Integrative analysis in head and neck cancer reveals distinct role of miRNome and methylome as tumour epigenetic drivers.

Head and neck cancer is the sixth most common malignancy worldwide, with the relatively low 5-year survival rate, mainly because it is diagnosed at a late stage. Infection with HPV is a well known aetiology, which affects the nature of these cancers and patients' survival. Besides, it is considered that the main driving force for this type of cancer could be epigenetics. In this study we aimed to find potential epigenetic biomarkers, by integrating miRNome, methylome, and transcriptome analyses. From the fresh head and neck cancer tissue samples, we chose a group for miRNome, methylome and transcriptome profiling, in comparison to adequate control samples. Bioinformatics analyses are performed in R v4.2.2. Count normalisation and group differential expression for mRNA and the previously obtained miRNA count data was performed with DESeq2 v1.36. Gene set enrichment analysis was performed and visualised using gProfiler2 v0.2.1 Identification of miRNA targets was performed by querying in miRTarBase using multiMiR v1.18.0. Annotation of CpG sites merging into islands was obtained from RnBeads.hg19 v1.28.0. package. For the integrative analysis we performed kmeans clustering using stats v4.2.2 package, using 8-12 clusters and nstart 100. We found that transcriptome analysis divides samples into cancers and controls clusters, with no relation to HPV status or cancer anatomical location. Differentially expressed genes (n = 2781) were predominantly associated with signalling pathways of tumour progression. We identified a cluster of genes under the control of the transcription factor E2F that are significantly underexpressed in cancer tissue, as well as T cell immunity genes and genes related to regulation of transcription. Among overexpressed genes in tumours we found those that belong to cell cycle regulation and vasculature. A small number of genes were found significantly differentially expressed in HPV-positive versus HPV-negative tumours (for example NEFH, ZFR2, TAF7L, ZNF541, and TYMS). In this comprehensive study on an overlapping set of samples where the integration of miRNome, methylome and transcriptome analysis were performed for head and neck cancer, we demonstrated that the majority of genes were associated exclusively with miRNome or methylome and, to a lesser extent, under the control of both epigenetic mechanisms.

NGR-bearing human adenovirus type 5 infects cells in flotillin- or caveolin-mediated manner depending on the NGR insertion site.

Human adenoviruses represent attractive candidates for the design of cancer gene therapy vectors. Modification of adenovirus tropism by incorporating a targeting ligand into the adenovirus capsid proteins allows retargeting of adenovirus towards the cells of interest. Human adenovirus type 5 (HAdV-C5) bearing NGR containing peptide (CNGRCVSGCAGRC) inserted into the fiber (AdFNGR) or the hexon (AdHNGR) protein demonstrated an increased transduction of endothelial cells showing expression of aminopeptidase N, also known as CD13, and αvß3 integrin both present on tumor vasculature, indicating that NGR-bearing adenoviruses could be used as tools for anti-angiogenic cancer therapy. Here we investigated how AdFNGR and AdHNGR infect cells lacking HAdV-C5 primary receptor, coxsackie and adenovirus receptor, and we showed that both AFNGR and AdHNGR enter cells by dynamin- and lipid raft-mediated endocytosis, while clathrin is not required for endocytosis of these viruses. We present evidence that productive infection of both AdFNGR and AdHNGR involves lipid rafts, with usage of flotillin-mediated cell entry for AdFNGR and limited role of caveolin in AdHNGR transduction efficiency. Lipid rafts play important role in angiogenesis and process of metastasis. Therefore, the ability of AdFNGR and AdHNGR to use lipid raft-dependent endocytosis, involving respectively flotillin- or caveolin-mediated pathway, could give them an advantage in targeting tumor cells lacking HAdV-C5 primary receptor.

Novel Tripodal Polyamine Tris-Pyrene: DNA/RNA Binding and Photodynamic Antiproliferative Activity.

