as therapeutic targets that could address the unmet medical needs of these patients.

The solid tumour microenvironment includes nerve fibres that arise from the peripheral nervous system(1,2). Recent work indicates that newly formed adrenergic nerve fibres promote tumour growth, but the origin of these nerves and the mechanism of their inception are unknown(1,3). Here, by comparing the transcriptomes of cancer-associated trigeminal sensory neurons with those of endogenous neurons in mouse models of oral cancer, we identified an adrenergic differentiation signature. We show that loss of TP53 leads to adrenergic transdifferentiation of tumour-associated sensory nerves through loss of the microRNA miR-34a. Tumour growth was inhibited by sensory denervation or pharmacological blockade of adrenergic receptors, but not by chemical sympathectomy of pre-existing adrenergic nerves. A retrospective analysis of samples from oral cancer revealed that p53 status was associated with nerve density, which was in turn associated with poor clinical outcomes. This crosstalk between cancer cells and neurons represents mechanism by which tumour-associated neurons are reprogrammed towards an adrenergic phenotype that can stimulate tumour progression, and is a potential target for anticancer therapy.

Neoadjuvant chemoradiotherapy followed by esophagectomy is the standard treatment for patients with locally advanced esophageal squamous cell carcinoma (ESCC). This study explored correlations of clinical factors and dose-volume histogram (DVH) parameters with postoperative cardiopulmonary complications and predicted their risk by establishing a nomogram model.Clinical and DVH parameters of ESCC patients who underwent trimodality treatment from 2002 to 2020 were collected. Postoperative cardiopulmonary complications were recorded. Logistic regression analysis was applied, and a nomogram model was constructed. Area under the receiver operating characteristic (AUC) curve, calibration curve, and decision curve analyses were performed to evaluate the performance of the nomogram.Of the 307 ESCC patients enrolled in this study, 65 (21.2%) experienced pulmonary complications and 57 (18.6%) experienced cardiac complications. The following six risk factors were identified as independent risk factors for pulmonary complications by multivariate logistic regression analyses in the integrated model: male sex (odds ratio [OR], 3.26; 95% confidence interval [CI], 1.27-9.70; P = 0.021), post-radiation therapy (RT) forced expiratory volume in 1 s (FEV1) (OR, 0.51; 95% CI 0.28-0.90; P = 0.023), mean lung dose (MLD) (OR, 1.13; 95% CI 1.01-1.28; P = 0.041), and pre-RT monocyte (OR, 8.36; 95% CI 1.23-11.7; P = 0.03). The AUC of this integrated model was 0.705 (95% CI 0.64-0.77). The paclitaxel and cisplatin (TP) concurrent chemotherapy regimen was the independent predictor of cardiac complication (OR, 2.50; 95% CI 1.22-5.55; P = 0.016).For ESCC patients who underwent trimodality treatment, male sex, post-RT FEV1, MLD, and pre-RT monocyte were confirmed as significant predictors of postoperative pulmonary complications. A nomogram model including six risk factors was further established. The independent predictor of cardiac complication was TP concurrent chemotherapy.

Mechanisms of lymph node invasion seem to play a prognostic role in pancreatic ductal adenocarcinoma (PDAC) after resection. However, the 8th edition of the TNM classification of the American Joint Committee on Cancer (AJCC) does not consider this. The aim of this study was to analyse the prognostic role of different mechanisms of lymph node invasion on PDAC. One hundred and twenty-two patients with resected PDAC were examined. We distinguished three groups: direct (per continuitatem, Nc) from the main tumour, metastasis (Nm) without any contact to the main tumour, and a mixed mechanism (Ncm). Afterwards, the prognostic power of the different groups was analysed concerning overall survival (OS). In total, 20 patients displayed direct lymph node invasion (Nc = 16.4%), 44 were classed as Nm (36.1%), and 21 were classed as Ncm (17.2%). The difference in OS was not statistically significant between N0 (no lymph node metastasis, n = 37) and Nc (p = 0.134), while Nm had worse OS than N0 (p < 0.001). Direct invasion alone had no statistically significant effect on OS (p = 0.885). Redefining the N0 stage by including Nc patients showed a more precise OS prediction among N stages (p = 0.001 vs. p = 0.002). Nc was more similar to N0 than to Nm; hence, we suggest a rethinking of TNM classification based on the mechanisms of lymph node metastases in PDAC. Overall, this novel classification is more precise.

