The Incidence of Mastocytosis in Patients with Unclear Cytopenia and/or Leucocytosis: The Hryd Study

Systemic mastocytosis (SM) is reported to be a rare myeloid disease. The true incidence is unknown as the diagnosis is often missed due to the diversity of symptoms and signs. In the HRYD study, we hypothesized that the reported incidence of around 1/100000/year is too low and the assumed underdiagnosis could be reduced if serum tryptase, although only a minor criterion for the diagnosis of SM, would be measured in patients (pts) with cytopenia and/or leukocytosis. Methods: HRYD was a prospective single-center trial. The protocol was approved by the local ethics committee. The primary endpoint was the incidence of SM in a consecutive cohort of 100 unselected pts with unclear cytopenia and/or leukocytosis at the University Hospital Halle. Pts with suspected or known SM or cutaneous mastocytosis were excluded. Serum tryptase levels were measured at initial presentation (normal < 11 ng/ml). The diagnosis and classification of SM were based on the 2016 WHO classification where a baseline tryptase level > 20 ng/mL is considered as a minor criterion for SM. Routine diagnostic work-up included medical history, physical examination, blood analyses (blood picture, differential count, liver and renal function, albumin, and further tests as needed), and if indicated a bone marrow biopsy and aspiration for cytology, immunophenotyping, karyotyping, and molecular diagnostics as appropriate. Mutational profiling from a peripheral blood sample with next-generation sequencing (NGS) was done if required. All pts gave informed consent. Results: 100 pts (51% males) > 18 years (y) were recruited between February, 2019 and June, 2020. Median age was 64.5 (range 18-88) y. Based on the final diagnosis, pts were allocated to five subgroups (group A: SM, group B: non-SM malignant hematological diseases, group C: benign hematological diseases, group D: non-hematological diseases, group E: unclear diagnosis at data-cut-off) (table 1). For the entire cohort, fatigue (n=46), weight loss (n=25), and dyspnea (n=15) were the most frequent symptoms at presentation. A non-SM malignant disease (group B) was most frequently diagnosed (n=49) followed by a non-hematological cause (n=24) (figure1). With a median age of 70 (31-88) y, pts in group B tended to be older compared to those in groups C and D. Serum tryptase > 20 ng/ml was detected in 9 pts (median level 53.4, range 21.6-200) ng/ml. SM was diagnosed in 4 (4%) pts (group A). The remaining 5 pts belonged to group B, n=4 and group D, n=1. A history of allergies, skin lesions, hepatomegaly, and/or splenomegaly were not predictive for the diagnosis of SM. The median tryptase level in SM pts was 163 (range 32.1-200) ng/ml. All pts in group A suffered from SM with associated hematologic neoplasm (SM-AHN). The AHN were MDS, n=1; MPN, n=1; AML, n=2. A KIT point mutation at codon 816 was detected in one of the SM-AHN pts. Overall, a diagnosis of SM could be excluded with a probability of 85.4% and a specificity of 95% if the tryptase level was < 20 ng/ml. On the other hand and although sensitivity was 100%, the positive predictive value of a serum tryptase > 20 ng/ml for SM was only 44.4%. WBC, Hb, and platelets did not correlate with tryptase levels or the final diagnosis in all groups. In group B, three pts with BCR-ABL positive CML were diagnosed. In contrast to SM-AHN pts, marked leukocytosis with a WBC >100x109/L was present in the CML pts. NGS was performed in 27 pts. A median of 3 (range 0-8) somatic mutations were detected. From a diagnostic point of view, the added value of NGS was met in 8/27 (30%) pts. Conclusions: In an unselected cohort of pts with undiagnosed cytopenia and/or leucocytosis, the incidence of SM-AHN was higher than that for BCR-ABL positive CML. Our data question the low reported epidemiological data to SM. Because of the prognostic and therapeutic consequences of a correct diagnosis and despite the low positive predictive value of an elevated tryptase for the diagnosis of SM, including the cheap serum tryptase test in the work-up of pts with unclear clinical scenarios and abnormal hematological parameters is a simple tool to increase awareness of physicians to SM as a differential diagnosis. SM is highly unlikely if the tryptase level is below 20 ng/ml. Disclosures Niederwieser: Amgen: Speakers Bureau; Daiichi: Research Funding; Cellectis: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau. Al-Ali:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

JAK2 Negative Erythrocytosis Associated with JAK2 GGCC_46/1 Haplotype, Calr rs1049481_G, and Normal Erythropoietin Level: Is This a New Entity?

