Six loci--CALR, EPOR, JUNB, JUND, CEA, and PRKCG--were assigned to bovine chromosomes using PCR-based hybrid somatic cell analysis. The five genes other than CALR are comparative mapping anchor loci. This study, together with the previous assignment of three anchor loci--INSR, LDLR, APOE--and four other genes--AMH, GPI, RYR1, LHB--defines the conserved synteny relationship between human chromosome 19 and cattle chromosomes 7 and 18. Genes on HSA 19p13.3-13.2 are conserved in cattle chromosome 7, while those on HSA19-q13.1-13.4 are conserved in cattle chromosome 18. In contrast, homologous genes from HSA19 are located on four different mouse chromosomes, namely MMU10, MMU8, MMU9, and MMU7. This is further evidence that syntenic conservation between cattle and human generally exceeds that observed between human and mouse.

In this study, members of family 2 GPCRs, one of the largest families of receptors in vertebrates, were isolated and characterized in the genome of the Japanese pufferfish, Fugu rubripes, and compared with the orthologous genes in other vertebrates. Phylogenetic analysis carried out with all vertebrate family 2 GPCR members indicated that CALR/CGRPR and CRF are the most divergent receptor group within this family and that the remaining members appear to originate from a common ancestral gene precursor.

Radiation-induced DNA damage initiates a series of overlapping responses that include DNA damage recognition and repair, induction of cell cycle checkpoints, senescence and/or apoptosis. This study assessed the DNA damage response and whole genome expression profile in two mammalian cell lines (HEK and U87) in response to (5-{4-methylpiperazin-1-yl}-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl] benzimidazole) DMA and ionizing radiation. DMA has been shown to act as a potent radiation protector, yielding significant levels of protection, i.e., 20.9% in HEK cells and 21.2% in U87 cells. Our findings revealed treatment with DMA significantly reduced γ-H2AX, 53BP1 and Rad51 foci formation after irradiation. MAP kinase, WNT signaling and p53 pathways were found to be activated in DMA-treated cells. In addition, the DNA damage response genes, HSP70, HSPD1, PRDX1, PRX, CALR, NPM, UBC, and SET showed differential regulation in DMA, DMA + radiation and radiation-treated cells. The data suggest that DMA-influenced repertoire of repair proteins, which are an indispensable part of the cell, interplay with each other to reduce DNA damage and maintain the genomic integrity of the cell.

We have investigated the distribution and morphology of hippocampal interneurons that express the neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the rat. For this study, we combined in situ hybridization for the detection of NGF and NT-3 mRNAs and immunocytochemistry against the calcium-binding proteins parvalbumin (PARV), calretinin (CALR), and calbindin (CALB). Whereas the majority of PARV+ interneurons expressed NGF mRNA, only subsets of CALR- and CALB-immunoreactive interneurons (23% and 24%, respectively) displayed NGF hybridization. Most CALB/NGF+ cells were located in the stratum oriens/alveus of the CA3-CA1 regions, suggesting that they may include the population of CALB+, hippocamposeptal, nonpyramidal neurons. Most of the nonspiny CALR/NGF+ neurons were located within or in the vicinity of the pyramidal layer and had faint CALR immunostaining and stellate, thin dendrites. Regarding the spiny CALR-immunoreactive cells, we found that most of these neurons in the hilus were NGF+, whereas only 59% of displayed NGF hybridization in the stratum lucidum of the CA3 region. A small subset of PARV- and CALR-immunoreactive cells expressed NT-3 mRNA (16% and 13%, respectively). NT-3 message was not found in the large basket cells of the dentate gyrus, whereas the distribution and morphology of CALR/NT-3+ cells were similar to those of nonspiny CALR/NGF+ cells. In fact, double in situ hybridization analysis confirmed that most NT-3+ neurons also expressed NGF mRNA, indicating coexpression of both neurotrophins in subpopulations of PARV+ and CALR+ neurons. Moreover, the level of NGF mRNA expression was higher in PARV+ neurons than in CALR- and CALB-immunoreactive interneurons, whereas NT-3 message was expressed similarly in PARV+ and CALR+ neurons. The present findings show a differential expression of NGF and NT-3 mRNAs in subsets of hippocampal interneurons and suggest that the expression of these transcripts depends on factors intrinsic to particular cell types.

BACKGROUND

To investigate the prevalence of calreticulin (CALR) mutations in JAK2- and MPL-non-mutated patients with suspected myeloproliferative neoplasm (MPN) from a large MPN clinic and confirm a diagnosis of MPN.

METHODS

JAK2/MPL-non-mutated patients from the Belfast City Hospital (BCH) with either of the MPNs - ET or MF - and diagnosed between 1988 and 2014 were selected for CALR screen. All cases were validated according to the WHO 2008 classification for MPNs. Statistical analysis was performed with Minitab 16 Statistical Software package. Exon 9 of CALR was amplified by PCR using genomic DNA, and mutations were detected by fragment analysis.