Renal cancer (RC) represents 3% of all cancers, with a 2% annual increase in incidence worldwide, opening the discussion about the need for screening. However, no established screening tool currently exists for RC. To tackle this issue, we assessed surface-enhanced Raman scattering (SERS) profiling of serum as a liquid biopsy strategy to detect renal cell carcinoma (RCC), the most prevalent histologic subtype of RC. Thus, serum samples were collected from 23 patients with RCC and 27 controls (CTRL) presenting with a benign urological pathology such as lithiasis or benign prostatic hypertrophy. SERS profiling of deproteinized serum yielded SERS band spectra attributed mainly to purine metabolites, which exhibited higher intensities in the RCC group, and Raman bands of carotenoids, which exhibited lower intensities in the RCC group. Principal component analysis (PCA) of the SERS spectra showed a tendency for the unsupervised clustering of the two groups. Next, three machine learning algorithms (random forest, kNN, naïve Bayes) were implemented as supervised classification algorithms for achieving discrimination between the RCC and CTRL groups, yielding an AUC of 0.78 for random forest, 0.78 for kNN, and 0.76 for naïve Bayes (average AUC 0.77 ± 0.01). The present study highlights the potential of SERS liquid biopsy as a diagnostic and screening strategy for RCC. Further studies involving large cohorts and other urologic malignancies as controls are needed to validate the proposed SERS approach.

Background Bladder cancer (BC) has the highest per-patient cost of all cancer types. Hence, we aim to develop a non-invasive, point-of-care tool for the diagnostic and molecular stratification of patients with BC based on combined microRNAs (miRNAs) and surface-enhanced Raman spectroscopy (SERS) profiling of urine. Methods Next-generation sequencing of the whole miRNome and SERS profiling were performed on urine samples collected from 15 patients with BC and 16 control subjects (CTRLs). A retrospective cohort (BC = 66 and CTRL = 50) and RT-qPCR were used to confirm the selected differently expressed miRNAs. Diagnostic accuracy was assessed using machine learning algorithms (logistic regression, naïve Bayes, and random forest), which were trained to discriminate between BC and CTRL, using as input either miRNAs, SERS, or both. The molecular stratification of BC based on miRNA and SERS profiling was performed to discriminate between high-grade and low-grade tumors and between luminal and basal types. Results Combining SERS data with three differentially expressed miRNAs (miR-34a-5p, miR-205-3p, miR-210-3p) yielded an Area Under the Curve (AUC) of 0.92 ± 0.06 in discriminating between BC and CTRL, an accuracy which was superior either to miRNAs (AUC = 0.84 ± 0.03) or SERS data (AUC = 0.84 ± 0.05) individually. When evaluating the classification accuracy for luminal and basal BC, the combination of miRNAs and SERS profiling averaged an AUC of 0.95 ± 0.03 across the three machine learning algorithms, again better than miRNA (AUC = 0.89 ± 0.04) or SERS (AUC = 0.92 ± 0.05) individually, although SERS alone performed better in terms of classification accuracy. Conclusion miRNA profiling synergizes with SERS profiling for point-of-care diagnostic and molecular stratification of BC. By combining the two liquid biopsy methods, a clinically relevant tool that can aid BC patients is envisaged.

Alterations in microRNAs (miRNAs) expression are causative in the initiation and progression of human cancers. The molecular events responsible for the widespread differential expression of miRNAs in malignancy are exemplified by their location in cancer-associated genomic regions, epigenetic mechanisms, transcriptional dysregulation, chemical modifications and editing, and alterations in miRNA biogenesis proteins. The classical miRNA function is synonymous with post-transcriptional repression of target protein genes. However, several studies have reported miRNAs functioning outside this paradigm and some of these novel modes of regulation of gene expression have been implicated in cancers. Here, we summarize key aspects of miRNA involvement in cancer, with a special focus on these lesser-studied mechanisms of action.

