Calreticulin (CALR) mutations were recently identified in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) devoid of JAK2 and MPL mutations. We evaluated the clinical, laboratory, and molecular features of a Taiwanese population of patients with ET. Among 147 ET patients, CALR mutations were detected in 33 (22.5 %), JAK2V617F in 94 (63.9 %), and MPL mutations in 4 (2.7 %). Sixteen (10.9 %) patients were negative for all three mutations (CALR, JAK2V617F, and MPL; triple negative). Interestingly, one patient with the type 2 CALR mutation also harbored a low allele burden (0.025 %) of JAK2V617F mutation. Furthermore, we found a novel CALR mutation, with the resultant protein sharing an identical amino acid sequence to the type 6 CALR mutant. Compared to those with JAK2 mutation, CALR-mutated ET patients were characterized by younger age, lower leukocyte count, higher platelet count, and decreased risk of thrombosis. CALR mutations had a favorable impact on thrombosis-free survival (TFS) for ET patients, whereas the respective TFS outcomes were similarly poorer in JAK2-mutated ET and PV patients. Multivariate analysis confirmed that younger age (<60 years), presence of CALR mutations, and a lower platelet count (<1,000 × 10(9)/L) were independently associated with a longer TFS in ET patients. The current study demonstrates that CALR mutations characterize a special group of ET patients with unique phenotypes that are not discrepant from those seen in Western countries.

Disease Overview: Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms respectively characterized by erythrocytosis and thrombocytosis; other disease features include leukocytosis, splenomegaly, thrombosis, bleeding, microcirculatory symptoms, pruritus, and risk of leukemic or fibrotic transformation. Diagnosis: Bone marrow morphology remains the cornerstone of diagnosis. In addition, the presence of JAK2 mutation is expected in PV while approximately 90% of patients with ET express mutually exclusive JAK2, CALR, or myeloproliferative leukemia mutations. In ET, it is most important to exclude the possibility of prefibrotic myelofibrosis. Survival: Median survivals are 14 years for PV and 20 years for ET; the corresponding values for younger patients are 24 and 33 years. Certain mutations (mostly spliceosome) and abnormal karyotype might compromise survival in PV and ET. Life-expectancy in ET is inferior to the control population. Driver mutations have not been shown to affect survival in ET. Risk of thrombosis is higher in JAK2-mutated ET. Leukemic transformation rates at 10 years are estimated at <1% for ET and 3% for PV. Thrombosis Risk: In PV, 2 risk categories are considered: high (age>> 60 years or thrombosis history present) and low (absence of both risk factors); in ET, 4 risk categories are considered: very low (age ≤ 60 years, no thrombosis history, JAK2 wild-type), low (same as very low but JAK2 mutation present), intermediate (age>> 60 years, no thrombosis history, JAK2 wild-type) and high (thrombosis history present or age>> 60 years with JAK2 mutation). Risk-Adapted Therapy: The main goal of therapy in both PV and ET is to prevent thrombohemorrhagic complications. All patients with PV require phlebotomy to keep hematocrit below 45% and once- or twice-daily aspirin (81 mg), in the absence of contraindications. Very low-risk ET might not require therapy while aspirin therapy is advised for low-risk disease. Cytoreductive therapy is recommended for high-risk ET and PV but it is not mandatory for intermediate-risk ET. First-line drug of choice for cytoreductive therapy, in both ET and PV, is hydroxyurea and second-line drugs of choice are interferon-α and busulfan. We do not recommend treatment with ruxolutinib in PV, unless in the presence of severe and protracted pruritus or marked splenomegaly that is not responding to the aforementioned drugs.

