Continuous Indexing of Fibrosis (CIF): improving the assessment and classification of MPN patients.

The grading of fibrosis in myeloproliferative neoplasms (MPN) is an important component of disease classification, prognostication and monitoring. However, current fibrosis grading systems are only semi-quantitative and fail to fully capture sample heterogeneity. To improve the quantitation of reticulin fibrosis, we developed a machine learning approach using bone marrow trephine (BMT) samples (n = 107) from patients diagnosed with MPN or a reactive marrow. The resulting Continuous Indexing of Fibrosis (CIF) enhances the detection and monitoring of fibrosis within BMTs, and aids MPN subtyping. When combined with megakaryocyte feature analysis, CIF discriminates between the frequently challenging differential diagnosis of essential thrombocythemia (ET) and pre-fibrotic myelofibrosis with high predictive accuracy [area under the curve = 0.94]. CIF also shows promise in the identification of MPN patients at risk of disease progression; analysis of samples from 35 patients diagnosed with ET and enrolled in the Primary Thrombocythemia-1 trial identified features predictive of post-ET myelofibrosis (area under the curve = 0.77). In addition to these clinical applications, automated analysis of fibrosis has clear potential to further refine disease classification boundaries and inform future studies of the micro-environmental factors driving disease initiation and progression in MPN and other stem cell disorders.

Bone marrow core biopsy in 508 consecutive patients with chronic myeloid leukemia: Assessment of potential value.

BACKGROUND: The diagnosis of chronic myeloid leukemia (CML) is based on characteristic clinical and laboratory findings and the presence of BCR/ABL1 in the blood and/or bone marrow (BM). The utility of BM core biopsy in the workup of patients with CML has been questioned. METHODS: The potential added value of BM biopsy versus aspiration in the workup of a single-institution series of 508 patients with CML at their initial presentation was systematically assessed. BM biopsy was considered essential when it was needed to establish the disease phase, often because blast counts derived from aspirate smears were misleading because the biopsy specimen was more representative of the disease. BM biopsy was considered helpful if it was needed for other nonessential reasons. RESULTS: In 127 patients (25%), BM biopsy was either essential (109 patients) or helpful (18 patients). Patients with accelerated-phase (AP) or blast-phase (BP) disease often required a biopsy related to essential reasons. High-grade myelofibrosis (MF) was more frequent in patients with AP/BP disease than patients with chronic-phase disease (P = .0005), and the identification of BP disease required a BM biopsy assessment in 75% of the patients (P = .001). A follow-up BM evaluation more often yielded inadequate aspirates in patients with inadequate BM aspirates at the time of their initial diagnosis. CONCLUSIONS: BM core biopsy remains valuable in the workup of 25% of patients with CML because it facilitates identification of the disease phase or MF. The initial grade of MF is associated with the disease stage and outcome after therapy. BM biopsy is, therefore, indicated for patients with CML who have AP/BP disease or other findings suggestive of progressive disease.

Quantitative assessment of JAK2 V617F and CALR mutations in Philadelphia negative myeloproliferative neoplasms.

BACKGROUND: Philadelphia negative myeloproliferative neoplasms (MPNs) are characterized by frequent mutations of driver genes including JAK2, CALR and MPL. While the influence of JAK2 V617F mutant allele burden on the clinical phenotype of MPN patients is well-described, the impact of CALR mutant allele burden on clinical features needs further investigation. PATIENTS AND METHODS: Quantitative assessment of JAK2 and CALR mutations was performed on diagnostic DNA samples from 425 essential thrombocythemia (ET) and 227 primary myelofibrosis patients using real-time quantitative PCR and fragment length analysis. Characterization of CALR mutations and detection of MPL mutations were performed by Sanger sequencing. RESULTS: Twelve novel CALR mutations have been identified. ET patients with CALRmut load exceeding the median value exhibited lower hemoglobin values (12.0 vs. 13.6 g/dL), higher LDH levels (510 vs. 351 IU/L) and higher rate of myelofibrotic transformation (19% vs. 5%). The CALRmut load was higher among ET patients presenting with splenomegaly compared to those without splenomegaly (50.0% vs. 43.5%). CONCLUSION: Our study confirms the clinical significance of driver mutational status and JAK2mut load in MPNs; in addition, unravels a novel clinical association between high CALRmut load and a more proliferative phenotype in ET.

