Myelofibrosis (MF) is a BCR-ABL1^- myeloproliferative neoplasm that arises from hematopoietic stem and progenitor cells frequently harboring a somatic driver mutation in 1 of 3 genes: JAK2, CALR, or MPL. The pathologic features of this hematologic malignancy include myeloproliferation, diffuse bone marrow fibrosis, and overactivation of the JAK-STAT pathway, resulting in enhanced inflammatory cytokine release. The common clinical manifestations of MF include systemic symptoms, abnormal peripheral blood count levels, and splenomegaly. However, it has become increasingly appreciated that significant clinical heterogeneity exists among patients with MF. Two distinct MF clinical phenotypes include the myeloproliferative and myelodepletive phenotype, with peripheral blood counts being the main discerning feature. Patients with the myeloproliferative phenotype will present with elevated peripheral blood counts and often experience significant constitutional symptoms and progressive splenomegaly. In contrast, patients with the myelodepletive phenotype will have low peripheral blood counts and will frequently require transfusion support. Current frontline therapies for MF, include ruxolitinib and fedratinib, which can exacerbate cytopenias and thereby pose an impediment to effective treatment of the myelodepletive patient. The present review discusses the clinical and prognostic implications of the myelodepletive phenotype and the therapeutic options and limitations for this subset of patients, representing an unmet clinical need.

Proteasomes are complex macromolecular structures existing in various forms to regulate a myriad of cellular processes. Besides degrading unwanted or misfolded proteins (proteostasis), distinct immune functions were ascribed for the immunoproteasome and thymoproteasome (TPr) complexes. For instance, antigen degradation during ongoing immune responses mainly relies on immunoproteasome activity, whereas intrathymic CD8 T-cell development requires peptide generation by the TPr complex. Despite these substantial differences, the functional contribution of the TPr to peripheral T-cell immunity remains ill-defined. We herein explored whether the use of mesenchymal stromal cells (MSCs) engineered to exhibit altered proteasomal activity through de novo expression of the TPr complex can be exploited as a novel anti-cancer vaccine capable of triggering potent CD8 T-cell activation. Phenotypic and molecular characterization of MSC-TPr revealed a substantial decrease in MHCI (H2-K^b and H2-D^d) expression along with elevated secretion of various chemokines (CCL2, CCL9, CXCL1, LIX, and CX3CL1). In parallel, transcriptomic analysis pinpointed the limited ability of MSC-TPr to present endogenous antigens, which is consistent with their low expression levels of the peptide-loading proteins TAP, CALR, and PDAI3. Nevertheless, MSC-TPr cross-presented peptides derived from captured soluble proteins. When tested for their protective capacity, MSC-TPr triggered modest anti-tumoral responses despite efficient generation of effector memory CD4 and CD8 T cells. In contrast, clodronate administration prior to vaccination dramatically enhanced the MSC-TPr-induced anti-tumoral immunity clearly highlighting a refractory role mediated by phagocytic cells. Thus, our data elute to a DC cross-priming-dependant pathway in mediating the therapeutic effect of MSC-TPr.

Keywords: antigen cross-presentation; cancer vaccine; clodronate; cross-priming; efferocytosis; mesenchymal stromal cell; thymoproteasome.

Background: Immunogenic cell death (ICD) is a tumor cell death involving both innate and adaptive immune responses. Given published findings that oxaliplatin, but not irinotecan, drives ICD, we investigated whether single nucleotide polymorphisms (SNPs) in the ICD pathway are associated with the efficacy of oxaliplatin-based chemotherapy in metastatic colorectal cancer (mCRC).

Methods: Two randomized clinical trials data were analyzed: discovery cohort, FOLFOX/bevacizumab arm (MAVERICC); validation cohort, FOLFOXIRI/bevacizumab arm (TRIBE); and two control cohorts, FOLFIRI/bevacizumab arms (both trials). Genomic DNA extracted from blood samples was genotyped. Ten SNPs in the ICD pathway were tested for associations with clinical outcomes.

Results: In total, 648 patients were included. In the discovery cohort, three SNPs were significantly associated with clinical outcomes in univariate analysis: CALR rs1010222 with progression-free survival (G/G vs any A, HR=0.61, 95% CI 0.43-0.88), ANXA1 rs1050305 with overall survival (OS) (A/A vs any G, HR=1.87, 95% CI 1.04-3.35), and LRP1 rs1799986 with OS (C/C vs any T, HR=1.69, 95% CI 1.07-2.70). Multivariate analysis confirmed the trend, but statistical significance was not reached. In the validation cohort, ANXA1 rs1050305, and LRP1 rs1799986 were validated to have the significant associations with clinical outcome. No significant associations of these SNPs were observed in the two control cohorts. Treatment-by-SNP interaction test confirmed the predictive values.

Conclusions: The predictive utility of ICD-related SNPs for the efficacy of oxaliplatin-based chemotherapy was demonstrated, warranting further validation studies to be translated into personalized treatment strategies using conventional cytotoxic agents in mCRC.

Keywords: adaptive immunity; gastrointestinal neoplasms; immunity; innate; translational medical research.

