The immunological death induction by EY-6 on the human tumor cell lines was screened. Human colon carcinoma (HCT15, HCT116), gastric carcinoma (MKN74, SNU668), and myeloma (KMS20, KMS26, KMS34) cells were died by EY-6 treatment with dose-dependent manner. CRT expression, a typical marker for the immunological death, was increased on the EY-6-treated colorectal and gastric cancer cells. Interestingly, the effects on the myeloma cell lines were complicated showing cell line dependent differential modulation. Cytokine secretion from the EY-6 treated tumor cells were dose and cell-dependent. IFN-gamma and IL-12 secretion was increased in the treated cells (200% to over 1000% of non-treated control), except HCT116, SNU668 and KMS26 cells which their secretion was declined by EY-6. Data suggest the potential of EY-6 as a new type of immuno-chemotherapeutics inducing tumor-specific cell death. Further studies are planned to confirm the efficacy of EY-6 including in vivo study.
Calreticulin ; Cell Death ; Cell Line ; Cell Line, Tumor ; Colon ; Humans ; Interleukin-12 ; Stomach Neoplasms

There is no gold diagnostic standard for BCR-ABL fusion gene negative chronic myeloproliterative neoplasm(cMPN). The following detection methods such as comprehensive bone marrow cell morphology, bone marrow pathology, genetic mutation, flow cytometry and immunohistochemical are needed to diagnose the BCR-ABL fusion gene positive cMPN. The JAK2 mutation can be used as a specific diagnostic criteria for polycythemia vera (PV), but there is no specific and sensitive indication for the JAK2 mutation-negative MPN. CALR mutation would be an indication in a certain extent. In this review, the CALR mutation detection, detection mean and its correlation with disease diagnosis and prognosis etc were summarized.
Bone Marrow ; Bone Marrow Cells ; Calreticulin ; Humans ; Mutation ; Myeloproliferative Disorders ; Prognosis

OBJECTIVETo study the clinicopathological significance of the expression of calreticulin (CRT) protein and mRNA in pancreatic ductal adenocarcinoma (PDAC).

METHODSThe expression of CRT protein in 33 paired paraffin embedded PDAC specimens and adjacent non-cancerous pancreatic tissues were detected by immunohistochemistry. Western blot and RT-PCR were used to examine the expression of CRT protein and mRNA in 12 paired fresh PDAC specimens and adjuvant non-cancerous pancreatic tissues. The relationship between the protein expression and clinicopathological features was analyzed.

RESULTSCRT expression was much higher in 33 PDAC tissues than that in paired adjacent non-cancerous pancreatic samples (t = 2.323, P = 0.027). CRT was over expressed in 16 PDAC tissues, but only in 8 adjuvant non-cancerous pancreatic tissues (48.5% vs. 24.2%). The expression of CRT protein had no correlation with tumor position (χ(2) = 1.588, P = 0.208), differentiation (χ(2) = 1.517, P = 0.218), TNM stage (χ(2) = 2.528, P = 0.112) and lymph node metastasis (χ(2) = 1.963, P = 0.161), but had statistic significancy with the prognosis of the patients (χ(2) = 4.080, P = 0.043). The median survival time in the patients with high expression of CRT protein was longer than that in the patients with low expression. The expression of CRT mRNA was higher in PDAC than that in non-cancerous tissues detected by RT-PCR (t = 2.539, P = 0.025), but no significant difference was found in protein level (t = 1.292, P = 0.223).

CONCLUSIONSCRT is up-regulated in PDAC and may be a prognosis factor for patients with PDAC.

Animals ; Calreticulin ; metabolism ; Male ; Muscle, Skeletal ; metabolism ; Oxidative Stress ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the prevalence of CARL gene mutations and the mutation types in patients with essential thrombocythemia (ET), and to compare the patients clinical characteristics of CALR mutation with JAK2 V617F, MPL W515K mutation patients and triple negative group.