Assessment of sites of marrow and extramedullary hematopoiesis by hybrid imaging in primary myelofibrosis patients.

We investigated noninvasive procedures by hybrid imaging to assess the sites of active or inactive hematopoiesis in patients with primary myelofibrosis (PMF). To this end, we used two radionuclides, technetium 99m ((99m) Tc) and indium 111-chloride ((111) In-Cl3 ), coupled with single-photon emission tomography/computed tomography (SPECT/CT). We studied five patients with PMF and one with secondary myelofibrosis (MF). The classical pattern of lower fixation of both tracers at the axial skeleton where the myelofibrotic process occurs and the reactivation of sites of active hematopoiesis at the distal skeleton were confirmed. Coupling both radionuclides to SPECT/CT imaging allowed for more precise visualization of the sites of extramedullary hematopoiesis as those observed in the spleen and liver. Splenic high uptake of (111) In-Cl3 coupled with SPECT/CT represents a pathognomonic feature of PMF. We conclude that, the hybrid imaging procedures that we studied might constitute an alternative noninvasive method for the screening of the whole-body marrow and, by this way, to assess the impact of targeted therapies in PMF patients in whom it is well known that the distribution of the hematopoietic active areas is disturbed. Hybrid imaging could also be useful for diagnostic purposes in cases of early PMF or in suspected cases of myelofibrosis secondary to polycythemia vera or essential thrombocythemia.

Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition.

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-ß. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit.

Assessment of bone marrow inflammation in patients with myelofibrosis: an 18F-fluorodeoxyglucose PET/CT study.

PURPOSE: Myelofibrosis is a haematopoietic stem cell neoplasm characterized by bone marrow inflammation, reactive marrow fibrosis and extramedullary haematopoiesis. The aim of this study was to determine if (18)F-FDG PET/CT can be used to noninvasively visualize and quantify the extent and activity of bone marrow involvement. METHODS: In 30 patients, the biodistribution of (18)F-FDG was analysed by measuring the standardized uptake value in the bone marrow compartment and spleen. Imaging findings were compared with laboratory, cytogenetic and histopathological data. RESULTS: Retention of (18)F-FDG was observed in bone marrow and spleen. Bone marrow involvement varied, ranging from mildly increased uptake in the central skeleton to extensive uptake in most parts of the skeleton. The extent of bone marrow involvement decreased over time from initial diagnosis (r s = -0.43, p = 0.019). Metabolic activity of the bone marrow decreased as the histopathological grade of fibrosis increased (r s = -0.37, p = 0.04). There was a significant positive correlation between the metabolic activity of the bone marrow and that of the spleen (p = 0.04). CONCLUSION: (18)F-FDG PET/CT is as a promising technique for the quantitation of bone marrow inflammation in myelofibrosis. Our data indicate that the intensity of bone marrow (18)F-FDG uptake decreases as bone marrow fibrosis increases. Further evaluation in prospective studies is required to determine the potential clinical impact and prognostic significance of PET.

Progressive burden of myelofibrosis in untreated patients: assessment of patient-reported outcomes in patients randomized to placebo in the COMFORT-I study.

Patient-reported outcomes (PROs) and spleen size in patients not receiving therapy (N=154) in COMFORT-I, a randomized, double-blind study of the JAK1/JAK2 inhibitor ruxolitinib in patients with intermediate-2 or high-risk myelofibrosis were evaluated. Baseline PROs indicated considerable disease burden. European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 scores, modified Myelofibrosis Symptom Assessment Form v2.0 Total Symptom Score, and Patient Reported Outcome Measurement Information System Fatigue scores worsened from baseline through week 24. At weeks 4 and 24, 18.3 and 40.2% of patients evaluated their condition as having worsened from baseline on the Patient Global Impression of Change questionnaire. Spleen volume and palpable length increased in most patients. These results demonstrate the progressive and debilitating effects of myelofibrosis. The consequences of delayed intervention should be assessed in the management of patients with myelofibrosis and treatment should be considered as clinically indicated for symptomatic relief or splenomegaly control.