Knockout (KO) of long non-coding RNAs (lncRNAs) enables functional characterization of this still poorly described group of transcripts. One of the most efficient and simplest methods to achieve complete KO of a lncRNA is by employing CRISPR/Cas gene editing. As most lncRNAs are not well annotated, their individual functional regions are often not defined, and the majority of the transcripts are not affected by single nucleotide mutations. Therefore, CRISPR/Cas KO is more challenging for lncRNAs as compared to KO of protein coding genes. Strategies for lncRNAs KO include complete removal of the entire gene, removal of the promoter and transcriptional start site, abolishing exon-exon junctions, or removing the transcriptional termination site. Here, we describe the methodology to perform CRISPR/Cas9 KO of lncRNAs in vitro using electroporation as the method of transfection of presynthesized single guide RNAs (sgRNAs) and Cas9 enzyme.

Surface-enhanced Raman scattering (SERS) is emerging as a novel strategy for biofluid analysis. In this review, we delineate four experimental SERS protocols that are frequently used for the profiling of biofluids: 1) liquid SERS for the detection of purine metabolites; 2) iodide-modified liquid SERS for the detection of proteins; 3) dried SERS for the detection of both purine metabolites and proteins; 4) resonant Raman for the detection of carotenoids. To explain the selectivity of each experimental SERS protocol, we introduce a heuristic model for the chemisorption of analytes mediated by adsorbed ions (adions) onto the SERS substrate. Next, we show that the promising results of SERS liquid biopsy stem from the fact that the concentration levels of purine metabolites, proteins and carotenoids are informative of the cellular turnover rate, inflammation, and oxidative stress, respectively. These processes are perturbed in virtually every disease, from cancer to autoimmune maladies. Finally, we review recent SERS liquid biopsy studies and discuss future steps that are required for translating SERS in the clinical setting.

The mechanisms that induce immunodeficiency after splenectomy remain unknown. The aim of this study was to measure the cytokine releasing capacity of the whole blood as an expression of the innate immunity after total (TS) and subtotal/partial splenectomy (S/PS) in order to assess the impact of splenectomy on the individual cytokine reactivity.We prospectively collected blood before (D0) and at multiple time points after splenectomy (7 days - D7, 30 days - D30, 90 days - D90, 180 days - D180, and 360 days - D360) and measured the cytokines releasing capacity of IL-6, TNF-alpha and IL-10 from whole blood under LPS stimulation which we normalized to the monocytes number.When analyzing all splenectomies at D0, D7 and D30, normalized ΔTNF-alpha significantly dropped after splenectomy (Splenectomy induces a decrease of the pro-inflammatory cytokine TNF-alpha and if splenic parenchyma is spared and the surgery is performed MI, this change is hindered.

In this study we aimed to develop nomogram models for predicting the overall survival (OS) and cancer-specific survival (CSS) of patients with metaplastic breast cancer (MBC). Data of patients diagnosed with MBC from 1973 to 2015 were collected from the Surveillance, Epidemiology, and End Results (SEER) database. Univariate and multivariate Cox analyses were performed to identify independent prognostic factors for OS and CSS of MBC patients. The obtained prognostic variables were combined to construct nomogram models for predicting OS and CSS in patients with MBC. Model performance was evaluated using concordance index (C-index) and calibration plots. Data from 1125 patients were collected and divided into a training (750) and a validation (375) cohort. The multivariate Cox model identified age, TNM stage, tumor size, and radiotherapy as independent covariates associated with OS and CSS. The nomogram constructed based on these covariates demonstrated excellent accuracy in estimating 3-, and 5-year OS and CSS, with a C-index of 0.769 (95% CI, 0.731-0.808) for OS and 0.761 (95% CI, 0.713-0.809) for CSS in the training cohort. In the validation cohort, the nomogram-predicted C-index was 0.738 (95%CI, 0.676-0.800) for OS and 0.747 (95%CI, 0.667-0.827) for CSS. All calibration curves exhibited good consistency between predicted and actual survival. The nomogram models established in this study may enhance the accuracy of prognosis prediction and therefore may improve individualized assessment of survival risks and enable constructive therapeutic suggestions.