METHODSThe mutations of CALR gene at extron 9 and MPL W515K in 150 ET patients were detected by PCR amplification followed by direct sequencing of genomic DNA, the JAK2 V617F mutation by using allele specific PCR.

RESULTS(1)The CALR mutations were found in 38 patients (25.3%) of 150 ET patients. A total of 4 types of CALR mutations were identified (type Ic.1092_1143del52bp, n=17; type II c.1154_1155insTTGTC, n=16; type III c.1094_1139del46bp, n=4; type IV c.1103_1136del34bp, n=1). (2)The incidence of JAK2 V617F and MPL W515K was 61.3% (92/150) and 2.7% (4/150), respectively. The frequency of CALR mutation was 70.4% (38/54) in 54 ET patients without JAK2 V617F and MPL W515K mutations. The co-occurrence of any two kinds of gene mutations was not detected. (3)The hemoglobin level and leukocyte counts of patients with CARL mutations were significantly lower than that in patients with JAK2 V617F mutations (P<0.05). The median age of patients with CALR mutation was significantly higher than that of triple negative patients (59 years vs 29.5 years, P<0.01). Cytogenetic analysis was performed in 147 patients, and there were 4 abnormal karyotype cases. CALR mutation incidence was significantly higher in abnormal karyotype cases than that in normal ones (75% vs 24.5%, P=0.019).

CONCLUSIONThe incidence of CALR mutations is high in ET patients without JAK2 V617F and MPL W515K mutations, and is associated with abnormal karyotype. CARL-mutated cases showed a significantly lower leucocyte and hemoglobin levels compared with JAK2 V617F mutated cases.

Myeloproliferative neoplasm(MPN) is clonal hematopoietic stem cell disorder characterized by abnormal proliferation and expansion of one or more myeloid lineages. BCR-ABL-negative MPN includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutations of JAK2, CALR and MPL genes are involved in the pathogenesis of MPN that provided a more complete molecular diagnostic standard for MPN. More and more new mutated genes related to prognosis of MPN were discovered in the past few years, at same time it was found that cytokines were also involved in the genesis and development of MPN. These provide a theoretical basis for the diagnosis, risk stratification and treatment management of MPN. In this article, the mechanisms of MPN-related cytokines and mutated genes in the genesis and development of disease and prognosis characteristics are summarized.
Calreticulin ; Cytokines ; Humans ; Janus Kinase 2 ; Mutation ; Myeloproliferative Disorders ; Thrombocythemia, Essential

OBJECTIVE: To investigate the clinical characteristics of essential thrombocytopenia (ET) patients with positive mutations including JAK2, CALR, MPL, or negative mutations. METHODS: A total of 66 newly diagnosed ET cases from January 2016 to December 2018 in Department of Hematology, Huaian No.1 People's Hospital affiliated to Nanjing Medical University were analyzed. Statistical analysis data included the patient's sex, age, symptoms, thrombosis and embolism events, spleen omegaly, platelet count (Plt), leukocyte (WBC) count, hemoglobin (Hb), fibrinogen (FIB), thrombus elastic diagram (TEG), serum potassium, blood glucose (GLU), lactate dehydrogenase (LDH), JAK2, CALR and MPL mutations, treatment options, and efficacy. RESULTS: All the patients were not MPL-positive, and divided in three groups: JAK2 mutation (46 cases, 69.7%), CALR mutation (9 cases, 13.6%) and gene negative mutation (11 cases, 16.7%) group. The average age of patients in the JAK2 mutation group was 63.2 years old, and significantly higher than that in the CALR mutation group (51.8 year) and gene negative group (50.2 year) (P＜0.05). Compared with the JAK2 mutation group and gene negative group, the CALR mutation group had lower WBC count (6.3×10/L vs 13.79×10/L) (P=0.003) (6.3×10/L vs 9.70×10/L) (P=0.009). Also the Hb level of patients in CALR mutation group was lower than the JAK2 mutation group (121.22 g/L vs 136.2 g/L) (P=0.036). However, there was higher tumor burden in the CALR mutation group, compared with the gene negative mutation group (300.11 U/L vs 227.4 U/L) (P=0. 033). There was no significant difference among the three groups, such as the Plt counts, serum potassium level, GLU level and FIB level (P＞0.05). In addition, thrombus and embolism appeared in 30.3% (20/66) cases. 18.2% (12/66) cases were complicated with hyperkalemia, which significantly correlated with Plt counts (r=0.518). TEG was performed in 34 patients, of which 41.2% (14/34) had abnormal TEG and 55.9% (19/34) were accompanied by Plt count ＞ 1 000 ×10/L, but there was no significant correlation between them (r=0.134). After routine clinical treatment, all the 66 cases achieved partial or complete hematological remission, but the disease usually repeated. Until now 4.5% (3/66) cases had been converted to myelofibrosis (MF) all with JAK2 mutation, but without advancing to acute myeloid leukemia. CONCLUSION ET patients with JAK2 mutation have higher incidence, moreover were in older age. However, the patients with CALR mutations display lower WBC count and Hb level, but higher tumor burden. In short, the multiple gene mutations of ET showed different clinical features closely relates with the prognosis, thus providing guidance for the clinical diagnosis and treatment.
Aged ; Calreticulin ; genetics ; Humans ; Janus Kinase 2 ; genetics ; Middle Aged ; Mutation ; Primary Myelofibrosis ; Thrombocythemia, Essential ; Thrombocytopenia

