There is mounting evidence that the immune system can spontaneously clear malignant lesions before they manifest as overt cancer, albeit this activity has been difficult to demonstrate in humans. The calreticulin (CALR) exon 9 mutations are driver mutations in patients with chronic myeloproliferative neoplasms (MPN), which are chronic blood cancers. The CALR mutations generate a neo-antigen that is recognized by patient T cells, and T cells isolated from a patient with a CALR-mutation can recognize and kill autologous CALR-mutant cells. Surprisingly, healthy individuals display frequent and strong T cell responses to the CALR neo-antigens too. Furthermore, healthy individuals display immune responses to all parts of the mutant CALR epitope, and the CALR neo-epitope specific responses are memory T cell responses. These data suggest that although healthy individuals might acquire a CALR mutation, the mutant cells can be eliminated by the immune system. Additionally, a small fraction of healthy individuals harbor a CALR exon 9 mutation. Four healthy individuals carrying CALR mutations underwent a full medical examination including a bone marrow biopsy after a median follow up of 6.2 years. None of these patients displayed any signs of CALR-mutant MPN. Additionally, all healthy individuals displayed strong CALR neo-epitope specific T cell responses suggesting that these healthy individuals retained their CALR-mutant cells in the editing stage for several years. Thus, we suggest that CALR-mutant MPN could be a disease model of cancer immuno-editing, as we have demonstrated that CALR-mutant MPN displays all three stages described in the theory of cancer immuno-editing.

Chronic neutrophilic leukaemia (CNL) is a rare type of myeloproliferative neoplasm (MPN) characterised by sustained leucocytosis (≥25×10(9)/L) with neoplastic proliferation of neutrophilic granulocytes in blood and bone marrow. In contrast to chronic myeloid leukaemia, the disease primarily involves neutrophilic lineage with persistent proliferation of mature forms of neutrophils. No consistent cytogenetic changes have been reported. Known recurrent genetic changes in other MPNs such as JAK2, MPL, CALR, BCR-ABL1, PDGFRA, PDGFRB and FGFR1 are mostly absent. Recently, mutations in colony stimulating factor 3 receptor (CSF3R) have been reported in high frequency in CNL. This discovery has provided more insight into its pathogenesis and opened up possible treatment options. In this article, we review the clinical findings, morphology, pathobiology and differential diagnosis of CNL and treatment implications of CSF3R mutations.

Myeloproliferative neoplasms (MPN) are the leading cause of splanchnic vein thrombosis (SVT). Janus kinase 2 gene (JAK2)V617F mutations are found in 80 to 90% of patients with SVT and MPN. Mutations of the calreticulin (CALR) gene have also been reported. However, as their prevalence ranges from 0 to 2%, the utility of routine testing is questionable. This study aimed to identify a group of patients with SVT at high risk of harboring CALR mutations and thus requiring this genetic testing.

CALR, JAK2V617F and thrombopoietin receptor gene (MPL) mutations were analysed in a test cohort that included 312 patients with SVT. Criteria to identify patients at high risk of CALR mutations in this test cohort was used and evaluated in a validation cohort that included 209 patients with SVT.

In the test cohort, 59 patients had JAK2V617F, five had CALR and none had MPL mutations. Patients with CALR mutations had higher spleen height and platelet count than patients without these mutations. All patients with CALR mutations had a spleen height ⩾16cm and platelet count >200×109/L. These criteria had a positive predictive value of 56% (5/9) and a negative predictive value of 100% (0/233) for the identification of CALR mutations. In the validation cohort, these criteria had a positive predictive value of 33% (2/6) and a negative predictive value of 99% (1/96).

CALR mutations should be tested in patients with SVT, a spleen height ⩾16cm, platelet count >200×109/L, and no JAK2V617F. This strategy avoids 96% of unnecessary CALR mutations testing. Lay summary: Mutations of the CALR gene are detected in 0 to 2% of patients with SVT, thus the utility of systematic CALR mutation testing to diagnose MPN is questionable. This study demonstrates that CALR mutations testing can be restricted to patients with SVT, a spleen height ⩾16cm, a platelet count >200×109/L, and no JAK2V617F. This strategy avoids 96% of unnecessary CALR mutations testing.