Simple Summary Due to the rising burden of pancreatic cancer and poor outcomes, a precise, post-operative cancer staging for further and individualized therapy is needed. In the latest cancer classification system, the lymph node invasion mechanism is not addressed. Due to different outcomes regarding the lymph node invasion, we suggest a rethinking of the current system. Abstract Mechanisms of lymph node invasion seem to play a prognostic role in pancreatic ductal adenocarcinoma (PDAC) after resection. However, the 8th edition of the TNM classification of the American Joint Committee on Cancer (AJCC) does not consider this. The aim of this study was to analyse the prognostic role of different mechanisms of lymph node invasion on PDAC. One hundred and twenty-two patients with resected PDAC were examined. We distinguished three groups: direct (per continuitatem, Nc) from the main tumour, metastasis (Nm) without any contact to the main tumour, and a mixed mechanism (Ncm). Afterwards, the prognostic power of the different groups was analysed concerning overall survival (OS). In total, 20 patients displayed direct lymph node invasion (Nc = 16.4%), 44 were classed as Nm (36.1%), and 21 were classed as Ncm (17.2%). The difference in OS was not statistically significant between N0 (no lymph node metastasis, n = 37) and Nc (p = 0.134), while Nm had worse OS than N0 (p < 0.001). Direct invasion alone had no statistically significant effect on OS (p = 0.885). Redefining the N0 stage by including Nc patients showed a more precise OS prediction among N stages (p = 0.001 vs. p = 0.002). Nc was more similar to N0 than to Nm; hence, we suggest a rethinking of TNM classification based on the mechanisms of lymph node metastases in PDAC. Overall, this novel classification is more precise.

Background Metaplastic breast cancer (MBC) is a rare type of breast cancer with an increasing incidence, we aim to develop clinical nomograms to predict the overall survival and cancer-specific survival for patients with MBC.MethodsPatients data were collected from the SEER database between 1973 and 2015. All included patients were randomly assigned into the training and validation sets. Univariate and multivariate Cox analysis were performed to identify independent prognostic factors of MBC. These essential prognostic variables were combined to construct nomogram models to predict overall survival (OS) and cancer-specific survival (CSS) in patients with MBC. Model performance was evaluated by concordance index (C-index) and calibration plots.ResultsA total of 1129 patients were collected and divided into the training (753) and validation (376) groups. The multivariate Cox model identified age, stage_ajcc, T stage, chemotherapy and radiotherapy as independent covariates associated with OS, while age, race, stage_ajcc, T stage, and radiotherapy were independent prognostic factors of CSS. The nomogram constructed based on these covariates demonstrated excellent accuracy in estimating 3-, and 5-year OS and CSS, with a C-index of 0.744 (95% CI, 0.701-0.787) for OS and 0.746 (95% CI, 0.695-0.797) for CSS in the training cohort. In the validation cohort, the nomogram-predicted C-index was in OS and 0.818 for OS (95% CI, 0.775-0.861) and 0.800 (95% CI, 0.747-0.853) for CSS. All calibration curves exhibited good consistency between predicted and actual survival.ConclusionsThese nomogram models established in this study can help to enhance the accuracy of prognostic prediction, which may thereby improve individualized assessment of survival risks and facilitate to provide constructive therapeutic suggestions.