OBJECTIVE

To investigate the feasibility and relibility of rapidly and accurately acquiring the informations of gene mutations in MPN patients by using self-designed custom MPN mutation-related multipe-PCR primer kit and next generation Ion Torrent PGM sequencing platform.

METHODS

The bone marrow samples of 10 MPN patients with JAK2V617F and/or CALR+, Ph- confirmed by sanger sequencing method were collected and were re-detected by using next generation Ion Torrent PGM sequencing method, then the consistence of results of above-mentioned 2 kinds of detection methods was compared.

RESULTS

In terms of JAK2V617F, MPL and CALR mutations, the results of Ion Torrent PGM sequencing were complete consistent with results of Sanger sequencing, except 52 bp deletion of CALR gene, which conld not be detected by next generation Ion Torrent PGM sequencing method in all bone marrow samples.

CONCLUSIONS

The detection of multiple gene mutations in MPN patients by Ion Torrent PGM sequencing platform is feasible and can meet the needs of clinical testing. This method can complete detection of all 23 mutetions within 1-2 days, moreover, possesses advantages of high sensitivity, specificity, rapidity, high throughput and low cost.

BACKGROUND

Mutations in the JAK2, CALR, and MPL genes have been shown to have prognostic value in essential thrombocythaemia (ET), but no clear association with morphological changes has been reported so far. We investigated the possible correlation between gene mutations and histopathological features in bone marrow (BM) biopsies of patients with ET.

METHODS

Marrow cellularity, fibrosis, and the number of total and dysmorphic megakaryocytes and clusters of megakaryocytes were compared to gene mutations in 90 cases of ET at diagnosis.

RESULTS

The JAK2V617F mutation was found in 58.9%, CALR in 28.9%, and MPL in 4.4% of the cases, and 7.8% were triple-negative. JAK2V617F-mutated ET showed a high BM cellularity, the lowest number of clusters of megakaryocytes and the highest number of dysmorphic megakaryocytes; CALR-mutated ET showed a reduced BM cellularity, many clusters of large megakaryocytes, and very few dysmorphic megakaryocytes; MPL-mutated ET showed the lowest BM cellularity, the highest number of clustered and large megakaryocytes, and the lowest number of dysmorphic megakaryocytes. Triple-negative ET cases had the highest BM cellularity.

CONCLUSIONS

Distinct morphological patterns were associated with gene mutations in ET, supporting the classification of ET into different subtypes.

A 63-year-old woman was referred to our department in 2015 because of anemia and thrombocytosis. MPL W515/K was positive, JAK-2V617F and CALR exon 9 were negative. Bone marrow(BM)biopsy led to a diagnosis of primary myelofibrosis (PMF)in the prefibrotic/early stage(Grade 1). BMbiopsy performed in 2016 showed overt fibrotic stage(Grade 2). She was classified according to the Dynamic International Prognostic Scoring System(DIPSS)as intermediate(Int)-Ⅱrisk. Ruxolitinib 10 mg daily was initiated. Ruxolitinib was suspended for hepatic dysfunction after the dose was increased to 15 mg. Subsequently, ruxolitinib was resumed at 10 mg. BM biopsy performed in 2017 showed progression of myelofibrosis(MF)to Grade 3. BM biopsy performed in 2018 showed improved to Grade 0-1, however, BM was fatty. Currently in 2019, she continues to be on ruxolitinib. Results of immunohistochemical staining of BM biopsy specimens for cytokines and CD34 suggested the role of cytokines in the pathogenesis of the PMF. It was speculated that ruxolitinib blocked the production of cytokines to ameliorate the MF and restore the hematopoietic function of the BM. Although the pathogenesis of the fatty marrow remained unclear, the possibility of involvement of ruxolitinib cannot be denied.

BACKGROUND Autoimmune myelofibrosis (AMF) is a rare clinicopathologic entity of bone marrow fibrosis that occurs in association with autoimmune disorders. Steroids are very effective for treatment of AMF and the disease has a good prognosis and should be distinguished from primary myelofibrosis. CASE REPORT A 49-year-old man with bleeding and petechial hemorrhage of the extremities presented to our institution. His platelet count was 1×10⁹/L. Bone marrow aspiration revealed a dry tap, and bone marrow biopsy confirmed small lymphocyte infiltration and increased reticular fibers, consistent with immune thrombocytopenia. Testing for mutations in JAK2, MPL, and CALR was negative. Because the patient had a history of Raynaud's phenomenon, he was suspected to have collagen disease. Anti-Sjögren's-syndrome-related antigen-A antibody testing, Schirmer's test, and fluorescein staining all came back positive, which led to a diagnosis of Sjögren's syndrome. Given the bone marrow findings, the patient also was diagnosed with AMF. Treatment with steroids resulted in an immediate improvement in his platelet count. CONCLUSIONS In the present case, treatment with steroids resulted in prompt improvement in platelet counts and subsequent marrow biopsy showed MF-0 reticulin fibrosis. Bone marrow fibrosis rarely is seen in association with autoimmune disease, and its significance and mechanism are still to be determined.