RESULTS

Of the 62 JAK2/MPL-non-mutated MPN patients screened, 57 had ET and 5 had MF; 34 patients (53.1%) carried CALR mutations. Three of 5 MF patients were CALR positive. Thirty-one ET patients (54.3%) harboured CALR mutation, whereas 26 (45.7%) were classified as 'triple negatives'.

CONCLUSIONS

Detection of CALR mutations in a cohort of JAK2/MPL-non-mutated patients with suspected MPN confirmed the diagnosis of MPN in around 53% of cases. This is lower than initially reported, but similar to subsequent studies. However, a sizable cohort of patients remains lacking a specific molecular marker.

The multifunctional calreticulin (CALR) was identified as a major calcium-binding protein of the endoplasmic reticulum before being recognized as a chaperone in the same place. Only later were activities of calreticulin outside the endoplasmic reticulum described that for example affect cell proliferation and the innate immune system. In the present work we have investigated those extracellular activities of CALR from the cancerogenic human liver fluke Opisthorchis viverrini (OvCALR), as they might be important in host/parasite interaction. We first demonstrate that OvCALR is released from the parasite and stimulates a specific humoral immune response. Recombinant OvCALR is then shown to suppress proliferation of primary endothelial cells, their motility and sprouting activities. The potential of OvCALR to interfere with the complement system is established, firstly by demonstrating its direct binding to C1q and, secondly by suppression of hemolysis of sensitized red blood cells. These findings suggest that OvCALR is an important parasite antigen that could modulate diverse host functions and support parasite survival.

A systematic review and meta-analysis were carried out to compare the clinical features and outcomes in calreticulin (CALR)-mutated and JAK2V617F patients of essential thrombocythemia (ET). Compared with JAK2V617F ET patients, CALR-mutated ET was associated with a clear increase in male predominance [OR 1.71 (95 % CI 1.28-2.28), P < 0.001, I(2)) = 51.6] and a significant decrease in thrombosis events [OR 0.40 (95 % CI 0.32-0.50), P < 0.001, I(2) = 0]. No difference was observed in hemorrhagic events [OR 0.86 (95 % CI 0.52-1.42), P = 0.558, I(2) = 0] or splenomegaly [OR 0.8 (95 % CI 0.55-1.14), P = 0.217 I (2) = 42.9]. CALR-mutated ET did not show better overall survival (OS) [HR 1.03 (95 % CI 0.74-1.44) P = 0.854, I(2) = 47.6] but showed better thrombosis-free survival (TFS) [HR 0.62 (0.44-0.87), P = 0.005, I(2) = 0] than JAK2V617F ET. In conclusion, CALR-mutated ET and JAK2V617F ET may represent two different subgroups of essential thrombocythaemia with respect to clinical features and outcomes.

Activated JAK-STAT signaling is central to the pathogenesis of BCR-ABL-negative myeloproliferative neoplasms (MPNs) and occurs as a result of MPN phenotypic driver mutations in JAK2, CALR, or MPL The spectrum of concomitant somatic mutations in other genes has now largely been defined in MPNs. With the integration of targeted next-generation sequencing (NGS) panels into clinical practice, the clinical significance of concomitant mutations in MPNs has become clearer. In this review, we describe the consequences of concomitant mutations in the most frequently mutated classes of genes in MPNs: (1) DNA methylation pathways, (2) chromatin modification, (3) RNA splicing, (4) signaling pathways, (5) transcription factors, and (6) DNA damage response/stress signaling. The increased use of molecular genetics for early risk stratification of patients brings the possibility of earlier intervention to prevent disease progression in MPNs. However, additional studies are required to decipher underlying molecular mechanisms and effectively target them.

We focused on the expression of Calreticulin (CALR) in oral squamous cell carcinoma (OSCC) on the basis of proteomic differential display analysis data. We used QR-32 cells in this study which is a regressive murine fibrosarcoma cell clone; and QRsP-11, a progressive malignant tumor cell clone originated from QR-32. CALR is an endoplasmic reticulum luminal Ca2+-binding chaperone protein, which is thought to affect the tumor behavior of various malignancies. This study was aimed to determine the usefulness of CALR as a prognostic factor in patients with OSCC. We investigated the expression of CALR in tissue samples taken from 111 OSCC patients by immunohistochemistry, and we also analyzed the relationship between CALR expression and patients' clinicopathological characteristics as well as patient survival. Positive immunohistchemical staining of CALR was observed in the cancer cell cytoplasm. Among 111 patients, high expression of CALR was observed in 44 patients (39.6%), whereas low expression was observed in 67 patients (60.4%). Significant association was found between CALR expression and T classification (P=0.0027), N classification (P=0.0219), stage (P=0.0013), and patient outcome (P=0.0014). Log-rank test showed that, there is a significant difference (P<0.0001) in 5-year survival rates between patients showing CALR high-expression (59.1%) and CALR low-expression (86.6%). According to our Multivariate analysis, reduced term survival of patients was correlated to high levels of CALR expression (P<0.0001). Our findings suggest that elevated expression of CALR might play an important role in tumor progression in OSCC, and could be considered as a useful prognostic factor in OSCC patients.