Background and purpose Despite definitive chemoradiotherapy (CRT) being a recommended therapeutic method for patients with T4b esophageal squamous cell carcinoma (ESCC), treatment response and complications remain unclear. Esophageal fistula is a severe CRT-related complication when treating locally advanced ESCC, but data on risk factors that lead to esophageal fistula formation are limited. The aim of this analysis is to characterize the outcomes of T4b ESCC treated by CRT and investigate the risk factors of esophageal fistula. Materials and methods We retrospectively analyzed 136 patients with clinically unresectable T4b ESCC who were treated with CRT. Response, survival, and complication rates, particularly the rate of esophageal fistula and its associated risk factors were analyzed. Results The median progression-free survival and overall survival (OS) of all patients were 7.9 (95% confidence interval [CI]: 6.1–9.7) and 12.2 months (95% [CI]: 8.9–15.4), respectively. The Kaplan–Meier curves showed that the 3- and 5-year OS rates were 29.9% and 20.2%, respectively. The incidence rate of esophageal fistulas was 30.1%. The median OS for patients with esophageal fistula was only 6.9 (95%[CI] = 6.0–7.8) months. The risk for developing esophageal fistulas was significantly high for ulcerative-type tumors (odds ratio [OR] = 3.202; 95%[CI] = 1.036–7.850, P = 0.011) and for those invading the bronchus/trachea (OR = 3.378; 95%[CI] = 1.223–9.332, P = 0.048). Conclusion We demonstrated that CRT for T4b ESCC patients has a curative potential, despite a high incidence of esophageal fistula, which was the main cause of treatment failure. The higher risk for fistula formation were tumors with ulceration or bronchus/trachea invasion.

Non-coding RNAs (ncRNAs) are essential players in many cellular processes, from normal development to oncogenic transformation. Initially, ncRNAs were defined as transcripts that lacked an open reading frame (ORF). However, multiple lines of evidence suggest that certain ncRNAs encode small peptides of less than 100 amino acids. The sequences encoding these peptides are known as small open reading frames (smORFs), many initiating with the traditional AUG start codon but terminating with atypical stop codons, suggesting a different biogenesis. The ncRNA-encoded peptides (ncPEPs) are gradually becoming appreciated as a new class of functional molecules that contribute to diverse cellular processes, and are deregulated in different diseases contributing to pathogenesis. As multiple publications have identified unique ncPEPs, we appreciated the need for assembling a new web resource that could gather information about these functional ncPEPs. We developed FuncPEP, a new database of functional ncRNA encoded peptides, containing all experimentally validated and functionally characterized ncPEPs. Currently, FuncPEP includes a comprehensive annotation of 112 functional ncPEPs and specific details regarding the ncRNA transcripts that encode these peptides. We believe that FuncPEP will serve as a platform for further deciphering the biologic significance and medical use of ncPEPs. The link for FuncPEP database can be found at the end of the Introduction Section.

The generation and organization of the invasion front shape of neoplasms is an intriguing problem. The intimate mechanism is not yet understood, but the prevailing theory is that it represents an example of morphogenesis. Morphogenesis requires the presence of specific molecules, known as morphogens (activators and inhibitors), which can diffuse and elicit dose-dependent responses in their target cells. Due to their ability to modulate most of the coding transcriptome, their well-established role in embryogenesis, and their capacity to rapidly move between neighboring and distant cells, we propose microRNAs as inhibitors that could shape the cancer invasion front. In order to explain the genesis of the tumor border, we use Alan Turing&rsquo, s reaction diffusion model, refined by Meinhardt and Gierer. This assumes the existence of an activator called a, and an inhibitor called h, which we hypothesize could be a freely moving microRNA. We used the fractal dimension as a measure of tumor border irregularity. We observed that the change in fractal dimension associates with variations in the diffusion coefficient of the activator (Da) or the inhibitor (Dh). We determined that the fractal dimension remains constant (i.e., the irregularity of the tumor border does not change) across a Dh interval, which becomes narrower as Da rises. We therefore conclude that a change in fractal dimension occurs when the balance between Da and Dh is disrupted. Biologically, this could be explained by a faulty distribution of the inhibitor caused by an abnormal density of the intercellular connection network. From a translational perspective, if experimentally confirmed, our observations can be used for a better diagnosis of cancer aggressiveness.