A novel tri-pyrene polyamine (TAL3PYR) bearing net five positive charges at biorelevant conditions revealed strong intramolecular interactions in aqueous medium between pyrenes, characterised by pronounced excimer fluorescence. A novel compound revealed strong binding to ds-DNA and ds-RNA, along with pronounced thermal stabilisation of DNA/RNA and extensive changes in DNA/RNA structure, as evidenced by circular dichroism. New dye caused pronounced ds-DNA or ds-RNA condensation, which was attributed to a combination of electrostatic interactions between 5+ charge of dye and negatively charged polynucleotide backbone, accompanied by aromatic and hydrophobic interactions of pyrenes within polynucleotide grooves. New dye also showed intriguing antiproliferative activity, strongly enhanced upon photo-induced activation of pyrenes, and is thus a promising lead compound for theranostic applications on ds-RNA or ds-DNA targets, applicable as a new strategy in cancer and gene therapy.

Methylation of Immune Gene Promoters in Oral and Oropharyngeal Cancer.

The proportion of oral and oropharyngeal squamous cell carcinoma (OOSCC) that can be attributed to human papillomavirus (HPV) infection is growing nowadays. A potential factor indicating the occurrence of HPV-positive OSCC is a change in the degree of methylation of gene promoters that play a key role in the immune response. In this study, we investigated the difference in the methylation of EDARADD, GBP4, HAVCR2, HLA DPB1, IL12RB1, MARCO, and SIGLEC12 gene promoters in samples of healthy oral mucosa versus samples of oral and oropharyngeal cancer. The presence of HPV infection in samples was examined earlier. To determine the difference in methylation of those gene promotors, isolated and bisulfite-modified DNA was analysed by the methylation-specific PCR method. The investigated gene promoters were found to be more hypomethylated in the oral and oropharyngeal cancer samples in comparison to normal tissue. The proportion of unmethylated gene promoters was similar in HPV-positive and HPV-negative cancers, although the data should be confirmed on a larger set of samples. To conclude, in samples of healthy oral mucosa, the investigated gene promoters were found to be methylated in a high percentage (73.3% to 100%), while in oral and oropharyngeal cancer samples, they were methylated in a low percentage (11.1% to 37%), regardless of HPV infection.

Head and Neck Cancer Patients' Survival According to HPV Status, miRNA Profiling, and Tumour Features-A Cohort Study.

Head and neck cancers (HNC) are a heterogeneous group of tumours mainly associated with tobacco and alcohol use and human papillomavirus (HPV). Over 90% of all HNC are squamous cell carcinomas (HNSCC). Sample material from patients diagnosed with primary HNSCC (n = 76) treated with surgery as primary treatment at a single centre were assessed for HPV genotype, miR-9-5p, miR-21-3p, miR-29a-3p and miR-100-5p expression levels. Clinical and pathological data were collected from medical records. Patients were enrolled between 2015 and 2019 and followed-up until November 2022. Overall survival, disease-specific survival and disease-free survival were assessed and correlated with clinical, pathological, and molecular data. Kaplan-Meier and Cox proportional hazard regression was used to assess different risk factors. In the study, male gender, HPV-negative HNSCC (76.3%) mostly located in the oral region (78.9%) predominated. Most patients had stage IV cancer (47.4%), and the overall survival rate was 50%. HPV was found not to affect survival, indicating that in this population, classic risk factors predominate. The presence of both perineural and angioinvasion was strongly associated with survival in all analyses. Of all miRNAs assessed, only upregulation of miR-21 was consistently shown to be an independent predictor of poor prognosis and may thus serve as a prognostic biomarker in HNSCC.

Para-N-Methylpyridinium Pyrenes: Impact of Positive Charge on ds-DNA/RNA and Protein Recognition, Photo-Induced Bioactivity, and Intracellular Localisation.