Somatic mutations of calreticulin (CALR) have been observed in many cases of essential thrombocythemia (ET) or primary myelofibrosis that harbor non-mutated Janus kinase 2 (JAK2). CALR mainly localizes within the endoplasmic reticulum lumen, but a small fraction of the total CALR pool is distributed over the cell surface. Cell surface CALR is known to transduce prophagocytic "eat me" signals to macrophages and acts as one of the important regulators for macrophage engulfment. In this study, we attempted to clarify whether mutant CALR may affect the threshold for macrophage engulfment and play an integral role in the pathogenesis of CALR-mutated ET. First, we compared the surface expression levels of CALR on hematopoietic stem and progenitor cells (HSPCs) and mature blood cells in patients with myeloproliferative neoplasms and found that the surface expression of mutant CALR did not change. Next, we compared the threshold for macrophage phagocytosis of each HSPC fraction and mature blood cells and found no significant change in the efficiency of macrophage engulfment. Our data suggest that CALR mutation does not affect sensitivity to phagocytosis by macrophages. Finally, we analyzed the phosphorylation statuses of molecules downstream of JAK2 at each HSPC level in patients with ET and found that CALR mutations activated the JAK-STAT pathway in a manner similar to that associated with JAK2 mutations. These results indicate that mutant CALR causes myeloproliferation because of the activation of JAK-STAT pathway and not by the inhibition of phagocytosis, which is similar to the myeloproliferation caused by JAK2 V617F mutation.

Calreticulin (CALR) exon 9 frameshift mutations, commonly detected in essential thrombocythemia (ET) and primary myelofibrosis patients, activate signal transducer and activator of transcription (STAT) proteins in the presence of Myeloproliferative Leukemia Virus (MPL) and induce ET in vivo. Loss of the KDEL motif, an endoplasmic reticulum retention signal, and generation of many positively charged amino acids (AAs) in the mutated C-terminus are thought to be important for disease induction. To test this hypothesis, we generated mice harboring a Calr frameshift mutation using the CRISPR/Cas9 system. Deletion of 19-base pairs in exon 9 (c.1099-1117del), designated the del19 mutation, induced loss of the KDEL motif and generated many positively charged AAs, similar to human mutants. Calr del19 mice exhibited mild thrombocytosis, slightly increased megakaryocytes, and mild splenomegaly. In vitro experiments revealed that the murine CALR del19 mutant had a weaker ability to combine with murine MPL than the human CALR del52 mutant. Consequently, STAT5 activation was also very weak downstream of the murine mutant and murine MPL, and may be the reason for the mild disease severity. In summary, loss of the KDEL motif and positively charged AAs in the C-terminus of CALR is insufficient for MPL binding and ET development.

Objective: To investigate the clinical manifestation, cytogenetics, gene mutations and prognostic factors of chronic neutrophilic leukemia (CNL) . Methods: 16 CNL cases, according to WHO (2016) -definition, were reviewed retrospectively. Identifications of the CSF3R, ASXL1, SETBP1, CALR and MPL mutations were performed by direct sequencing. JAK2 V617F mutation was detected by AS-PCR. Results: Of the 16 CNL patients, the median age was 64 (43-80) years with a male predominance of 75% (12/16) . The median hemoglobin was 114 (81-154) g/L, with median WBC of 41.20 (26.05-167.70) (10(9)/L and median PLT of 238 (91-394) ×10(9)/L.The median level of marrow fibrosis (MF) was 1 (0-3) degree. There was no other cytogenetic abnormalities except t (1;7) (p32;q11) , +21 and 14ps+ for each. All the 16 CNL patients harbored CSF3R T618I mutation. ASXL1 mutations were identified in 81% (13/16) , while SETBP1 mutations were confirmed in 63% (10/16) . The CALR K385fs*47 mutation was found. There was no mutation in JAK2 V617F or MPL in the above 16 patients. The median overall survival (OS) of patients presented with WBC≥50×10(9)/L at diagnosis (11 months) was significantly shorter than of WBC<50×10(9)/L (39 months, P=0.005) . Conclusion: CSF3R T618I mutation was specific for CNL. The median OS of CNL patients was 24 months, and WBC≥50×10(9)/L at diagnosis was an unfavorable prognostic factor.