BACKGROUNDEssential thrombocythemia is a subgroup of myeloproliferative neoplasms. Previous studies identified mutations of JAK2, CALR, and MPL that are closely related with the pathogenesis of myeloproliferative neoplasms. All these mutations contribute to the hyperactivation of JAK2/STAT pathway. However, a small proportion of essential thrombocythemia patients does not display such mutations. The pathogenesis of "triple-negative" form of essential thrombocythemia remains unknown.

OBJECTIVETo investigate the clinical characteristics of triple-negative essential thrombocythemia and related mutation genes.

METHODSTo identify the mutations associated with triple-negative essential thrombocythemia, next-generation sequencing was used to conduct targeted sequencing of 360 genes in samples from 68 patients.

RESULTSAt least one missense mutation was detected in all the patients and all the detected genes. After screening the data, it was observed that 10 genes with the 10 highest mutation were follows: FLT3, SH2B3, ASXL1, ADAMTS1, TET2, TP53, EGFR, CUX1, GATA2, and MPL.When only rare genes (i.e., with a frequency in Asian populations lower than 5%, as estimated by the 1000 Genomes Project) were analyzed, the most frequently mutated genes in the patients were TET2 (33.82%), SH2B3(29.41%), and ASXL1 (23.53%). Our study identified some mutations that did not previously reported. Although all these mutations need further validation, high incidence rates may indicate relevance of the respective mutations to essential thrombocythemia pathogenesis. Some of the detected mutations have been previously reported; these mutations were also found in a large proportion of our subjects.

CONCLUSIONwhole-exon sequencing can provide a higher level of accuracy for gene mutation analysis and assist in identifying mutations that contribute to illustrate the pathogenesis of essential thrombocythemia.

OBJECTIVETo detect the JAK2, CALR and MPL gene mutations in patients with BCR/ABL1 negative chronic myeloproliferative diseases（BCR/ABL1-CMPD）and to evaluate their diagnostic value.

METHODSTwo hundred and eight cases of BCR/ABL1-CMPD comprising of 146 cases of essential thrombocythemia（ET）, 37 cases of polycythemia vera（PV）and 25 cases of primary myelofibrosis（PMF）from March 2012 to December 2015 were enrolled in the BCR/ABL1-CMPD, while 124 cases of secondary thrombocythemia and 73 cases of secondary polycythemia were enrolled in the control group. The genomic DNA and total RNA Were isolated from bone marrow or peripheral blood, then the exons 12 to 20 of JAK2 gene, exon 10 of MPL gene and exons 3 to 9 of CALR gene were analyzed by using DNA sequencing.