Combining different approaches (resequencing of portions of 54 obesity candidate genes, literature mining for pig markers associated with fat deposition or related traits in 77 genes, and in silico mining of porcine expressed sequence tags and other sequences available in databases), we identified and analyzed 736 SNP within candidate genes to identify markers associated with back fat thickness (BFT) in Italian Large White sows. Animals were chosen using a selective genotyping approach according to their EBV for BFT (276 with most negative and 279 with most positive EBV) within a population of ≈ 12,000 pigs. Association analysis between the SNP and BFT has been carried out using the MAX test proposed for case-control studies. The designed assays were successful for 656 SNP: 370 were excluded (low call rate or minor allele frequency <5%), whereas the remaining 286 in 212 genes were taken for subsequent analyses, among which 64 showed a P(nominal) value <0.1. To deal with the multiple testing problem in a candidate gene approach, we applied the proportion of false positives (PFP) method. Thirty-eight SNP were significant (P(PFP) < 0.20). The most significant SNP was the IGF2 intron3-g.3072G>A polymorphism (P(nominal) < 1.0E-50). The second most significant SNP was the MC4R c.1426A>G polymorphism (P(nominal) = 8.0E-05). The third top SNP (P(nominal) = 6.2E-04) was the intronic TBC1D1 g.219G>A polymorphic site, in agreement with our previous results obtained in an independent study. The list of significant markers also included SNP in additional genes (ABHD16A, ABHD5, ACP2, ALMS1, APOA2, ATP1A2, CALR, COL14A1, CTSF, DARS, DECR1, ENPP1, ESR1, GH1, GHRL, GNMT, IKBKB, JAK3, MTTP, NFKBIA, NT5E, PLAT, PPARG, PPP2R5D, PRLR, RRAGD, RFC2, SDHD, SERPINF1, UBE2H, VCAM1, and WAT). Functional relationships between genes were obtained using the Ingenuity Pathway Analysis (IPA) Knowledge Base. The top scoring pathway included 19 genes with a P(nominal) < 0.1, 2 of which (IKBKB and NFKBIA) are involved in the hypothalamic IKKβ/NFκB program that could represent a key axis to affect fat deposition traits in pigs. These results represent a starting point to plan marker-assisted selection in Italian Large White nuclei for BFT. Because of similarities between humans and pigs, this study might also provide useful clues to investigate genetic factors affecting human obesity.

Disease overview: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by stem cell-derived clonal myeloproliferation that is often but not always accompanied by JAK2, CALR or MPL mutation, abnormal cytokine expression, bone marrow fibrosis, anemia, splenomegaly, extramedullary hematopoiesis (EMH), constitutional symptoms, cachexia, leukemic progression and shortened survival.

Diagnosis is based on bone marrow morphology. The presence of JAK2, CALR or MPL mutation is supportive but not essential for diagnosis; approximately 90% of patients carry one of these mutations and 10% are "triple-negative." None of these mutations are specific to PMF and are also seen in essential thrombocythemia (ET). According to the revised 2016 World Health Organization (WHO) classification and diagnostic criteria, "prefibrotic" PMF (pre-PMF) is distinguished from "overtly fibrotic" PMF; the former might mimic ET in its presentation and it is prognostically relevant to distinguish the two. Risk stratification: The Dynamic International Prognostic Scoring System-plus (DIPSS-plus) uses eight predictors of inferior survival: age >65 years, hemoglobin <10 g/dL, leukocytes >25 × 109 /L, circulating blasts ≥1%, constitutional symptoms, red cell transfusion dependency, platelet count <100 × 109 /L and unfavorable karyotype (i.e., complex karyotype or sole or two abnormalities that include +8, -7/7q-, i(17q), inv(3), 5/5q-, 12p-, or 11q23 rearrangement). The presence of 0, 1, "2 or 3" and ≥4 adverse factors defines low, intermediate-1, intermediate-2 and high-risk disease with median survivals of approximately 15.4, 6.5, 2.9 and 1.3 years, respectively. Most recently, DIPSS-plus-independent adverse prognostic relevance has been demonstrated for certain mutations including ASXL1 and SRSF2 whereas patients with type 1/like CALR mutations, compared to their counterparts with other driver mutations, displayed significantly better survival. Risk-adapted therapy: Observation alone is a reasonable treatment strategy for asymptomatic low or intermediate-1 DIPSS-plus risk disease, especially in the absence of high-risk mutations. All other patients with high or intermediate-2 risk disease, or those harboring high-risk mutations such as ASXL1 or SRSF2, should be considered for stem cell transplant, which is currently the only treatment modality with the potential to favorably modify the natural history of the disease. Non-transplant candidates should be encouraged to participate in clinical trials, since the value of conventional drug therapy, including the use of JAK2 inhibitors, is limited to symptoms palliation and reduction in spleen size. Specifically, JAK2 inhibitors have not been shown to induce complete clinical or cytogenetic remissions or significantly affect JAK2/CALR/MPL mutant allele burden. Splenectomy is considered for drug-refractory splenomegaly. Involved field radiotherapy is most useful for post-splenectomy hepatomegaly, non-hepatosplenic EMH, PMF-associated pulmonary hypertension and extremity bone pain. Am. J. Hematol. 91:1262-1271, 2016. © 2016 Wiley Periodicals, Inc.