Introduction. The JAK2 haplotype known as "GGCC or 46/1 haplotype" consists of a germline combination of single nucleotide polymorphisms (SNPs) that are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPNs) positive for both JAK2 V617 and exon 12 mutations. It has been reported a significant association between JAK2 negative erythrocytosis and the simultaneous occurrence of JAK2 haplotype GGCC_46/1 and CALR rs1049481_G allele, mapping in the 3' UTR of the gene. In the present study, we investigated the presence of JAK2 haplotype GGCC_46/1 and CALR rs1049481_G allele in a more extensive series of erythrocytosis patients and evaluated a possible correlation with serum erythropoietin (EPO) level. Methods. Fifty-nine erythrocytosis patients, diagnosed at our Center from 2009 to 2020, negative for canonical JAK2 mutations, and secondary causes, were included in this study. Forty-nine (83%) out of 59 cases met only two major WHO 2016 diagnostic criteria for polycythemia vera (PV); the remaining 10 cases were "JAK2 negative PV", as they also showed the minor criterion with subnormal serum EPO. Genomic DNA from all patients was extracted from peripheral blood granulocytes and analyzed for the presence of both SNPs. The occurrence of the JAK2 haplotype was investigated by analyzing rs10974944 tagging SNP, using distal JAK2 intron 12 primers (forward: 5'-CTGTGCAGTCCAAACCCATG-3' and reverse: 5'-TTCTCTGCTTGCTAGTGGGT-3'). This amplification generated a PCR product of 272 bp, revealing a C/G substitution by Sanger sequencing. PCR investigated the occurrence of the G/T allele in the SNP rs1049481 in the CALR gene with primers CALR-UTR-F 5'-GACAAGGAGGATGATGAGGACAAA-3' and CALR-UTR-R 5'- AAAAATGAAAGTTCTCGAGTCTCACAGA-3' generating a 205 bp product. In silico data from 2504 healthy individuals of the 1000G Project (1000G) were used as a control group. Fisher's exact test was used to compare the differences of both SNPs and the frequency of their alleles between cases and controls, respectively; the c-square test was employed for evaluating the distribution of JAK2 GGCC haplotype in erythrocytosis patients and controls. Results. Thirty-eight (64.4%) and 21 (35.6%) cases resulted in being positive and negative for the JAK2 GGCC haplotype, respectively. The JAK2 GGCC haplotype occurred to be associated with erythrocytosis as a statistically significant difference in frequency was detected as respect to 2504 healthy individuals from the 1000G Project (p=0.0013). This finding was also confirmed considering the Genome Aggregation Database (gnomAD v2.1.1, https://gnomad.broadinstitute.org/)(p=0.0024). The association was also demonstrated in terms of allelic frequency (p=0.0131) and genotype distribution (p=0.0030). Regarding CALR rs1049481 SNP, a significant difference in the CALR rs1049481_G allelic rate was confirmed in our cohort compared to 1000G controls (p=0.0360). Overall, the association between JAK2 GGCC haplotype and CALR rs1049481_G was observed in 28/59 (47.5%) erythrocytosis cases compared to healthy individuals (628/2504, 25%), and this difference resulted in being statistically significant (p=0.0002). Based on the EPO level, erythrocytosis patients were divided into two groups: normal (49 cases) or subnormal (10 cases). Interestingly, the simultaneous presence of JAK2 GGCC haplotype and CALR rs1049481_G was statistically significantly associated with the erythrocytosis group showing normal EPO (p=0.0133). Conclusions. This study suggests that the JAK2 GGCC haplotype and the presence of the CALR rs1049481_G allele were significantly associated with erythrocytosis cases, negative for canonical JAK2 mutations. Our findings are in line with recent literature evidence showing that germline predisposition factors such as SNPs and haplotypes may play a crucial role in MPN pathogenesis. Moreover, this study shows that patients showing two major WHO 2016 diagnostic criteria (erythrocytosis and panmyelosis) without JAK2 mutations and with normal EPO levels can benefit from the search for germline polymorphisms combination in JAK2 and CALR driver genes for a better diagnostic classification. Therefore, the presence of these polymorphisms could represent a novel minor criterion for the diagnosis of "JAK2 negative PV". Disclosures No relevant conflicts of interest to declare.

