A clinical-molecular prognostic model to predict survival in patients with post polycythemia vera and post essential thrombocythemia myelofibrosis.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms with variable risk of evolution into post-PV and post-ET myelofibrosis, from now on referred to as secondary myelofibrosis (SMF). No specific tools have been defined for risk stratification in SMF. To develop a prognostic model for predicting survival, we studied 685 JAK2, CALR, and MPL annotated patients with SMF. Median survival of the whole cohort was 9.3 years (95% CI: 8-not reached-NR-). Through penalized Cox regressions we identified negative predictors of survival and according to beta risk coefficients we assigned 2 points to hemoglobin level <11 g/dl, to circulating blasts ⩾3%, and to CALR-unmutated genotype, 1 point to platelet count <150 × 109/l and to constitutional symptoms, and 0.15 points to any year of age. Myelofibrosis Secondary to PV and ET-Prognostic Model (MYSEC-PM) allocated SMF patients into four risk categories with different survival (P<0.0001): low (median survival NR; 133 patients), intermediate-1 (9.3 years, 95% CI: 8.1-NR; 245 patients), intermediate-2 (4.4 years, 95% CI: 3.2-7.9; 126 patients), and high risk (2 years, 95% CI: 1.7-3.9; 75 patients). Finally, we found that the MYSEC-PM represents the most appropriate tool for SMF decision-making to be used in clinical and trial settings.

Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.

A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype.

Minimal morphological criteria for defining bone marrow dysplasia: a basis for clinical implementation of WHO classification of myelodysplastic syndromes.

The World Health Organization classification of myelodysplastic syndromes (MDS) is based on morphological evaluation of marrow dysplasia. We performed a systematic review of cytological and histological data from 1150 patients with peripheral blood cytopenia. We analyzed the frequency and discriminant power of single morphological abnormalities. A score to define minimal morphological criteria associated to the presence of marrow dysplasia was developed. This score showed high sensitivity/specificity (>90%), acceptable reproducibility and was independently validated. The severity of granulocytic and megakaryocytic dysplasia significantly affected survival. A close association was found between ring sideroblasts and SF3B1 mutations, and between severe granulocytic dysplasia and mutation of ASXL1, RUNX1, TP53 and SRSF2 genes. In myeloid neoplasms with fibrosis, multilineage dysplasia, hypolobulated/multinucleated megakaryocytes and increased CD34+ progenitors in the absence of JAK2, MPL and CALR gene mutations were significantly associated with a myelodysplastic phenotype. In myeloid disorders with marrow hypoplasia, granulocytic and/or megakaryocytic dysplasia, increased CD34+ progenitors and chromosomal abnormalities are consistent with a diagnosis of MDS. The proposed morphological score may be useful to evaluate the presence of dysplasia in cases without a clearly objective myelodysplastic phenotype. The integration of cytological and histological parameters improves the identification of MDS cases among myeloid disorders with fibrosis and hypocellularity.

The number of prognostically detrimental mutations and prognosis in primary myelofibrosis: an international study of 797 patients.

We recently defined a high-molecular risk category (HMR) in primary myelofibrosis (PMF), based on the presence of at least one of the five 'prognostically detrimental' mutated genes (ASXL1, EZH2, SRSF2 and IDH1/2). Herein, we evaluate the additional prognostic value of the 'number' of mutated genes. A total of 797 patients were recruited from Europe (n=537) and the Mayo Clinic (n=260). In the European cohort, 167 (31%) patients were HMR: 127 (23.6%) had one and 40 (7.4%) had two or more mutated genes. The presence of two or more mutations predicted the worst survival: median 2.6 years (hazard ratio (HR) 3.8, 95% confidence interval (CI) 2.6-5.7) vs. 7.0 years (HR 1.9, 95% CI 1.4-2.6) for one mutation vs 12.3 years for no mutations. The results were validated in the Mayo cohort and prognostic significance in both cohorts was independent of International Prognostic Scoring System (IPSS; HR 2.4, 95% CI 1.6-3.6) and dynamic IPSS (DIPSS)-plus (HR 1.9, 95% CI 1.2-3.1), respectively. Two or more mutations were also associated with shortened leukemia-free survival (HR 6.2, 95% CI 3.5-10.7), also Mayo validated. Calreticulin mutations favorably affected survival, independently of both number of mutations and IPSS/DIPSS-plus. We conclude that the 'number' of prognostically detrimental mutations provides added value in the combined molecular and clinical prognostication of PMF.