We tested 357 Chinese with primary myelofibrosis for mutations in CALR, JAK2 and MPL. CALR mutations were detected in 76 subjects (21%). There were 24 (32%) type-1 (L367fs*46) and 49 (64%) type-2 (K385fs*47) mutations. Seventy-two of 168 subjects (43%) without a JAK2 or MPL mutation had a CALR mutation. Subjects with a type-2 CALR mutation had lower hemoglobin concentrations (P=0.001), lower WBC counts (P<0.001), a higher percentage of blood blasts (P=0.009), and higher conventional (P<0.001) and Chinese-adjusted Dynamic International Prognostic Scoring System (P<0.001) scores compared with subjects with JAK2 mutations. Subjects with a type-2 CALR mutation were also likely to have abnormal platelet levels (<100 × 10(9)/L, P=0.01 or >450 × 10(9)/L, P=0.042) and no splenomegaly (P=0.004). Type-2 CALR mutation or no detectable mutation was an independent high-risk factor for survival in multivariate analyses. These data suggest the ratio between type-1 and type-2 mutations is reversed in Chinese with primary myelofibrosis compared with populations of subjects with primary myelofibrosis of predominately European descent. The unfavorable prognostic impact of CALR mutations in Chinese with primary myelofibrosis is only seen in those with type-2 mutations. These data underscore the need to evaluate the prognostic impact of genetic mutations in different populations.

Philadelphia-negative classical myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2016 revision of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues includes new criteria for the diagnosis of these disorders. Somatic mutations in the 3 driver genes, that is, JAK2, CALR, and MPL, represent major diagnostic criteria in combination with hematologic and morphological abnormalities. PV is characterized by erythrocytosis with suppressed endogenous erythropoietin production, bone marrow panmyelosis, and JAK2 mutation. Thrombocytosis, bone marrow megakaryocytic proliferation, and presence of JAK2, CALR, or MPL mutation are the main diagnostic criteria for ET. PMF is characterized by bone marrow megakaryocytic proliferation, reticulin and/or collagen fibrosis, and presence of JAK2, CALR, or MPL mutation. Prefibrotic myelofibrosis represents an early phase of myelofibrosis, and is characterized by granulocytic/megakaryocytic proliferation and lack of reticulin fibrosis in the bone marrow. The genomic landscape of MPNs is more complex than initially thought and involves several mutant genes beyond the 3 drivers. Comutated, myeloid tumor-suppressor genes contribute to phenotypic variability, phenotypic shifts, and progression to more aggressive disorders. Patients with myeloid neoplasms are at variable risk of vascular complications, including arterial or venous thrombosis and bleeding. Current prognostic models are mainly based on clinical and hematologic parameters, but innovative models that include genetic data are being developed for both clinical and trial settings. In perspective, molecular profiling of MPNs might also allow for accurate evaluation and monitoring of response to innovative drugs that target the mutant clone.

The recent discovery of somatically acquired CALR mutations in a substantial proportion of patients with myeloproliferative neoplasms has provided a new marker of clonal disease, advancing both diagnosis and prognosis in these previously difficult to characterise disorders. The mutations, which can be challenging to detect on a routine basis, are heterogeneous insertions/deletions (indels) in exon 9 with mutant allele burden that vary substantially between patients. We evaluated four genetic screening methods for their ability to detect a series of different CALR mutations; Sanger sequencing, fragment analysis PCR, high resolution melt (HRM) and targeted next generation sequencing (NGS). The limit of detection (LoD) of each assay was tested using serial dilution series made with DNA from CALR positive sample DNA and a cell line, MARIMO, found to carry a heterozygous 61 nucleotide CALR deletion. All methods were capable of detecting each mutation; HRM and fragment analysis PCR were better at detecting low mutation levels compared to Sanger sequencing but targeted NGS had the lowest LoD at a 1% mutation burden.