Are the minor high bleeding risk criteria of the academic research consortium truly minor? Insights from a high-volume tertiary care pci centre

Background The Academic Research Consortium (ARC) has recently published a consensus-based definition to identify patients at high bleeding risk (HBR), reflected by a BARC 3 or 5 bleeding rate of ≥4% at 1 year after percutaneous coronary intervention (PCI). The HBR criteria included in the definition are divided into minor and major categories, with patients deemed to be at HBR if they fulfill at least one major or two minor criteria. As a result, patients who present with only one minor criterion are categorized as non-HBR. Purpose To compare the differences in baseline characteristics and 1-year bleeding and ischaemic outcomes between non-HBR patients undergoing PCI that present with only one minor HBR criterion versus those that do not fulfill any HBR criteria. Methods The study population consisted of all consecutive patients who underwent PCI with stent implantation in a single high-volume centre from January 2014 to December 2017. Patients were classified as non-HBR if they did not fulfill at least one major or two minor ARC-HBR criteria. The outcomes of interest were major bleeding (composite of peri-procedural and post-discharge bleeding), all-cause death, and myocardial infarction (MI) at 1 year. The Kaplan-Meier method was used for time-to-event analyses, with comparative risks being assessed using Cox regression. Results Of the 9,623 patients included in the analysis, 5,345 were classified as non-HBR. Within the non-HBR patients, 2,078 (38.9%) presented with only one minor HBR criterion and 3,267 (61.1%) presented with no HBR criteria. Non-HBR patients with one minor criterion were more often female, significantly older, with a higher burden of comorbidities such as diabetes mellitus, hypertension and hyperlipidaemia, and more likely to have multivessel disease as well as a history of prior MI and revascularisation, while non-HBR patients with no criteria were more likely to be smokers and have a higher BMI. Distribution of the minor HBR criteria within the group presenting with one minor criterion are illustrated in the figure. Non-HBR patients with only one minor criterion had a numerically higher rate of major bleeding compared to non-HBR patients with no criteria (3.6% vs. 2.9%, p=0.09). While the rate of all-cause death was significantly higher in the group with only one minor criterion (1.2% vs. 0.4%, p=0.004), there was no difference in the rate of MI between the two groups (2.1% vs. 1.9%, p=0.83). Hazard ratios comparing the two groups are presented in the figure. Conclusions Non-HBR patients presenting with only one minor criterion had a numerically higher rate of post-PCI bleeding and significantly higher mortality compared to those without any criteria. Nonetheless, the major bleeding rates of both groups at 1 year were less than the 4% cutoff to qualify as HBR according to the ARC definition, thereby supporting their inclusion as “minor” criteria in the recent ARC-HBR definition. Figure 1 Funding Acknowledgement Type of funding source: None

Validation of high bleeding risk criteria and definition as proposed by the academic research consortium for high bleeding risk