JAK2V617F and CALR mutated essential thrombocythemia (ET) patients have different clinical characteristics, with lower thrombosis risk in patients with CALR mutations. To elucidate the mechanism for this lower risk we studied platelet function in ET patients with either JAK2V617F or a CALR mutation. Platelet activation state was similar in ET and healthy controls at baseline using P-selectin and PAC1 flow cytometry analysis. However, CALR mutated platelets were significantly less activated following ADP stimulation compared to control or JAK2 mutated platelets (p<0.0001). In live-cell imaging of platelet attachment to immobilized fibrinogen by Interference Reflection Microscopy (IRM), the number of attached CALR mutated platelets was lower compared to control and JAK2 mutated platelets, with lower fractions of platelets achieving the fully spread state (90%, 78% and 54% of adherent cells for control, JAK2 and CALR mutated subjects, respectively). Compared to controls, ET patients, regardless of the mutation type, had increased numbers of immature platelets (IP) and leukocyte platelet aggregates (LPA), as well as plasma sP-selectin. These were all correlated with the platelet count and not to the state of platelet activation. We also found that intracellular free Ca²⁺ was increased in resting ET compared to control platelets. CALR had a more dispersed localization in activated ET platelets compared to healthy controls, and mutated CALR interact physically with TpoR in CALR mutated platelets. We hypothesize that defects in platelet activation and spreading in CALR mutated patients can explain, at least in part, the lower thrombotic tendency in CALR mutated ET patients. This article is protected by copyright. All rights reserved.

Somatic mutations in JAK2, MPL and CALR are recurrently identified in most of the cases with Philadelphia chromosome negative myeloproliferative neoplasms (MPNs). We applied four molecular genetic methods for identification of CALR exon 9 mutations, including high resolution melt (HRM) analysis, Sanger sequencing, semiconductor target genes sequencing and whole exome sequencing. A total of 78 patients with myeloid malignancies were included in the study. We identified 14 CALR exon 9 mutated cases out of 78 studied patients with myeloid malignancies. All mutated patients were diagnosed with MPN being either PMF (n = 7) or ET (n = 7). Nine cases had type 1 mutations and 5 cases had type 2 mutations. CALR exon 9, MPL exon 10 and JAK2 p. V617F were mutually exclusive. There were no statistically significant differences in the hematological parameters between the cases with CALR and JAK2 or MPL mutations. Notably, all four techniques were fully concordant in the detection of CALR mutations. This is one of the few reports on the CALR mutations frequency in South-eastern populations. Our study shows that the frequency and patterns of these mutations is identical to those in the patients' cohorts from Western countries. Besides we demonstrated the utility of four different methods for their detection.

The myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+), chronic myeloid leukemia, or negative: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph negative cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene (CALR) were detected in 56-88% of JAK2/MPL-negative patients affected by ET or PMF. The most frequent mutations in CARL gene are type-1 and 2. Currently, CALR mutations are evaluated by sanger sequencing. The evaluation of CARL mutations increases the diagnostic accuracy in patients without other molecular markers and could represent a new therapeutic target for molecular drugs.We developed a novel detection assay in order to identify type-1 and 2 CALR mutations by PNA directed PCR clamping. Seventy-five patients affected by myeloproliferative neoplasms and seven controls were examined by direct DNA sequencing and by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than sanger sequencing and it could be applied even in laboratory not equipped for more sophisticated analysis. Interestingly, we report here a case carrying both type 1 and type2 mutations in CALR gene.