Assessment of bone marrow fibrosis and angiogenesis in monitoring patients with multiple myeloma.

The aim of our study was to emphasize the importance of accurate and standardized techniques for detailed monitoring of the microenvironment in multiple myeloma (MM). Bone marrow fibrosis, angiogenesis, and plasma cell infiltrates in bone marrow biopsy (BMB) samples at the time of diagnosis and on completion of therapy were analyzed for 42 patients with newly diagnosed MM. Computerized image analysis was used for all slides stained with anti-CD138 and anti-CD34. The patients with fibrosis in pretreatment BMB samples had significantly higher microvessel density (MVD) and plasma cell infiltrates. In posttreatment BMB samples, nonresponders had a significantly higher frequency and grade of fibrosis and higher values of MVD, total vascular area, and plasma cell percentage. The overall survival of nonresponders and patients with increased marrow fibrosis in posttreatment BMB samples was significantly shorter. The obtained results confirm that complex morphologic examination of the bone marrow microenvironment during the monitoring of MM can provide better prognostic significance.

Inability of immunomorphometric assessment of angiogenesis to distinguish primary versus secondary myelofibrosis.

OBJECTIVE: To explore the utility of bone marrow (BM) angiogenesis in differentiating primary myelofibrosis (PMF) from secondary myelofibrosis (MF). STUDY DESIGN: CD34 immunostaining was performed on BM biopsies from 21 PMFs, 23 non-PMF myeloproliferative neoplasms (MPN) with associated MF, 20 secondary MF samples, and 10 nonfibrotic controls. Microvessel density (MVD) and microvessel surface area (MSA), along with blood and BM findings were compared between the groups. RESULTS: The post-MPN MF cases included chronic myeloid leukemia-MF and polycythemia vera-MF. Etiologies of secondary MF were metastatic carcinomas, non-MPN hematologic malignancies, tuberculosis, autoimmune MF, and osteopetrosis. Megakaryocytic clustering was the most frequent and intrasinusoidal hematopoiesis the most specific feature of PMF. Higher reticulin grade, collagenization, and osteomyelosclerosis were commoner in PMF. MVD and MSA were significantly increased in fibrotic marrows regardless of etiology. Although mean MVD as well as MSA were highest in PMF, extensive overlaps among groups and marked heterogeneity in the secondary MF group rendered them of limited utility in the differential diagnosis. CONCLUSION: Enhanced angiogenesis is not entirely specific for PMF. Overlaps with secondary MF limits its differential diagnostic utility. Pathogenetically, our findings suggest that enhanced angiogenesis is a secondary paraneoplastic stromal response shared by various unrelated conditions.

Nonclinical safety assessment of a synthetic peptide thrombopoietin agonist: effects on platelets, bone homeostasis, and immunogenicity and the implications for clinical safety monitoring of adverse bone effects.

RWJ-800088 is a novel, potent polyethylene glycol (PEG)-conjugated thrombopoietin (TPO) mimetic that increases platelet levels and protects against thrombocytopenia. A nonclinical safety program was customized for this peptide that takes into account its protein-like structure, synthetic chemical nature, agonist pharmacologic activity, and mode of administration. In repeat-dose toxicity studies, the salient findings were dose-related increases in circulating platelet counts, mean platelet volume, and megakaryocytes in the bone marrow with no antibody formation. Reversible myelofibrosis and hyperostosis were observed in rats, but not dogs, when the circulating platelet levels exceeded 3× those of vehicle controls. The bone effects were due to the exaggerated pharmacologic effect and excessive stimulation and elevation of megakaryocytes by TPO, which results in intramedullary proliferation of fibroblasts and mesenchymal cells followed by osseous metaplasia. These findings support the use of platelet elevations of >3× as a stopping criterion to prevent potential adverse bone-related effects in humans.