The calcium-binding proteins calbindin D-28k (CalB) and calretinin (CalR) have been shown to be useful markers of neuronal subpopulations located mainly in layers II-III of the neocortex of a variety of species, including human. Double labeling immunocytochemical studies of CalB, CalR, and GABA in experimental animals have shown that CalB and CalR are present in separate subpopulations of neurons. However, there are no studies of colocalization of these calcium-binding proteins and GABA in the human neocortex. The principal goal of the present work was to investigate the degree of colocalization of these substances in layers II-III of the human temporal neocortex, using a postembedding immunocytochemical method. The patterns of staining for CalB, CalR, and GABA in the human cortex were similar to those found in monkey neocortex. However, the degree of colocalization for certain combinations was different from that reported in the monkey and other experimental animals. A relatively large proportion of CalB- and CalR-immunoreactive cells (approximately 71% and 74%, respectively) were found to be immunoreactive for GABA. However, the degree of colocalization of CalB with CalR was low (between 4% and 6%). Thus, our quantitative and qualitative data suggest that these calcium-binding proteins are present in similar cortical circuits in all primates, but that in the human neocortex, there might be additional GABAergic and perhaps also non-GABAergic interneurons with unique chemical characteristics.

Major progress has been recently made in understanding the molecular pathogenesis of myeloproliferative neoplasms (MPN). Mutations in one of four genes-JAK2, MPL, CALR, and CSF3R-can be found in the vast majority of patients with MPN and represent driver mutations that can induce the MPN phenotype. Hyperactive JAK/STAT signaling appears to be the common denominator of MPN, even in patients with CALR mutations and the so-called "triple-negative" MPN, where the driver gene mutation is still unknown. Mutations in epigenetic regulators, transcription factors, and signaling components modify the course of the disease and can contribute to disease initiation and/or progression. The central role of JAK2 in MPN allowed development of small molecular inhibitors that are in clinical use and are active in almost all patients with MPN. Advances in understanding the mechanism of JAK2 activation open new perspectives of developing the next generation of inhibitors that will be selective for the mutated forms of JAK2.

OBJECTIVE

We investigated mutation profiles of CALR, JAK2, and MPL in 199 Korean patients with myeloproliferative neoplasms (MPNs).

METHODS

In total, 199 patients with MPN (54 primary myelofibrosis [PMF], 79 essential thrombocythemia [ET], 58 polycythemia vera [PV], and eight MPN-unclassifiable [MPN-U]) and 4 patients with acute panmyelosis with myelofibrosis (APMF) were retrospectively subjected to Sanger sequencing for CALR, JAK2, and MPL.

RESULTS

The overall frequency of CALR mutations was 12.6% (type 1 mutation, 16 patients; type 2 mutation, nine patients): most frequent in MPN-U (37.5%), followed by ET (17.7%) and PMF (14.8%). CALR mutations were not found in PV or APMF. CALR and JAK2 or MPL mutations were mutually exclusive. In PMF, the CALR mutations were associated with lower levels of leukocytes, lower bone marrow cellularity, and higher number of megakaryocytes. Patients with CALR-mutated ET more frequently progressed to the accelerated or blast phases compared with patients with JAK2 mutations. CALR mutations were frequently observed in the JAK2-negative MPNs, most frequently in MPN-U.

CONCLUSIONS

The prognostic significance of CALR mutations likely differs among the MPN subtypes.

Myeloproliferative neoplasms (MPNs) are stem cell-derived clonal myeloid malignancies characterized by a unique somatic mutational profile since three mutually exclusive mutations (JAK2V617F, MPL, and CALR) sustain the great majority of the cases. However, clinical observation that autoimmune diseases may predispose to MPNs, autoimmune disorders or autoimmune phenomena may be associated with MPNs, and genetic constitutional variants that predispose to autoimmune disorders or inflammatory phenomena also predispose to MPNs raises a hypothesis that there might be an autoimmune/inflammatory basis underlying the pathogenesis of MPNs. Recent studies have documented that MPNs are characterized by an abnormal activity of key cells of the immune system, for example, increase in monocyte/macrophage compartment, altered regulatory T cell frequency, expansion of myeloid-derived suppressor cells, and CD4/natural killer cell dysfunction. This review is focused on summarizing recent advances in our understanding of immunological defects in MPNs with accompanying translational implications.