The aim of the study was to evaluate selected angiogenic factors in patients with essential thrombocythemia (ET) depending on JAK2V617F, calreticulin gene (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations. Sixty ET patients and 20 healthy volunteers were enrolled in the study. The following tests were performed: vascular endothelial growth factor- A (VEGF-A), soluble vascular endothelial growth factor receptor-1 (sVEGFR-1),soluble vascular endothelial growth factor receptor-2 (sVEGFR-2), platelet-derived growth factor( PDGF-BB), and stromal-derived factor-1α (SDF-1α). We observed an increased PDGF-BB level in patients with ET compared to the controls. Patients with CALR mutation had significantly higher concentration of PDGF-BB and lower concentration of SDF-1α than patients with JAK2V617F mutation. High concentration of PDGF-BB and low concentration of SDF-1α in patients with CALR(+) ET may indicate a contribution of these chemokines in disturbed Ca2+ metabolism in platelets.

The biological and molecular events that underlie bone marrow fibrosis in patients with myelodysplastic syndromes are poorly understood, and its prognostic role in the era of the Revised International Prognostic Scoring System (IPSS-R) is not yet fully determined. We have evaluated the clinical and biological events that underlie bone marrow fibrotic changes, as well as its prognostic role, in a well-characterized prospective patient cohort (n=77) of primary MDS patients. The degree of marrow fibrosis was linked to parameters of erythropoietic failure, marrow cellularity, p53 protein accumulation, WT1 gene expression, and serum levels of CXCL9 and CXCL10, but not to other covariates including the IPSS-R score. The presence of bone marrow fibrosis grade 2 or higher was associated with the presence of mutations in cohesin complex genes (31.5% vs. 5.4%, p=0.006). By contrast, mutations in CALR, JAK2, PDGFRA, PDGFRB,and TP53 were very rare. Survival analysis showed that marrow fibrosis grade 2 or higher was a relevant significant predictor for of overall survival, and independent of age, performance status, and IPSS-R score in multivariate analysis.

Calbindin (CALB) is well established as immunohistochemical marker for intrinsic primary afferent neurons in the guinea pig gut. Its expression by numerous human enteric neurons has been demonstrated but little is known about particular types of neurons immunoreactive for CALB. Here we investigated small and large intestinal wholemount sets of 26 tumor patients in order to evaluate (1) the proportion of CALB⁺ neurons in the total neuron population, (2) the colocalization of CALB with calretinin (CALR), somatostatin (SOM) and vasoactive intestinal peptide (VIP) and (3) the morphology of CALB+ neurons. CALB+ neurons represented a minority of myenteric neurons (small intestine: 31%; large intestine: 25%) and the majority of submucosal neurons (between 72 and 95%). In the submucosa, most CALB⁺ neurons co-stained for CALR and VIP (between 69 and 80%) or for SOM (between 20 and 3%). In the myenteric plexus, 85% of CALB+ neurons did not co-stain with the other markers investigated. An unequivocal correlation between CALB reactivity and neuronal morphology was found for myenteric type III neurons in the small intestine: uniaxonal neurons with long, slender and branched dendrites were generally positive for CALB. Since also other neurons displayed occasional CALB reactivity, this protein is not suited as an exclusive marker for type III neurons.

Although next-generation sequencing (NGS) has helped characterize the complex genomic landscape of myeloid malignancies, its clinical utility remains undefined. This has resulted in variable funding for NGS testing, limiting its accessibility. At our center, targeted sequencing (TAR-SEQ) using a 54-gene NGS myeloid panel is offered to all new patients referred for myeloid malignancies, as part of a prospective observational study. Here, we evaluated the diagnostic, prognostic, and potential therapeutic utility of clinical grade TAR-SEQ in the routine workflow of 179 patients with myeloproliferative neoplasms (MPN). Of 13 patients with triple negative (TN) MPN, who lacked driver mutations in JAK2, CALR, and MPL, TAR-SEQ confirmed clonal hematopoiesis in 8 patients. In patients with intermediate-risk myelofibrosis (MF), TAR-SEQ helped optimize clinical decisions in hematopoietic cell transplant (HCT)-eligible patients through identifying a high molecular risk (HMR) mutation profile. The presence of an HMR profile favored HCT in 9 patients with intermediate-1 risk MF. Absence of an HMR profile resulted in a delayed HCT strategy in 10 patients with intermediate-2 risk MF, 7 of which were stable at the last follow-up. Finally, TAR-SEQ identified patients with various targetable mutations in IDH1/2 (4%), spliceosome genes (28%), and EZH2 (7%). Some of these patients can be potential candidates for future targeted therapy trials. In conclusion, we have demonstrated that TAR-SEQ improves the characterization of TN MPN, can be integrated in clinical practice as an additional tool to refine decision making in HCT, and has the potential to identify candidates for future targeted therapy trials.