RESULTSamong 146 ET patients, the JAK2, CALR or MPL mutations were found in: 138 cases（94.5%）including 86 cases with JAK2V617F mutation（58.9%）and 2 cases（1.4%）with exon 12 of JAK2 mutations. CALR mutations were detected in 41 cases（28.1%）, among them type 1（c.1092_1143del）in 22 cases, type 2（c.1154_1155insTTGTC）in 11 cases, and type 5（c. 1091_1142del）, type 8（c.1104_1137del）, type 41(c.1107_1137del）, type 42(c.1125_1125del）in one case respectively. In addition, 4 cases were detected withother mutations of the CALR gene（c.1107_1115del, c.1111_1144 del, c.1101 A>C, c.1112_1117del）. Moreover, 9 cases harbored MPL mutations（6.2%）. Secondly, 31 patients were detected with JAK2V617F mutation（83.8%）in 37 cases of PV, and JAK2 exon 12 mutations were found in 2 cases（5.4%）. Besides, CALR mutations were detected in 2 cases（5.4%）, including 1 case of type I, the other of novel mutation of CALR. Thirdly, 19 in 25 cases of PMF were detected with JAK2V617F mutation（76%）, 2 cases with CALR mutations（8%）. 4 patients（16%）, JAK2, CALR or MPL mutations were not detected, but among them 3 cases were found harboring other genetic abnormalities. Fourthly, no mutations of JAK2, MPL and CALR genes were detected in 124 patients with secondary thrombocytosis and 73 cases with secondary polycythemia.

CONCLUSIONCombined detection of JAK2, CALR and MPL gene mutations can cover the vast majority of patients with BCR/ABL1-negative myeloproliferative neoplasms. For higher frequencies of the mutations of CALR in ET patients, CALR mutation can be used as a new diagnostic marker in ET patients with JAK2 and MPL wild type.

OBJECTIVETo analyze the mutation rate and clinical characteristics of CALR, MPL W515K and JAK2 V617F genes in patients with primary thrombocythemia (PT).

METHODSFifty-six patients with PT were selected as the research objects in our hospital. The CALR and MPL W515K gene mutations were determined by genomic DNA-PCR direct sequencing of the PCR products, and the JAK2 V617F gene mutation was detected by allele specific PCR method.

RESULTSAmong the 56 patients with PT there were 14 cases of CALR gene mutation with the incidence rate of 25%, including 6 cases of type I, 5 cases of type II and 3 cases of type III. The sex, age, platelet(Plt) count, white blood cell (WBC) count and hemoglobin (Hb) level in the type I case of CALR gene mutation all were not significantly different from that in type II and III(all P>0.05); the WBC level in type III group significantly increased in comparison of type II group (P<0.05), while the sex, age, Hb and Plt levels showed no significant difference between the type III and type II groups (P>0.05). There were 3 cases of MPL W515K gene mutation with the incidence rate of 5.36%; 21 cases of JAK2 V617F gene mutation with the incidence rate of 37.50%. There were 13 cases of CALR gene mutation in negative patients with MPL W515K and JAK2 V617F (18 cases) with 72.22% incidence rate (13/18), and there was no cases of 1 or 2 gene mutations coexisted. The levels of Hb and WBC in peripheral blood of patients with CALR mutation were significantly lower than those of JAK2 V617F mutation (both P<0.05). In 56 cases, there were 3 cases of abnormal karyotype, with the incidence rate of 5.36%. The mutation rate of CALR gene in abnormal karyotypes (66.67%) was significantly higher than that of normal karyotypes (20.75%) (P<0.01).

CONCLUSIONThe incidence of JAK2 V617F gene mutation increases in the patients with primary thrombocythemia; CALR mutation rate is higher in the patients with negative MPL W515K and JAK2 V617F gene mutation, which may closely correlate with abnormal karyotype; the levels of peripheral Hb and WBC in PT the patients with CALR gene mutation are significantly lower than those in patients with JAK2 V617F mutation.