Mutations and prognosis in primary myelofibrosis.

Patient outcome in primary myelofibrosis (PMF) is significantly influenced by karyotype. We studied 879 PMF patients to determine the individual and combinatorial prognostic relevance of somatic mutations. Analysis was performed in 483 European patients and the seminal observations were validated in 396 Mayo Clinic patients. Samples from the European cohort, collected at time of diagnosis, were analyzed for mutations in ASXL1, SRSF2, EZH2, TET2, DNMT3A, CBL, IDH1, IDH2, MPL and JAK2. Of these, ASXL1, SRSF2 and EZH2 mutations inter-independently predicted shortened survival. However, only ASXL1 mutations (HR: 2.02; P<0.001) remained significant in the context of the International Prognostic Scoring System (IPSS). These observations were validated in the Mayo Clinic cohort where mutation and survival analyses were performed from time of referral. ASXL1, SRSF2 and EZH2 mutations were independently associated with poor survival, but only ASXL1 mutations held their prognostic relevance (HR: 1.4; P=0.04) independent of the Dynamic IPSS (DIPSS)-plus model, which incorporates cytogenetic risk. In the European cohort, leukemia-free survival was negatively affected by IDH1/2, SRSF2 and ASXL1 mutations and in the Mayo cohort by IDH1 and SRSF2 mutations. Mutational profiling for ASXL1, EZH2, SRSF2 and IDH identifies PMF patients who are at risk for premature death or leukemic transformation.

The JAK2 V617F mutation in patients with cerebral venous thrombosis.

BACKGROUND: It is currently unclear whether or not cerebral venous thrombosis, such as splanchnic venous thrombosis, can be the first manifestation of an underlying myeloproliferative neoplasm. OBJECTIVE: To determine the prevalence of the JAK2 V617F mutation in patients with a first episode of cerebral venous thrombosis. PATIENTS: In this retrospective cohort study, patients with cerebral venous thrombosis were tested for the JAK2 V617F mutation and were followed until the development of a myeloproliferative neoplasm or censored at the end of follow-up. RESULTS: Ten of 152 patients (6.6%) carried the JAK2 V617F mutation. Three of them had known acquired risk factors for thrombosis, and five had thrombophilia. Six patients met the diagnostic criteria for myeloproliferative neoplasm at the time of cerebral venous thrombosis, and three additional patients developed the disease during the follow-up (median duration 7.8 years, range 6 months to 21.3 years), giving an annual incidence of 0.26% patient-years (95% confidence interval 0.05-0.64). The last patient has no evidence of disease after 3 years of follow-up. Patients without the JAK2 V617F mutation at the time of cerebral venous thrombosis were retested at the end of the follow-up and remained negative, with normal blood counts (log-rank test χ(2) : 159 [P<0.0001]). CONCLUSIONS: Cerebral venous thrombosis can be the first symptom of a myeloproliferative neoplasm. Patients with cerebral venous thrombosis can carry the JAK2 V617F mutation, irrespective of blood count.

A prospective study of 338 patients with polycythemia vera: the impact of JAK2 (V617F) allele burden and leukocytosis on fibrotic or leukemic disease transformation and vascular complications.

We studied the relationship between JAK2 (V617F) mutant allele burden and clinical phenotype, disease progression and survival in patients with polycythemia vera (PV). The percentage of granulocyte mutant alleles was evaluated using a quantitative real-time polymerase chain reaction-based allelic discrimination assay. Of the 338 patients enrolled in this prospective study, 320 (94.7%) carried the JAK2 (V617F) mutation. Direct relationships were found between mutant allele burden and hemoglobin concentration (P=0.001), white blood cell count (P=0.001), spleen size (P=0.001) and age-adjusted bone marrow cellularity (P=0.002), while an inverse relationship was found with platelet count (P<0.001). During the study period, eight patients progressed to post-PV myelofibrosis (MF) (all carrying >50% mutant alleles), while 10 patients developed acute myeloid leukemia (AML). The mutant allele burden was significantly related to the risk of developing myelofibrosis (P=0.029) and retained its significant effect also in multivariable analysis (P=0.03). By contrast, the risk of developing AML as well as that of thrombosis was not significantly related to mutant allele burden. Leukocytosis did not affect thrombosis, MF, leukemia or survival. In conclusion, a JAK2 (V617F) allele burden >50% represents a risk factor for progression to MF in PV.