Calreticulin (CALR), a Ca(2+) binding chaperone of the endoplasmic reticulum (ER) and mainly involved in Ca(2+) storage and signaling. In this study, we report the molecular characterization and immune responses of CALR homolog from disk abalone (AbCALR). The full length AbCALR cDNA (1837 bp) had an ORF of 1224 bp. According to the multiple alignments analysis, N- and P-domains were highly conserved in all the selected members of CALRs. In contrast, the C-domain which terminated with the characteristic ER retrieval signal (HDEL) was relatively less conserved. The phylogenetic analysis showed that all the selected molluscan homologs clustered together. Genomic sequence of AbCALR revealed that cDNA sequence was dispersed into ten exons interconnected with nine introns. AbCALR mRNA expression shows the significant (P < 0.05) up-regulation of AbCALR transcripts in hemocytes upon bacterial (Listeria monocytogenes and Vibrio parahaemolyticus), viral (Viral haemorrhagic septicaemia virus; VHSV) and immune stimulants (LPS and poly I:C) challenges at middle and/or late phases. These results collectively implied that AbCALR is able to be stimulated by pathogenic signals and might play a potential role in host immunity.

Objective: To evaluate the efficacy and tolerability of ruxolitinib combined with prednisone, thalidomide and danazol for treatment of in myelofibrosis (MF). Methods: Patients of MF according to the WHO 2016 criteria, received ruxolitinib (RUX) combined with prednisone, thalidomide and danazol (PTD). The response, changes of blood counts and adverse events were evaluated. Results: Six PMF and one post-ET MF patients were enrolled. Four patients presented JAK2V617F mutation, one CALR mutation, one MPL mutation, one triple-negative. Responses per IWG-MRT criteria were clinical improvement in 5 patients, stable disease in 2 ones, spleen response in 6 ones. All of 7 patients were symptomatic responses, four patients achieved at least 50% improvement from baseline on MPN-SAF TSS. Three patients initially treated with RUX alone, all of 3 patients experienced treatment-associated anemia and thrombocytopenia. Then these 3 patients received RUX combined with PTD, both hemoglobin and platelet increased significantly. Four patients initially treated with RUX combined with PTD. Increased levels of hemoglobin and platelet were seen in all of 7 patients received RUX combined with PTD with maximum increased hemoglobin of 30(18-54) g/L and maximum increased platelets of 116(13-369)×10(9)/L, respectively from baseline. The treatment dose of RUX increased due to improved platelet count in 3 patients. The frequent non-hematologic adverse events grade 1-2 were constipation, abdominal distension, crura edema and increased ALT. Conclusions: RUX combined with PTD for treatment of MF may modulate initial hematologic toxicity observed when RUX alone, and may increase response due to improved levels of hemoglobin or platelet.

In the present article, a patient with incidental findings in computerized tomography (CT) of cavernous transformation of the splanchnic veins, thrombosis of the splenic and portal veins, esophagus and gastric varicose veins and splenomegaly is presented. The CT was performed due to mild chronic normocytic anemia known for two years and the elevated level of LDH (Lactic dehydrogenase). Although usually such incidental findings without cirrhosis do not necessitate anticoagulation therapy according to the literature, in cases of myeloproliferative diseases, anticoagulation is required in order to prevent thrombus propagation. The Calreticulin (CALR) mutation is associated with more bleeding tendency and less thrombotic manifestations while the Janus kinase 2 V617F (JAK-2) mutation increases the risk of thrombosis. In the present article, we present the case report and review the relevant literature.

OBJECTIVE

In our previous study, we have built a nine-gene (GPC3, HGF, ANXA1, FOS, SPAG9, HSPA1B, CXCR4, PFN1, and CALR) expression detection system based on the GeXP system. Based on peripheral blood and GeXP, we aimed to analyze the results of genes expression by different multi-parameter analysis methods and build a diagnostic model to classify hepatocellular carcinoma (HCC) patients and healthy people.

METHODS

Logistic regression analysis, discriminant analysis, classification tree analysis, and artificial neural network were used for the multi-parameter gene expression analysis method. One hundred and three patients with early HCC and 54 age-matched healthy normal controls were used to build a diagnostic model. Fifty-two patients with early HCC and 34 healthy people were used for validation. The area under the curve, sensitivity, and specificity were used as diagnostic indicators.

RESULTS

Artificial neural network of the total nine genes had the best diagnostic value, and the AUC, sensitivity, and specificity were 0.943, 98%, and 85%, respectively. At last, 52 HCC patients and 34 healthy normal controls were used for validation. The sensitivity and specificity were 96% and 86%, respectively.

CONCLUSIONS

Multi-parameter analysis methods may increase the diagnostic value compared to single factor analysis and they may be a trend of the clinical diagnosis in the future.

We describe a patient with MDS/MPN with ring sideroblasts and thrombocytosis who had deletions of long arm of chromosome 5 (5q-) and chromosome 20 (20q-). Molecular studies showed an exon 9, frame shift mutation in the calreticulin (CALR) gene, and absence of mutations in JAK2, MPL, SETBP1 or SF3B1. Treatment with lenalidomide resulted in durable clinical remission which has lasted 2 years.