Aims To validate the set of clinical and biochemical criteria proposed by consensus by the Academic Research Consortium (ARC) for High Bleeding Risk (HBR) for the identification of HBR patients. These criteria were categorized into major and minor, if expected to carry in isolation, respectively, ≥4% and <4% Bleeding Academic Research Consortium (BARC) 3 or 5 bleeding risk within 1-year after percutaneous coronary intervention (PCI). High bleeding risk patients are those meeting at least 1 major or 2 minor criteria. Methods and results All patients undergoing PCI at Bern University Hospital, between February 2009 and September 2018 were prospectively entered into the Bern PCI Registry (NCT02241291). Age, haemoglobin, platelet count, creatinine, and use of oral anticoagulation were prospectively collected, while the remaining HBR criteria except for planned surgery were retrospectively adjudicated. A total of 16 580 participants with complete ARC-HBR criteria were included. After assigning 1 point to each major and 0.5 point to each minor criterion, we observed for every 0.5 score increase a step-wise augmentation of BARC 3 or 5 bleeding rates at 1 year ranging from 1.90% among patients fulfilling no criterion, through 4.01%, 5.98%, 7.42%, 8.60%, 12.21%, 12.29%, and 17.64%. All major and five out of six minor criteria, conferred in isolation a risk for BARC 3 or 5 bleeding at 1 year exceeding 4% at the upper limit of the 95% confidence intervals. Conclusion All major and the majority of minor ARC-HBR criteria identify in isolation patients at HBR.

Thrombocytosis and essential thrombocythaemia

The term thrombocytosis refers to a platelet count elevated above 450 × 109/litre, which can be (1) primary—including essential thrombocythaemia, chronic myeloid leukaemia, polycythaemia vera, and myelodysplastic syndromes; or (2) secondary—including iron deficiency, infection, blood loss, and malignancy. Essential thrombocythaemia: aetiology—the JAK2 V617F missense mutation typical of polycythaemia vera is found in about 50% of cases. In addition, 10% of patients have a mutation in the thrombopoietin receptor gene, MPL, and 30% have a mutation in calreticulin (CALR). Approximately 10% of patients have none of these mutations and are referred to as ‘triple negative’ essential thrombocythaemia. Diagnosis requires all of the following four major criteria: (1) platelet count greater than 450 × 109/litre; (2) bone marrow biopsy showing proliferation mainly of the megakaryocyte lineage with increased numbers of enlarged, mature megakaryocytes with hyperlobulated nuclei without a significant increase or left shift in neutrophil granulopoiesis or erythropoiesis and very rarely minor (grade 1) increase in reticulin fibres; (3) failure to meet the criteria for other myeloproliferative neoplasms; and (4) presence of JAK2, CALR, or MPL mutations. Alternatively, diagnosis can be met when the first three major criteria are present and the one minor criterion, namely the presence of another clonal marker or absence of evidence for reactive thrombocytosis. Treatment requires risk stratification based on the age of the patient and any prior history of thrombosis, with treatment being reserved for those at a high risk of developing complications and not introduced simply on the basis of platelet counts alone unless there is extreme thrombocytosis (>1500 × 109/litre). Therapies include low-dose aspirin and cytoreduction.

Debridement, antibiotics, and implant retention for periprosthetic knee infections: a pooling analysis of 1266 cases

Background The debridement, antibiotics, and implant retention (DAIR) procedure is an established therapeutic option for periprosthetic knee infections (PKI). However, the efficacy and the indication for this procedure are still controversial. Methods All the relevant literatures were systematically reviewed and analyzed. The present study aimed to assess the success rate of DAIR in the management of PKI, identify the factors associated with prognosis of DAIR, and establish a simple algorithm for predicting a high success rate of DAIR. Results Totally, 33 studies with 1266 cases were included. The overall success rate following DAIR in the management of PKI was 57.11%. In the subgroup analyses, the factors of “the time from symptoms to debridement was < 3 weeks” and “the bacterial species other than methicillin-resistant Staphylococcus aureus” significantly improved the success rate of DAIR and thus were defined as the major criteria. The statistically insignificant factors of “the open debridement and liner exchange” and “the comorbidity of rheumatoid arthritis” were set as the minor criteria. The success rate of DAIR for PKI meeting all the major criteria and no less than one minor criterion was 80.98%, which was significantly higher than the overall success rate of DAIR (p < 0.05). Conclusion PKI cases meeting two major criteria and no less than one minor criterion may confer a high success rate of DAIR. This simple algorithm may contribute to identifying the appropriate PKI patient for DAIR treatment and predicting the prognosis of DAIR.