The 2- and 2,7- substituted para-N-methylpyridinium pyrene cations show high-affinity intercalation into ds-DNAs, whereas their non-methylated analogues interacted with ds-DNA/RNA only in the protonated form (at pH 5), but not at physiological conditions (pH 7). The fluorescence from non-methylated analogues was strongly dependent on the protonation of the pyridines; consequently, they act as fluorescence ratiometric probes for simultaneous detection of both ds-DNA and BSA at pH 5, relying on the ratio between intensities at 420 nm (BSA specific) and 520 nm (DNA specific), whereby exclusively ds-DNA sensing could be switched-off by adjustment to pH 7. Only methylated, permanently charged pyrenes show photoinduced cleavage of circular DNA, attributed to pyrene-mediated irradiation-induced production of singlet oxygen. Consequently, the moderate toxicity of these cations against human cell lines is strongly increased upon irradiation. Detailed studies revealed increased total ROS production in cells treated by the compounds studied, accompanied by cell swelling and augmentation of cellular complexity. The most photo-active 2-para-N-methylpyridinium pyrene showed significant localization at mitochondria, its photo-bioactivity likely due to mitochondrial DNA damage. Other derivatives were mostly non-selectively distributed between various cytoplasmic organelles, thus being less photoactive.

Impact of the Histidine-Triazole and Tryptophan-Pyrene Exchange in the WHW Peptide: Cu(II) Binding, DNA/RNA Interactions and Bioactivity.

In three novel peptidoids based on the tryptophan-histidine-tryptophan (WHW) peptide, the central histidine was replaced by Ala-(triazole), and two derivatives also had one tryptophan replaced with pyrene-alkyls of different lengths and flexibility. Pyrene analogues show strong fluorescence at 480-500 nm, attributed to intramolecular exciplex formation with tryptophan. All three peptidoids bind Cu2+ cation in water with strong affinity, with Trp- Ala-(triazole)-Trp binding comparably to the parent WHW, and the pyrene analogues even stronger, demonstrating that replacement of histidine with triazole in peptides does not hamper Cu2+ coordination. The studied peptidoids strongly bind to ds-DNA and ds-RNA, whereby their complexes with Cu2+ exhibit distinctively different interactions in comparison to metal-free analogues, particularly in the stabilization of ds-DNA against thermal denaturation. The pyrene peptidoids efficiently enter living cells with no apparent cytotoxic effect, whereby their red-shifted emission compared to the parent pyrene allows intracellular confocal microscopy imaging, showing accumulation in cytoplasmic organelles. However, irradiation with 350 nm light resulted in evident antiproliferative effect on cells treated with micromolar concentrations of the pyrene analogues, presumably attributed to pyrene-induced production of singlet oxygen and consecutive cellular damage.

Diethynylarene-linked bis(triarylborane)cations as theranostic agents for tumor cell and virus-targeted photodynamic therapy.

We recently reported diethynylarene-linked bis(triarylborane) tetracations which show remarkable fluorimetric and Raman-SERS sensing of DNA/RNA. In the current study, we show that they exhibit promising photodynamic therapy (PDT)-based biological activity on human cell lines and adenovirus type 5 (HAdV5), acting as theranostic agents. All compounds efficiently enter living cells showing negligible antiproliferative activity. Bis-thiophene- and anthracene- analogues bind non-covalently to HAdV5 virus with high affinity, the anthracene-analogue itself causing a moderate antiviral effect, i.e., decreased ability of the virus to infect human cells. Irradiation of bis-thiophene- and anthracene- analogues with visible light (400-700 nm) caused a very rapid (within 1 min) and strong increase in cytotoxicity, as well as an order of magnitude increase in antiviral activity, attributed to the formation of reactive oxygen species (ROS). Photochemical studies of the compounds revealed that, upon irradiation, they produce singlet oxygen, which correlates with the observed light-induced bioactivity.

Methyl Viologens of Bis-(4'-Pyridylethynyl)Arenes - Structures, Photophysical and Electrochemical Studies, and their Potential Application in Biology.