Deletions of the transcription factor Ikaros in myeloproliferative neoplasms.

Transformation to acute leukemia is a major complication of myeloproliferative neoplasms (MPNs), however, the genetic changes leading to transformation remain largely unknown. We screened nine patients with post-MPN leukemia for chromosomal aberrations using microarray karyotyping. Deletions on the short arm of chromosome 7 (del7p) emerged as a recurrent defect. We mapped the common deleted region to the IKZF1 gene, which encodes the transcription factor Ikaros. We further examined the frequency of IKZF1 deletions in a total of 29 post-MPN leukemia and 526 MPN patients without transformation and observed a strong association of IKZF1 deletions with post-MPN leukemia in two independent cohorts. Patients with IKZF1 loss showed complex karyotypes, and del7p was a late event in the genetic evolution of the MPN clone. IKZF1 deletions were observed in both undifferentiated and differentiated myeloid cell types, indicating that IKZF1 loss does not cause differentiation arrest but rather renders progenitors susceptible to transformation, most likely through chromosomal instability. Induced Ikzf1 haploinsufficiency in primary murine progenitors resulted in elevated Stat5 phosphorylation and increased cytokine-dependent growth, suggesting that reduced expression of IKZF1 is sufficient to perturb growth regulation. Thus, IKZF1 loss is an important step in the leukemic transformation of a subpopulation of MPN patients.

Blood p50 evaluation enhances diagnostic definition of isolated erythrocytosis.

BACKGROUND: High oxygen-affinity haemoglobin variants and 2,3-diphosphoglycerate (2,3-DPG) deficiency are inherited diseases generating low tissue oxygen tension and erythropoietin-driven erythrocytosis, that characterizes the clinical phenotype of patients. Level of blood p50 (the oxygen tension at which haemoglobin is 50% saturated) is used to recognize these conditions. OBJECTIVES: To define the clinical utility of blood p50 measurement in the diagnosis of isolated erythrocytosis. SUBJECTS AND DESIGN: Venous blood p50 measurement was included in the diagnostic work-up of 102 consecutive patients with isolated erythrocytosis besides blood cell count, arterial oxygen saturation, serum erythropoietin measurement and screening for JAK2 mutations. SETTING: Haematological Outpatient Section at University Hospital. RESULTS: Seven patients had relative erythrocytosis. Among 95 patients with absolute erythrocytosis, 4 (4.2%) had decreased p50 level. The extended study of family members revealed a familial inheritance. Two families had haemoglobin variants already described as Haemoglobin Malmö and Haemoglobin San Diego. In one family, the proband had a new high oxygen-affinity haemoglobin variant (Haemoglobin Safi) resulting from the transversion C-->A at codon 81 of the alpha2-globin gene. In the last family, a deficiency of 2,3-DPG was found. Within the 91 patients with normal p50 values, 46 (51%) had secondary erythrocytosis, 13 (14%) polycythemia vera and 32 (35%) idiopathic erythrocytosis. CONCLUSIONS: This study suggests that the investigation of blood p50 level may be useful to define diagnosis in patients with isolated erythrocytosis.

Cord blood in vitro expanded CD41 cells: identification of novel components of megakaryocytopoiesis.

BACKGROUND: Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets. AIM: To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out. METHODS: Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the dchip and pam algorithms. RESULTS: A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the 'MK-core' isolated by informatics tools. CONCLUSIONS: This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.

Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome.