Gastric cancer is one of the most common cancers in Asian countries. Searching for reliable biomarkers involving the development of gastric cancer is important for clinical practice. Quantitative proteomics has become an important method contributed to the discovery of novel diagnostic or therapeutic targets for the management of cancer. Here, we identified differently expressed proteins in gastric cancer and normal gastric tissues by using the high resolution mass spectrometer. Among the total of 2280 identified proteins, 87 were differentially expressed between gastric cancer and normal gastric tissues. Notably, several significant proteins are in the PDGF-B signaling pathway, including peroxiredoxin5 (PRDX5), S100A6, calreticulin (CALR) and cathepsin D (CTSD), which were validated by western blot. Furthermore, upstream regulators including PDGF-B, PDGFR-β, Akt, eIF4E and p70s6K were found significantly increased in the gastric cancer tissues. In addition, silencing of PRDX5 and PDGF-B suppressed the proliferation of gastric cancer cells in vitro. The administration of exogenous PDGF-BB recovered the reduced expression of PDGF-B signaling pathway in PDGF-B knockdown cells. Taken together, our findings suggested that PDGF-B signaling pathway plays an important role in the regulation of gastric cancer proliferation and the inhibition of this pathway may be a potential approach for treatment of gastric cancer.

To identify intrinsic differences in cartilage gene expression profiles between wild-type- and Dio2-/--mice, as a mechanism to investigate factors that contribute to prolonged healthy tissue homeostasis.

Previously generated microarray-data (Illumina MouseWG-6 v2) of knee cartilage of wild-type and Dio2 -/- -mice were re-analyzed to identify differential expressed genes independent of mechanical loading conditions by forced treadmill-running. RT-qPCR and western blot analyses of overexpression and knockdown of Calr in mouse chondro-progenitor cells (ATDC5) were applied to assess the direct effect of differential Calr expression on cartilage deposition.

Differential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC>> |1,5|) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044). Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition.

We here demonstrate that the beneficial homeostatic state of articular cartilage in Dio2-/- mice is accompanied with significant lower expression of Calr. Functional analyses further showed that upregulation of Calr expression could act as an initiator of cartilage destruction. The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.

Accumulating evidence of population association studies support the hypothesis that the high heritability of major psychiatric disorders is a combination of relatively common alleles of modest effect, and rare alleles some with relatively larger effects. We have previously reported low frequency mutations in the proximal promoter of the human calreticulin (CALR) gene that co-occur with the spectrum of major psychiatric disorders. One of those mutations at -205C>T (rs556992558) was detected in an isolate case of schizoaffective disorder. In the current study, the functional implication of mutation -205T is studied in the human neuronal cell lines LAN-5, BE(2)-C and HEK-293. In contrast with other mutations in the promoter region which increase gene expression activity, the -205T mutation significantly decreased gene expression in those cell lines in comparison with the wild-type -205C nucleotide (p < 0.000001, p < 0.0005, and p < 0.017, respectively). Treatment of the cell lines with the mood-stabilizing drug, valproic acid (VPA) resulted in differential gene expression activity in the mutant -205T versus the wild-type -205C construct. VPA increased gene expression activity in both constructs, while a significantly higher expression activity was observed in the mutant construct (p < 0.01), indicative of the creation of a positive effector binding site for VPA as a result of the -205T mutation. We conclude that deviation from normalcy in the level of CALR in either direction is associated with major psychiatric disorders.

Recent emerging evidence indicates that changes in gene expression levels are linked to human evolution. We have previously reported a human-specific nucleotide in the promoter sequence of the calreticulin (CALR) gene at position -220C, which is the site of action of valproic acid. Reversion of this nucleotide to the ancestral A-allele has been detected in patients with degrees of deficit in higher brain cognitive functions. This mutation has since been reported in the 1000 genomes database at an approximate frequency of <0.0004 in humans (rs138452745). In the study reported here, we present update on the status of rs138452745 across evolution, based on the Ensembl and NCBI databases. The DNA pulldown assay was also used to identify the proteins binding to the C- and A-alleles, using two cell lines, SK-N-BE and HeLa. Consistent with our previous findings, the C-allele is human-specific, and the A-allele is the rule across all other species (N=38). This nucleotide resides in a block of 12-nucleotides that is strictly conserved across evolution. The DNA pulldown experiments revealed that in both SK-N-BE and HeLa cells, the transcription repressor BEN domain containing 3 (BEND3) binds to the human-specific C-allele, whereas the nuclear factor I (NFI) family members, NF1A, B, C, and X, specifically bind to the ancestral A-allele. This binding pattern is consistent with a previously reported decreased promoter activity of the C-allele vs. the A-allele. We propose that there is a link between binding of BEND3 to the CALR rs138452745 C-allele and removal of NFI binding site from this nucleotide, and the evolution of human-specific higher brain functions. To our knowledge, CALR rs138452745 is the first instance of enormous nucleotide conservation across evolution, except in the human species.