A series of bis-(4'-pyridylethynyl)arenes (arene=benzene, tetrafluorobenzene, and anthracene) were synthesized and their bis-N-methylpyridinium compounds were investigated as a class of π-extended methyl viologens. Their structures were determined by single crystal X-ray diffraction, and their photophysical and electrochemical properties (cyclic voltammetry), as well as their interactions with DNA/RNA were investigated. The dications showed bathochromic shifts in emission compared to the neutral compounds. The neutral compounds showed very small Stokes shifts, which are a little larger for the dications. All of the compounds showed very short fluorescence lifetimes (<4 ns). The neutral compound with an anthracene core has a quantum yield of almost unity. With stronger acceptors, the analogous bis-N-methylpyridinium compound showed a larger two-photon absorption cross-section than its neutral precursor. All of the dicationic compounds interact with DNA/RNA; while the compounds with benzene and tetrafluorobenzene cores bind in the grooves, the one with an anthracene core intercalates as a consequence of its large, condensed aromatic linker moiety, and it aggregates within the polynucleotide when in excess over DNA/RNA. Moreover, all cationic compounds showed highly specific CD spectra upon binding to ds-DNA/RNA, attributed to the rare case of forcing the planar, achiral molecule into a chiral rotamer, and negligible toxicity toward human cell lines at ≤10 µM concentrations. The anthracene-analogue exhibited intracellular accumulation within lysosomes, preventing its interaction with cellular DNA/RNA. However, cytotoxicity was evident at 1 µM concentration upon exposure to light, due to singlet oxygen generation within cells. These multi-faceted features, in combination with its two-photon absorption properties, suggest it to be a promising lead compound for development of novel light-activated theranostic agents.

Human Adenovirus Type 26 Induced IL-6 Gene Expression in an αvß3 Integrin- and NF-κB-Dependent Manner.

The low seroprevalent human adenovirus type 26 (HAdV26)-based vaccine vector was the first adenovirus-based vector to receive marketing authorization from European Commission. HAdV26-based vaccine vectors induce durable humoral and cellular immune responses and, as such, represent a highly valuable tool for fighting infectious diseases. Despite well-described immunogenicity in vivo, the basic biology of HAdV26 still needs some refinement. The aim of this study was to determine the pro-inflammatory cytokine profile of epithelial cells infected with HAdV26 and then investigate the underlying molecular mechanism. The expression of studied genes and proteins was assessed by quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Confocal microscopy was used to visualize HAdV26 cell uptake. We found that HAdV26 infection in human epithelial cells triggers the expression of pro-inflammatory cytokines and chemokines, namely IL-6, IL-8, IL-1ß, and TNF-α, with the most pronounced difference shown for IL-6. We investigated the underlying molecular mechanism and observed that HAdV26-induced IL-6 gene expression is αvß3 integrin dependent and NF-κB mediated. Our findings provide new data regarding pro-inflammatory cytokine and chemokine expression in HAdV26-infected epithelial cells, as well as details concerning HAdV26-induced host signaling pathways. Information obtained within this research increases our current knowledge of HAdV26 basic biology and, as such, can contribute to further development of HAdV26-based vaccine vectors.

The Revolving Door of Adenovirus Cell Entry: Not All Pathways Are Equal.

Adenoviruses represent exceptional candidates for wide-ranging therapeutic applications, from vectors for gene therapy to oncolytics for cancer treatments. The first ever commercial gene therapy medicine was based on a recombinant adenovirus vector, while most recently, adenoviral vectors have proven critical as vaccine platforms in effectively controlling the global coronavirus pandemic. Here, we discuss factors involved in adenovirus cell binding, entry, and trafficking; how they influence efficiency of adenovirus-based vectors; and how they can be manipulated to enhance efficacy of genetically modified adenoviral variants. We focus particularly on endocytosis and how different adenovirus serotypes employ different endocytic pathways to gain cell entry, and thus, have different intracellular trafficking pathways that subsequently trigger different host antiviral responses. In the context of gene therapy, the final goal of the adenovirus vector is to efficiently deliver therapeutic transgenes into the target cell nucleus, thus allowing its functional expression. Aberrant or inefficient endocytosis can impede this goal, therefore, it should be considered when designing and constructing adenovirus-based vectors.