Erythroid dysplasia is the pathologic hallmark of myelodysplastic syndromes (MDS). To develop a quantitative flow-cytometry approach to its evaluation, we analyzed the expression of CD71, CD105, cytosolic H-ferritin (HF), cytosolic L-ferritin (LF) and mitochondrial ferritin (MtF) in erythroblasts from 104 MDS patients, 69 pathologic control patients and 19 healthy subjects. Six-parameter, 4-color flow cytometry was employed, and data were expressed as mean fluorescence intensity. Compared with pathologic and healthy controls, MDS patients had higher expression of HF (P < 0.001) and CD105 (P < 0.001), and lower expression of CD71 (P < 0.001). MtF was specifically detected in MDS with ringed sideroblasts, and there was a close relationship between its expression and Prussian blue staining (r = 0.89, P < 0.001). In vitro cultures of myelodysplastic hematopoietic progenitors showed that both HF and MtF were expressed at a very early stage of erythroid differentiation, and that MtF expression is specifically related to mitochondrial iron loading. A classification function based on expression levels of HF, CD71 and CD105 allowed us to correctly classify > 95% of MDS patients. This flow-cytometry approach provides an accurate quantitative evaluation of erythroid dysplasia and allows a reliable diagnosis of sideroblastic anemia, and may therefore be a useful tool in the work-up of patients with MDS.

Mutations in the XPC gene in families with xeroderma pigmentosum and consequences at the cell, protein, and transcript levels.

Xeroderma pigmentosum (XP)-C is one of the more common complementation groups of XP, but causative mutations have thus far been reported for only six cases (S. G. Khan et al., J. Investig. Dermatol., 115: 791-796, 1998; L. Li et al., Nat. Genet., 5: 413-417, 1993). We have now extended this analysis by investigating the genomic and coding sequence of the XPC gene, the level of expression of the XPC transcript and the status of the XPC protein in 12 unrelated patients, including all of the 8 Italian XP-C cases identified thus far and in 13 of their parents. Eighteen mutations were detected in the open reading frame of the XPC gene, 13 of which are relevant for the pathological phenotype. The mutations are distributed across the gene, with no indication of any hotspots or founder effects. Only 1 of the 13 relevant changes is a missense mutation, the remainder causing protein truncations as a result of nonsense mutations (3), frameshifts (6), deletion (1) or splicing abnormalities (2). These findings indicate that the XPC gene is not essential for cell proliferation and viability and that mutations causing minor structural alterations may not give an XP phenotype and may not, therefore, be identified clinically. XP13PV was the only patient carrying a missense mutation (Trp690Ser on the paternal allele). This was also the only patient in which the XPC transcript was present at a normal level and the XPC protein was detectable, although at a lower than normal level. No quantitative alterations in the transcript or protein levels were detected in the XP-C heterozygous parents. However, the expression of the normal allele predominated in all of them, except the father of XP13PV, which suggests the existence of a possible mechanism for monitoring the amount of the XPC protein.

Diet treatment of branched chain ketoaciduria studied by proton magnetic resonance spectroscopy.

A novel nuclear magnetic resonance method is proposed for the diagnosis and follow-up of patients affected by branched chain ketoaciduria. The method allows quantitation of the branched chain amino acids (BCAA's) such as leucine, isoleucine and valine and of related keto- and hydroxy acids by means of a single spectrum. The method implies short time of analysis, as opposed to the very long time required by the techniques currently in use (amino acid analyzer combined with gaschromatography/mass spectrometry of keto- and hydroxyacids), it is easy and suitable for adjustements of the dietary treatment even on a daily basis. The case of a 15 days old newborn child, presenting muscular hypertonicity was unambiguously diagnosed in few minutes by means of one single NMR spectrum of urine. More interestingly, NMR spectra of serum in the following days were suitable for quantitating amino-, and keto acids as well as other metabolites of relevance in the follow up of the dietary treatment of the disease. After a diet lacking of BCAA's, to eliminate keto acids, a low BCAA diet was introduced, that succeeded in keeping the serum levels of the three amino acids within the normal range, while dropping the related keto acids.

[Posterior cranial fossa hemorrhage in term newborn infants: clinical and ultrasonographic aspects in 2 cases]. / L'emorragia della fossa cranica posteriore nel neonato a termine: aspetti clinici ed ecografici di due casi.

Haemorrhages of the posterior cranial fossa were diagnosed by cerebral sonography, sometimes these lesions were misinterpreted by the examination. We report two cases of posterior fossa haemorrhages in the term newborn without ultrasonography signs of this pathology. In a case some days after haemorrhagical damage, the signs of past haemorrhage were demonstrated.