Azimilide exhibited antiarrhythmic activity in several rodent models of ventricular arrhythmias. In the mouse chloroform model, azimilide provided limited efficacy by the i.p. route (50% at 100 mg/kg versus 20% by vehicle), and no efficacy by the oral route (300 mg/kg). In a rat model in which arrhythmias are induced by ligation and reperfusion of the left descending coronary artery (CALR model), azimilide provided dose-dependent (1-18 mg/kg) efficacy by the intravenous route. The estimated dose that suppressed ventricular fibrillation (VF) was 5.0 mg/kg i.v. At 18 mg/kg i.v. azimilide also partially suppressed ventricular tachyarrhythmia (VT) and extrasystoles (VES). Rats dosed orally (100 mg/kg) were fully protected from VF. In isolated guinea pig hearts exposed to 1 microM ouabain, azimilide at 10 microM prevented the VT and VF seen in 69% and 23%, respectively, of control hearts. In anesthetized guinea pigs, azimilide at 10 and 30 mg/kg i.v. increased the dose of ouabain required to induce VES. While sematilide, dofetilide, and E-4031 significantly increased sensitivity to the arrhythmogenic actions of ouabain (by lowering the dose that caused VF), azimilide did not. Azimilide's antiarrhythmic profile in these rodent models differs from that of other class III agents, since azimilide had less efficacy in the mouse chloroform model, could suppress VT and VES as well as VF in the CALR rat model, and protected from or did not aggravate cardiac glycoside-induced arrhythmias in guinea pigs. These results demonstrating the antiarrhythmic efficacy of azimilide in the intact animal suggest that the compound has a different profile than other class III agents.

Many genetic risk factors have been identified for causing venous thromboembolism (VTE). Most of them affect the function of natural anticoagulant pathways, particularly the protein C system, although recent studies suggest a role of components of the hematopoietic pathway in the etiology of venous thrombosis. In this case-control study, we aimed to determine the frequency of prothrombin G20210A and factor V Leiden (FVL) G1691A polymorphisms and protein C, protein S, and antithrombin III deficiencies in the East Algerian population and to investigate whether these genetic factors are associated with VTE. On the other hand, our study tends to evaluate the status of JAK2V617F and calreticulin (CALR) mutations among these cases. The participants consisted of 121 cases with VTE and 146 healthy controls. Polymorphisms of FVL G1691A and prothrombin G20210A were genotyped by polymerase chain reaction (PCR) restriction fragment length polymorphism. JAK2-V617F and calreticulin mutations were analyzed by quantitative PCR and PCR followed by capillary electrophoresis sequencing, respectively. Protein C, protein S, and antithrombin levels were determined and then hereditary deficiencies were identified. Of all cases and controls, none was a carrier of the antithrombin III deficiency, prothrombin gene G20210A, and CALR mutations. Only 1 case reported having a positive JAK2 mutation (mutant allele burden was 15%). The FVL mutation (GA/AA) was found in 14 (11.6%) cases and 2 (1.4%) controls and it was significantly different between both the groups ( P = .001). Deficiencies of protein S and protein C were detected in 17 (18.8%) cases. The univariate analysis resulted in a significant impact of FVL (odds ratio [OR] = 9.4, 95% confidence interval [CI] = 2.1-42.3; P = .003) and of protein S deficiency (OR = 16.9, 95% CI =2.1-132.8, P = .007) on the VTE status. Both factors stayed significant after adjustment for sex and age. The OR of the protein C deficiency was slightly elevated (OR = 6.4, 95% CI = 0.7-55.5), but it did not reach the level of statistical significance ( P = .091), and it was therefore not considered as a risk factor. In conclusion, coagulant factor V gene G1691A mutation and protein S deficiency constitute important genetic risk factors in patients with VTE in Eastern Algeria. The somatic mutation of JAK2 V617F and CALR mutations are less frequent causes of VTE, thus routine testing for these mutations is not recommended.