Fluorimetric and CD Recognition between Various ds-DNA/RNA Depends on a Cyanine Connectivity in Cyanine-guanidiniocarbonyl-pyrrole Conjugate.

Two novel isosteric conjugates of guanidiniocarbonyl-pyrrole and 6-bromo-TO (thiazole orange) were prepared, differing only in linker connectivity to cyanine (benzothiazole nitrogen vs. quinoline nitrogen). The quinoline analog was significantly more susceptible to aggregation in an aqueous medium, which resulted in induced circular dichroism (ICD; λ = 450-550 nm) recognition between A-T(U) and G-C basepair containing polynucleotides. The benzothiazole-isostere showed pronounced (four-fold) fluorimetric selectivity toward ds-RNA in comparison to any ds-DNA, at variance to its quinoline-analogue fluorescence being weakly selective to GC-DNA. Preliminary screening on human tumor and normal lung cell lines showed that both dyes very efficiently enter living cells and accumulate in mitochondria, causing moderate cytotoxic effects, and thus could be considered as lead compounds toward novel theragnostic mitochondrial dyes.

DNA Methylome Distinguishes Head and Neck Cancer from Potentially Malignant Oral Lesions and Healthy Oral Mucosa.

There is a strong need to find new, good biomarkers of head and neck squamous cell carcinoma (HNSCC) because of the bad prognoses and high mortality rates. The aim of this study was to identify the potential biomarkers in HNSCC that have differences in their DNA methylome and potentially premalignant oral lesions, in comparison to healthy oral mucosa. In this study, 32 oral samples were tested: nine healthy oral mucosae, 13 HNSCC, and 10 oral lesions for DNA methylation by the Infinium MethylationEPIC BeadChip. Our findings showed that a panel of genes significantly hypermethylated in their promoters or specific sites in HNSCC samples in comparison to healthy oral samples, which are mainly oncogenes, receptor, and transcription factor genes, or genes included in cell cycle, transformation, apoptosis, and autophagy. A group of hypomethylated genes in HNSCC, in comparison to healthy oral mucosa, are mainly involved in the host immune response and transcriptional regulation. The results also showed significant differences in gene methylation between HNSCC and potentially premalignant oral lesions, as well as differently methylated genes that discriminate between oral lesions and healthy mucosa. The given methylation panels point to novel potential biomarkers for early diagnostics of HNSCC, as well as potentially premalignant oral lesions.

In-vitro toxicity of molybdenum trioxide nanoparticles on human keratinocytes.

Molybdenum trioxide (MoO3) nanoparticles (NPs) embedded in polymer films have been proposed as a cheap way of producing antibacterial coatings on external surfaces. Recently, we synthesized MoO3 nanowires in a unique shape and degree of anisotropy, which enables their fast water dissolution and quick antimicrobial reaction. Potential human health risks following the exposure to MoO3 NPs however need to be assessed prior their wide use. We therefore, investigated the biological effect of these newly synthesized MoO3 NPs on the human keratinocyte cell line HaCaT, used here as a model for the human skin. Exposure of HaCaT cells to 1 mg/mL MoO3 NPs concentration for 1 h showed no effect on cell survival, had no influence on reactive oxygen species production, expression of proteins involved in antioxidant defense, secretion of pro-inflammatory cytokines, nor induced DNA damage. Interestingly however, ERK and p38 MAP kinases were activated, and upon longer time exposure, induced a moderate release of the pro-inflammatory cytokine interleukin 6, increased DNA damage and increased the level of caspase independent cell death. Our study indicates that exposing HaCaT cells to antibacterial MoO3 NPs water-based solution in durations less than 1 h exhibits no cytotoxicity, but rather triggers cell signalling involved in cell survival and inflammation; which should be taken into consideration when evaluating MoO3 NPs for medical applications.