A myeloid neoplasm-relevant 27-gene panel was used for next-generation sequencing of bone marrow or whole blood DNA in 182 patients with primary myelofibrosis (PMF). DNA sequence variants/mutations other than JAK2/CALR/MPL were detected in 147 patients (81%), with the most frequent being ASXL1 (36%), TET2 (18%), SRSF2 (18%), and U2AF1 (16%); furthermore, 35%, 26%, 10%, and 9% of the patients harbored 1, 2, 3, or 4 or more such variants/mutations, respectively. Adverse variants/mutations were identified by age-adjusted multivariable analysis of impact on overall survival or leukemia-free survival and included ASXL1, SRSF2, CBL, KIT, RUNX1, SH2B3, and CEBPA; their combined prevalence was 56%. Adverse variants/mutations were associated with inferior overall survival (median, 3.6 vs 8.5 years; P < .001) and leukemia-free survival (7-year risk, 25% vs 4%; P < .001), and the effect on survival was independent of both the Dynamic International Prognostic Scoring System Plus and JAK2/CALR/MPL mutational status, with respective hazard ratios of 2.0 (95% confidence interval [CI], 1.3-3.1) and 2.9 (95% CI, 1.9-4.4). Additional prognostic information was obtained by considering the number of adverse variants/mutations; median survivals in patients with zero (n = 80), 1 or 2 (n = 93), or 3 or more (n = 9) adverse variants/mutations were 8.5, 4, and 0.7 years, respectively (P < .001). Additional data were obtained on pattern of mutation co-segregation and phenotypic correlation, including significant associations between U2AF1 and JAK2 mutations (P = .04) and U2AF1 mutations and anemia (P = .003) and thrombocytopenia (P = .006). We conclude that DNA variants/mutations other than JAK2/CALR/MPL are prevalent in PMF and are qualitatively and quantitatively relevant in predicting overall and leukemia-free survival.

JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non- BCR-ABL1 myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations ( CALR and MPL) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targeting JAK2V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In contrast, the strong anti-inflammatory effects of the JAK inhibitors appear as a very promising therapeutic approach for many inflammatory and auto-immune diseases.

Most biotherapeutic drugs are recombinant monoclonal antibodies which are mostly produced in monoclonal cell lines derived from Chinese hamster ovary (CHO) cells. Various clones expressing a monoclonal recombinant antibody were analyzed and a correlation of the antibody concentration and the relative mRNA level of calreticulin (CALR), glucose-regulated protein 78 and 94 kDa (GRP78, GRP94) and spliced X-box binding protein 1 (XPB1) was observed. By means of these results we were motivated to establish a novel selection system based on endoplasmic reticulum (ER) stress, which allows the rapid identification and isolation of high-expressing clones out of a pool mainly consisting of low- and medium-producing cells. Several ER stress responsive elements were tested with the aid of a recombinase mediated cassette exchange (RMCE) procedure. Very surprisingly, only GRP78 reporter constructs were strongly stimulated upon antibody expression. Furthermore we found that GRP78 reporter constructs are very suitable to reflect the level of antibody expression (IgG) in recombinant CHO cells. Based on these results, it is concluded, that the novel ER stress based selection system developed during this study is suitable to identify and isolate clones with a high level of antibody expression.

The role of antiplatelet therapy as primary prophylaxis of thrombosis in low-risk essential thrombocythemia has not been studied in randomized clinical trials. We assessed the benefit/risk of low-dose aspirin in 433 patients with low-risk essential thrombocythemia (271 with a CALR mutation, 162 with a JAK2(V617F) mutation) who were on antiplatelet therapy or observation only. After a follow up of 2215 person-years free from cytoreduction, 25 thrombotic and 17 bleeding episodes were recorded. In CALR-mutated patients, antiplatelet therapy did not affect the risk of thrombosis but was associated with a higher incidence of bleeding (12.9 versus 1.8 episodes per 1000 patient-years, P=0.03). In JAK2(V617F)-mutated patients, low-dose aspirin was associated with a reduced incidence of venous thrombosis with no effect on the risk of bleeding. Coexistence of JAK2(V617F)-mutation and cardiovascular risk factors increased the risk of thrombosis, even after adjusting for treatment with low-dose aspirin (incidence rate ratio: 9.8; 95% confidence interval: 2.3-42.3; P=0.02). Time free from cytoreduction was significantly shorter in CALR-mutated patients with essential thrombocythemia than in JAK2(V617F)-mutated ones (median time 5 years and 9.8 years, respectively; P=0.0002) and cytoreduction was usually necessary to control extreme thrombocytosis. In conclusion, in patients with low-risk, CALR-mutated essential thrombocythemia, low-dose aspirin does not reduce the risk of thrombosis and may increase the risk of bleeding.