Dispersal of Glioblastoma (GBM) renders localized therapy ineffective and is a major cause of recurrence. Previous studies have demonstrated that Dexamethasone (Dex), a drug currently used to treat brain tumor-related edema, can also significantly reduce dispersal of human primary GBM cells from neurospheres. It does so by triggering α5 integrin activity, leading to restoration of fibronectin matrix assembly (FNMA), increased neurosphere cohesion, and reduction of neurosphere dispersal velocity (DV). How Dex specifically activates α5 integrin in these GBM lines is unknown. Several chaperone proteins are known to activate integrins, including calreticulin (CALR). We explore the role of CALR as a potential mediator of Dex-dependent induction of α5 integrin activity in primary human GBM cells. We use CALR knock-down and knock-in strategies to explore the effects on FNMA, aggregate compaction, and dispersal velocity in vitro, as well as dispersal ex vivo on extirpated mouse retina and brain slices. We show that Dex increases CALR expression and that siRNA knockdown suppresses Dex-mediated FNMA. Overexpression of CALR in GBM cells activates FNMA, increases compaction, and decreases DV in vitro and on explants of mouse retina and brain slices. Our results define a novel interaction between Dex, CALR, and FNMA as inhibitors of GBM dispersal.

Objective As the calreticulin (CALR) mutation frequency is significantly associated with essential thrombocythemia (ET) and primary myelofibrosis (PMF), this mutation may be an important biomarker in patients with ET and PMF. Methods We performed a literature search until April 2015 and obtained 21 relevant studies. The outcome was pooled as the effect size by using the Stata software program. Results The CALR mutation frequencies in patients with ET and PMF were 19% and 22%, respectively. The CALR mutation ratio in Asian patients with ET was 23% and higher than that in European-American patients (16%). Moreover, the mutation ratio in Asian patients with PMF was lower (21%) than that in European-American patients (23%). A slight trend toward fibrotic transformation was found in ET with CALR mutations, whereas leukemic transformation was not significant in patients with ET or PMF with CALR mutations. Conclusion CALR mutations significantly influence the incident of ET as demonstrated by the meta-analysis.

Myeloproliferative neoplasms (MPNs) are associated with somatic mutations of genes including JAK2, CALR, or MPL in hematopoietic stem cells. Various glomerular lesions are known to be involved in MPN-related glomerulopathy, including mesangial hypercellularity, segmental sclerosis, features of chronic thrombotic microangiopathy, and intracapillary hematopoietic cell infiltration. Renal extramedullary hematopoiesis (EMH) is uncommon, but it is reported to occur in the setting of MPN; however, to our knowledge, there have been no reports of renal EMH with pathologically verified mutations. We report the case of a 65-year-old woman with MPN who had a CALR mutation and developed nephrotic syndrome. Kidney biopsy showed the typical findings of MPN-related glomerulopathy. CALR mutation-specific immunostaining of the kidney revealed immunopositive cells in the EMH lesion of the interstitium, indicating that renal EMH was caused by CALR-mutated cells. Based on these findings, we diagnosed nephrotic syndrome caused by MPN-related glomerulopathy. After initiation of steroid therapy, the patient's proteinuria gradually decreased and she achieved an incomplete remission. Additionally, the patient was prescribed the JAK inhibitor ruxolitinib and maintained incomplete remission. There is no established treatment for MPN-related glomerulopathy; therefore, further studies are needed to elucidate its pathophysiology.

BACKGROUND

The 2008 WHO criteria for the diagnosis and classification of myeloproliferative neoplasms (MPN) rely in part upon the assessment of mutations in JAK2 and MPL genes. Recently, mutations in calreticulin (CALR) have been identified in MPN lacking JAK2 and MPL mutations. We have validated a sensitive fragment analysis assay to detect CALR mutations.