HPV-driven oropharyngeal squamous cell cancer in Croatia - Demography and survival.

OBJECTIVES: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Main HNSCC risk factors are tobacco, alcohol, and high-risk human papillomavirus (HPV). HPV+ oropharyngeal squamous cell cancer (OPSCC) usually have different etiology, increasing incidence and often show an improved survival when compared to HPV-negative cases. The objective of the current study was to retrospectively examine the influence of HPV on the survival of OPSCC patients in a non-Western population setting. MATERIALS AND METHODS: We determined the presence of HPV DNA and/or RNA in 99 formalin-fixed paraffin embedded (FFPE) tissue samples of OPSCC patients treated between 2002 and 2015. Patients were compared based on laboratory, demographic, clinical, life style characteristics and survival. RESULTS: HPV RNA was found in 29.3% cases. However, groups based on HPV data (either RNA, DNA or retrospectively collected p16 staining) did not show significant differences. Overall, 5-year survival was 30% with minimal influence of the HPV positivity. CONCLUSIONS: The HPV influence on survival of OPSCC patients is not identical between populations probably due to other factors overshadowing the HPV effect. This should be taken into account when treatment or policy decisions are made in each particular setting.

Genome-wide miRNA profiling reinforces the importance of miR-9 in human papillomavirus associated oral and oropharyngeal head and neck cancer.

Head and neck cancer is the sixth most common malignancy worldwide, predominantly developing from squamous cell epithelia (HNSCC). The main HNSCC risk factors are tobacco, excessive alcohol use, and the presence of human papillomavirus (HPV). HPV positive (+) cancers are etiologically different from other HNSCC and often show better prognosis. The current knowledge regarding HNSCC miRNA profiles is still incomplete especially in the context of HPV+ cancer. Thus, we analyzed 61 freshly collected primary oral (OSCC) and oropharyngeal (OPSCC) SCC samples. HPV DNA and RNA was found in 21% cases. The Illumina whole-genome small-RNA profiling by next-generation sequencing was done on 22 samples and revealed 7 specific miRNAs to HPV+ OSCC, 77 to HPV+ OPSCC, and additional 3 shared with both; 51 miRNAs were specific to HPV- OPSCC, 62 to HPV- OSCC, and 31 shared with both. The results for 9 miRNAs (miR-9, -21, -29a, -100, -106b, -143 and -145) were assessed by reverse transcription-quantitative polymerase chain reaction on the whole study population. The data was additionally confirmed by reanalyzing publicly available miRNA sequencing Cancer Genome Atlas consortium (TCGA) HNSCC data. Cell signaling pathway analysis revealed differences between HPV+ and HPV- HNSCC. Our findings compared with literature data revealed extensive heterogeneity of miRNA deregulation with only several miRNAs consistently affected, and miR-9 being the most likely HPV related miRNA.

Cervical HPV type-specific pre-vaccination prevalence and age distribution in Croatia.