The WHO 2008 definition of chronic neutrophilic leukemia (CNL) is based on clinical and laboratory parameters but not on molecular abnormalities. Mutations in CSF3R, SETBP1 and CALR are reported in patients with chronic neutrophilic leukemia (CNL). However, because CNL is rare, there are few large studies of this issue. We sequenced these genes in 14 patients who met the WHO-criteria of CNL. 8 subjects had CSF3R(T618I), 6 SETBP1 mutations and 1 a CALR mutation. Our data suggest mutation analysis of CSF3R, SETBP1 and CALR should be included in the diagnostic criteria for CNL. These data may also have therapy implications.

OBJECTIVE

Biomarkers for the diagnosis and prognosis of gliomas are lacking. To elucidate new diagnostic and prognostic targets, a routine method is used to evaluate differences between the protein profile of normal and tumor cells. The object of the current study was to investigate novel differentially expressed proteins and their roles in gliomas.

METHODS

Differences in the protein profile were compared using 2D polyacrylamide gel electrophoresis using C6 glioma cells and rat astrocytes. The mRNA and protein expression of ANXA2, PGAM1, and CALR were analyzed in glioma tissues and normal brain tissues. The expression of ANXA2 in the U87 glioma cell line was interrupted using short interfering RNA duplexes, and the role of ANXA2 in the migration and invasiveness of glioma cells was assessed. The expression of ANXA2, PGAM1, and CALR was examined further by immunohistochemical analysis using 130 glioma samples obtained in patients, and their prognostic roles in gliomas were evaluated using Kaplan-Meier and Cox regression analyses.

RESULTS

Significantly higher expression levels of ANXA2 and PGAM1 and a lower level of CALR were found in glioma samples than in the normal brain samples. ANXA2, PGAM1, and CALR expression correlated with the grade and survival of patients with gliomas. Multivariate analysis further revealed that ANXA2 was an independent prognostic marker for glioma. After ANXA2 expression was suppressed using short interfering RNA, U87 cells had decreased migratory and invasive capabilities in vitro.

CONCLUSIONS

Protein expression alterations in ANXA2, PGAM1, and CALR were found in gliomas, and ANXA2 provided a novel prognostic value.

Haemonchus contortus, a gastrointestinal parasite of sheep and goat feeds on the blood of its host and causes bleeding at the biting site. In this report, we demonstrate that the Ca2+ binding protein, calreticulin (CalR), is present in excretory/secretory products of adult worms. The secreted CalR enhanced plasma coagulation time. Using recombinant fragments, this property has been mapped to C-terminal part of the molecule which has binding sites for Ca2+ as well as clotting factors. Complement protein C1q bound to immobilized CalR and C1q dependent lysis of sensitized sheep erythrocytes was inhibited by CalR, a function mapped to N-domain of the protein. Factor X and a 24 kDa polypeptide derived from prothrombin but not prothrombin bound to immobilized CalR. The binding site for 24 kDa polypeptide in the CalR molecule has been localized in the P-domain. Our results suggest at least two functions for secreted CalR: first, to prevent blood clotting by binding to Ca2+ and clotting factors thus enabling parasite to feed on the host blood and second to modulate the host immune response by binding to complement C1q thereby facilitating parasite's survival within the host.

BACKGROUND

Vascular events in essential thrombocythemia (ET) are associated with advanced age and thrombosis history. Recent information suggests additional effect from the presence of specific mutations.

OBJECTIVE

To examine the influence of age and thrombosis history on the reported association between mutational status and thrombosis-free survival in ET.

METHODS

Analysis was performed using a Mayo Clinic cohort of 300 ET patients, and key findings were reanalyzed by including additional 102 Italian patients.