METHODS

Genomic DNA from peripheral blood, bone marrow, and FFPE bone marrow clot preparations from 52 MPN specimens with known JAK2 and MPL mutation status and 29 non-MPN specimens was analyzed. CALR mutation testing was performed by fragment length analysis, and the results were confirmed by sequencing. Accuracy, precision, sensitivity, specificity, and robustness of the assay were determined.

RESULTS

Forty specimens (32 JAK2+, 2 JAK2-/MPL+, and 6 JAK2-/MPL-) were negative for CALR mutations. Twelve specimens had CALR mutations including 52 bp deletion (5), 5 bp insertion (6), and a novel 9 bp deletion (1). This 9 bp inframe deletion occurring at an allelic burden of 50% would delete three amino acids. One specimen with a 52 bp deletion also had JAK2 V617F mutation. All 29 non-MPN specimens were negative for CALR mutations. The assay accurately identified the mutation status of all 52 MPN specimens and had a coefficient of variation <3% for the fragment size and mutant peaks with a sensitivity of 5% for indels.

CONCLUSIONS

Fragment analysis is an accurate and sensitive method for the detection of CALR indels. The novel 9 bp deletion is likely a germline variant. Consequence of coexisting JAK2 V617F and CALR mutations requires careful interpretation.

Bipolar neurons (BNs) in the vestibular ganglion (VG) connect vestibular hair cells with the central nervous system (CNS). Disturbed function and cell loss in central vestibular target areas or in the vestibular periphery involve BNs either retro- or anterogradely. However, the impact of central vestibular disturbances or hair cell defects on the maintenance of BNs is poorly understood. In the present study the volume of the VG, the size and total number of BNs, and the number of BNs expressing the calcium-binding protein calretinin (Calr) were quantified stereologically in the cerebellar mutants purkinje cell degeneration (pcd/pcd), weaver (wv/wv), and Lurcher (Lc/+), and in the vestibular mutants jerker (je/je), shaker-1 (sh/sh), and Varitint-waddler (Va/+). In all the different mutant mice investigated the total number of BNs did not differ from that of wildtypes. In contrast, the number of Calr-positive BNs was significantly reduced in je/je (23%) and sh/sh (33%) mutants. Reduced cell size was apparent in sh/sh mutants and the volume of the VG significantly decreased in je/je mice. Calr was virtually absent from calyx endings in the vestibular periphery of je/je, sh/sh, and Va/+ mutants, whereas in wildtypes and cerebellar mutants many calyces displayed intense Calr labeling. These results imply that the survival of BNs is apparently unaffected by the peripheral and central target defects found in the mutants investigated. Whether the decrease in Calr expression may reflect biochemical adaptations in response to input disturbances or a specific loss of large BNs is discussed.

BACKGROUND

Essential 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.

OBJECTIVE

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

METHODS

To 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.

RESULTS

At 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.

CONCLUSIONS

whole-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.

Next-generation sequencing (NGS)-based mutation panels profile multiple genes simultaneously, allowing the reporting of numerous genes while saving labor and resources. However, one drawback of using NGS is that the turnaround time is often longer than conventional single gene tests. This delay can be problematic if molecular results are required to guide therapy in patients with clinically aggressive diseases, such as acute myeloid leukemia. To overcome this limitation, we developed a novel custom platform designated as Ultra-rapid Reporting of GENomic Targets (URGENTseq), an integrated solution that includes workflow optimization and an innovative custom bioinformatics pipeline to provide targeted NGS results on fresh peripheral blood and bone marrow samples within an actionable time period. URGENTseq was validated for clinical use by determining mutant allelic frequency and minimum coverage in silico to achieve 100% concordance for all positive and negative calls between the URGENTseq and conventional sequencing approach. URGENTseq enables the reporting of selected genes useful for immediate diagnosis (CALR, CSF3R, JAK2, KRAS, MPL, NPM1, NRAS, SF3B1) and treatment decisions (IDH1, IDH2) in hematologic malignancies within 48 hours of specimen collection. In addition, we summarize the molecular findings of the first 272 clinical test results performed using the URGENTseq platform.