The main etiological factor of precancerous lesion and invasive cervical cancer are oncogenic human papillomaviruses types (HPVs). The objective of this study was to establish the distribution of the most common HPVs in different cervical lesions and cancer prior to the implementation of organized population-based cervical screening and HPV vaccination in Croatia. In this study, 4,432 cervical specimens, collected through a 16-year period, were tested for the presence of HPV-DNA by polymerase chain reaction (PCR) with three sets of broad-spectrum primers and type-specific primers for most common low-risk (LR) types (HPV-6, 11) and the most common high-risk (HR) types (HPV-16, 18, 31, 33, 45, 52, 58). Additional 35 archival formalin-fixed, paraffin embedded tissue of cervical cancer specimens were analyzed using LiPA25 assay. The highest age-specific HPV-prevalence was in the group 18-24 years, which decreased continuously with age (P<0.0001) regardless of the cytological diagnosis. The prevalence of HR-HPV types significantly increased (P<0.0001) with the severity of cervical lesions. HPV-16 was the most common type found with a prevalence (with or without another HPV-type) of 6.9% in normal cytology, 15.5% in atypical squamous cells of undetermined significance, 14.4% in low-grade squamous intraepithelial lesions, 33.3% in high-grade squamous intraepithelial lesions, and 60.9% in cervical cancer specimens (P<0.0001). This study provides comprehensive and extensive data on the distribution of the most common HPV types among Croatian women, which will enable to predict and to monitor the impact of HPV-vaccination and to design effective screening strategies in Croatia.

An accurate, simple prognostic model consisting of age, JAK2, CALR, and MPL mutation status for patients with primary myelofibrosis.

In most patients with primary myelofibrosis, one of three mutually exclusive somatic mutations is detected. In approximately 60% of patients, the Janus kinase 2 gene is mutated, in 20%, the calreticulin gene is mutated, and in 5%, the myeloproliferative leukemia virus gene is mutated. Although patients with mutated calreticulin or myeloproliferative leukemia genes have a favorable outcome, and those with none of these mutations have an unfavorable outcome, prognostication based on mutation status is challenging due to the heterogeneous survival of patients with mutated Janus kinase 2. To develop a prognostic model based on mutation status, we screened primary myelofibrosis patients seen at the MD Anderson Cancer Center, Houston, USA, between 2000 and 2013 for the presence of Janus kinase 2, calreticulin, and myeloproliferative leukemia mutations. Of 344 primary myelofibrosis patients, Janus kinase 2V617F was detected in 226 (66%), calreticulin mutation in 43 (12%), and myeloproliferative leukemia mutation in 16 (5%); 59 patients (17%) were triple-negatives. A 50% cut-off dichotomized Janus kinase 2-mutated patients into those with high Janus kinase 2V617F allele burden and favorable survival and those with low Janus kinase 2V617F allele burden and unfavorable survival. Patients with a favorable mutation status (high Janus kinase 2V617F allele burden/myeloproliferative leukemia/calreticulin mutation) and aged 65 years or under had a median survival of 126 months. Patients with one risk factor (low Janus kinase 2V617F allele burden/triple-negative or age >65 years) had an intermediate survival duration, and patients aged over 65 years with an adverse mutation status (low Janus kinase 2V617F allele burden or triple-negative) had a median survival of only 35 months. Our simple and easily applied age- and mutation status-based scoring system accurately predicted the survival of patients with primary myelofibrosis.

Role of neoplastic monocyte-derived fibrocytes in primary myelofibrosis.

Primary myelofibrosis (PMF) is a fatal neoplastic disease characterized by clonal myeloproliferation and progressive bone marrow (BM) fibrosis thought to be induced by mesenchymal stromal cells stimulated by overproduced growth factors. However, tissue fibrosis in other diseases is associated with monocyte-derived fibrocytes. Therefore, we sought to determine whether fibrocytes play a role in the induction of BM fibrosis in PMF. In this study, we show that BM from patients with PMF harbors an abundance of clonal, neoplastic collagen- and fibronectin-producing fibrocytes. Immunodeficient mice transplanted with myelofibrosis patients' BM cells developed a lethal myelofibrosis-like phenotype. Treatment of the xenograft mice with the fibrocyte inhibitor serum amyloid P (SAP; pentraxin-2) significantly prolonged survival and slowed the development of BM fibrosis. Collectively, our data suggest that neoplastic fibrocytes contribute to the induction of BM fibrosis in PMF, and inhibiting fibrocyte differentiation with SAP may interfere with this process.