RESULTS

Among 300 Mayo patients with ET (median age 55 yr, 60% females), mutational frequencies were 53% JAK2, 32% CALR, 3% MPL, and 12% JAK2, CALR and MPL wild type. One hundred and six (35%) patients experienced arterial (n = 75) or venous (n = 43) events, before (n = 55) or after (n = 71) diagnosis. In univariate analysis, compared to JAK2-mutated cases, JAK2, CALR and MPL wild type (HR 0.31, 95% CI 0.11-0.86), and CALR-mutated (0.53, 95% CI 0.30-0.92) patients displayed better thrombosis-free survival. JAK2, CALR, and MPL wild type remained significant (P = 0.03; HR 0.32, 95% CI 0.11-0.9) during multivariable analysis that included age (P = 0.01) and thrombosis history (P = 0.0006); a favorable impact from CALR mutations was of borderline significance (P = 0.1; HR 0.62, 95% CI 0.35-1.1), but became significant (P = 0.02) when multivariable analysis including thrombosis history (P = 0.02) was performed on patients younger than 60 yr of age.

CONCLUSIONS

The favorable impact of mutational status on thrombosis-free survival in ET might be most evident for JAK2, CALR, and MPL wild type patients, whereas the favorable effect from CALR mutations might be confined to young patients.

UNASSIGNED

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by stem cell-derived clonal myeloproliferation that is often but not always accompanied by JAK2, CALR, or MPL mutations; additional disease features include bone marrow stromal reaction including reticulin fibrosis, abnormal cytokine expression, anemia, hepatosplenomegaly, extramedullary hematopoiesis (EMH), constitutional symptoms, cachexia, leukemic progression, and shortened survival.

METHODS

Diagnosis of PMF is based on bone marrow morphology. Presence of JAK2, CALR, or MPL mutation, expected in ∼ 90% of the patients, is supportive but not essential for diagnosis. The revised 2016 World Health Organization (WHO) classification system distinguishes "prefibrotic" from "overtly fibrotic" PMF; the former might mimic ET in its presentation and it is prognostically relevant to distinguish the two.

UNASSIGNED

Two new prognostic systems for PMF have recently been introduced: GIPSS (genetically inspired prognostic scoring system) and MIPSS70+ version 2.0 (mutation- and karyotype-enhanced international prognostic scoring system). GIPSS is based exclusively on mutations and karyotype. MIPSS70+ version 2.0 utilizes both genetic and clinical risk factors. GIPSS features four and MIPSS70+ version 2.0 five risk categories. MIPSS70+ version 2.0 requires an online score calculator (http://www.mipss70score.it) while GIPPS offers a lower complexity prognostic tool.

UNASSIGNED

Observation alone is advised for MIPSS70+ version 2.0 "low" and "very low" risk disease (estimated 10-year survival 56%-92%); allogeneic stem cell transplant is the preferred treatment of choice for "very high" and "high" risk disease (estimated 10-year survival 0-13%); treatment-requiring patients with intermediate-risk disease (estimated 10-year survival 30%) are best served by participating in clinical trials. All other treatment approaches, including the use of JAK2 inhibitors, are mostly palliative and should not be used in the absence of clear treatment indications. Conventional treatment for anemia includes androgens, prednisone, thalidomide and danazol, for symptomatic splenomegaly hydroxyurea and ruxolitinib and for constitutional symptoms ruxolitinib. Splenectomy is considered for drug-refractory splenomegaly and involved field radiotherapy for nonhepatosplenic EMH and extremity bone pain.

The earliest generated cells of the mammalian cerebral cortex form the preplate layer (PPL). The subsequently born cortical plate (CP) cells split this layer into the superficial layer I (LI) and the deep subplate (SP). The cellular and molecular mechanisms that underlie this event are unclear. To investigate the role of the cyclin-dependent kinase 5 (Cdk5) and its activator p35 in preplate splitting, we used Nissl staining, carbocyanine dye tracing, cell birthdating, and immunohistochemistry for calretinin (CalR) in p35 and Cdk5 knockout mice. Our data demonstrated changes in early cortical lamination and aberrant thalamic axon trajectories in these mice. Specifically, LI was thicker, and cell-dense and thalamic axons did not accumulate in the SP layer before invading the CP. Instead, they grew past the SP and more superficial cortical layers and coursed obliquely toward the pial surface. This behavior has been previously observed in reeler mice and suggests a defect in PPL splitting. CalR immunohistochemistry and bromo-deoxyuridine birthdating confirmed the abnormality in position of the earliest generated cortical cells of mutants. These observations suggest that the p35/Cdk5 pathway plays a role in preplate splitting in addition to regulating layer formation.