Bladder cancer (BC) is the tenth most frequent cancer worldwide. Due to the need for recurrent cystoscopies and the lack of non-invasive biomarkers, BC is associated with a high management burden. In this respect, small non-coding RNAs (sncRNAs) have been investigated in urine as possible biomarkers for BC, but in plasma their potential has not yet been defined. The expression levels of sncRNAs contained in plasma extracellular vesicles (EVs) from 47 men with BC and 46 healthy controls were assessed by next-generation sequencing. The sncRNA profiles were compared with urinary profiles from the same subjects. miR-4508 resulted downregulated in plasma EVs of muscle-invasive BC patients, compared to controls (adj-p = 0.04). In World Health Organization (WHO) grade 3 (G3) BC, miR-126-3p was upregulated both in plasma EVs and urine, when compared to controls (for both, adj-p &lt, 0.05). Interestingly, two sncRNAs were associated with the risk class: miR-4508 with a downward trend going from controls to high risk BC, and piR-hsa-5936 with an upward trend (adj-p = 0.04 and adj-p = 0.05, respectively). Additionally, BC cases with low expression of miR-185-5p and miR-106a-5p or high expression of miR-10b-5p showed shorter survival (adj-p = 0.0013, adj-p = 0.039 and adj-p = 0.047, respectively). SncRNAs from plasma EVs could be diagnostic biomarkers for BC, especially in advanced grade.

Hereditary spherocytosis (HS) is a common inherited disease affecting the erythrocyte membrane. Total splenectomy (TS) is effective in reducing hemolysis and decreasing the need of transfusions, but total removal of the spleen represents a potential risk factor for infectious and non-infectious complications. On the other hand, subtotal splenectomy (STS) could be an alternative therapy for HS. The aim of this study is to establish which surgical approach has the best outcome in HS. All patients (n = 63) receiving splenectomy for HS between 2002 and 2016 from one institution were retrospectively reviewed. Hemoglobin and reticulocytes levels during preoperative and postoperative follow-up periods were compared. Additionally, a meta-analysis was performed analyzing data regarding hemoglobin and reticulocytes levels from several available studies. At 1-year follow-up, our clinical data showed that mean hemoglobin levels increased after TS from (mean ± SD) 9.77 ± 1.82 to 11.88 ± 2.08 g/dl, while after STS from 8.98 ± 1.66 to 11.87 ± 1.38 g/dl. At 3-year and 5-year follow-up after TS, we observed an increase from 9.77 ± 1.82 to 13.59 ± 2.03 and 13.46 ± 1.64 g/dl, respectively. At 3-year and 5-year follow-up after STS in our cohort, we observed an increase from 8.98 ± 1.66 to 13.21 ± 1.95 and 13.68 ± 1.65 g/dl, respectively. The meta-analysis (for a follow-up period of 1 year) showed that the hemoglobin levels increased with 2.61 g/dl (95% CI 2.15–3.08 g/dl; p

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription–quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options The work in GAC’s laboratory was partly supported by NIH/National Center for Advancing Translational Sciences grant UH3TR00943-01, the NIH/National Cancer Institute (NCI) grant 1 R01 CA182905-01, U54 grant UPR/MDACC Partnership for Excellence in Cancer Research 2016 Pilot Project, Team Department of Defense grant CA160445P1, a Ladies Leukemia League grant, a Chronic Lymphocytic Leukemia Moonshot Flagship project, a Sister Institution Network Fund 2017 grant, and the Estate of C. G. Johnson, Jr. EFM was supported in part by award number P50 CA140388 from the NCI and by the NIH Clinical Research Loan Repayment Program. AMS was supported by Fundação para a Ciência e a Tecnologia through fellowship SFRH/BD/85968/2012. ZJZ’s work was supported by grants from the MPN Foundation and the Oklahoma Center for the Advancement of Science and Technology. DG, MPD, and IBN were supported in part by a Programul Opera¿ional Competitivitate grant nr35/01.09.2016, ID 37_796, titled “Clinical and economical impact of personalized targeted anti-mi-croRNA therapies in reconverting lung cancer chemoresistance” (CANTEMIR). NSDA was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, BEPE 2016/09349-4.

ObjectiveTo investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target.DesignFLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases.ResultsFLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis.ConclusionsBased on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.