Diagnostic value of micro-CT in comparison with histology in the qualitative assessment of historical human skull bone pathologies.

Cases of pathologically changed bone might constitute a diagnostic pitfall and frequently need histological methods to be etiologically properly evaluated. With micro-computed tomography (microCT), a new epoch of 2D and 3D imaging has been launched. We evaluated the diagnostic investigation of this analytical method versus well established histological investigations of historical human bone. Pathological changes due to various etiologies (infectious, traumatic, endocrinological, neoplasia) observed in autopsy-based macerated human skulls (Galler Collection, Natural History Museum Basel, Switzerland) were investigated by microCT and compared with histological thin ground sections using polarized light. Micro-CT images visualize the architecture of the bone with high spatial resolution without preparation or destruction of the sample in the area to be sectioned. Changes in the bone surfaces as well as alterations of the diploë can be assessed. However, morphological patterns caused by reactive response, such as typical arrangements of collagen fibers, can only be visualized by the microscopic investigation of thin ground sections using polarized light. A great advantage of microCT is the high number of slices obtained so that spatial differences within the areas of the specimen become visible. Micro-CT is a valuable tool for the diagnosis of vestiges of skull bone diseases. Its advantages over histology are the fast, automated image acquisition and the fact that the specimen is not completely destroyed. Only excision of the area to be scanned is necessary, if the specimen is too large to be scanned as a whole. Further, the 3D visualization of the micro-architecture allows an easy orientation within the sample, for example, for the choice of the location of the histological slices. However, the need to differentiate woven from lamellar bone still makes histology an indispensable method.

[Evaluation of V617F mutation of JAK2 in negative chromosome Philadelphia chronic myeloproliferative disorders]. / Valoración de la mutación V617F del gen JAK2 en síndromes mieloproliferativos crónicos con cromosoma Filadelfia negativo.

BACKGROUND AND OBJECTIVE: Polycythemia vera (PV) and essential thrombocytemia (ET) are chronic myeloproliferative diseases (MPD) characterized by overactive hemopoiesis. A single point mutation of JAK2 (Val617Phe) has been detected in PV, ET and myelofibrosis (MF). The aim of this work was to investigate the JAK2 mutation in patients with MPD and to compare the results to those of the endogenous formation of BFU-E erythroid colonies (EEC). Finally, different sources of hematopoietic cells to obtain DNA were evaluated. PATIENTS AND METHOD: In this work 146 patents were studied (81 MPD: 27 PV, 28 ET, 11 MF and 15 with myeloid chronic leukemia). Moreover, 28 patients showed secondary polycythemias or reactive thrombocytosis, 8 MPD/myelodysplastic syndromes and 29 other disorders. In 54 patients, EEC were also evaluated. Peripheral blood cells were used as source of DNA in 122 patients, bone marrow in 33, cells from BFU-E in 14 and cells from EEC in 24 patients. Their DNA samples were analyzed using an allele-specific polimerase chain reaction methodology. RESULTS: The JAK2 mutation was present in 96% of PV patients, 59% of ET and 63.6% of MF. None of the remaining patients showed this mutation. Diagnostic agreement was excellent between EEC and the mutation (kappa index = 0.93; 97% positive agreement and 95% negative agreement). DNA was obtained in 119 out of 122 samples from peripheral blood, in all patients with bone marrow, and in 50% of patients with BFU-E or EEC. In 7 cases, samples from different cell sources were studied. Their results were identical. CONCLUSIONS: The V617F mutation of JAK2 is present in most of PV patients and half of those with MF or ET. There is an excellent concordance with the EEC results.

European consensus on grading bone marrow fibrosis and assessment of cellularity.

Quantification of characteristic bone marrow biopsy features includes basic parameters such as cellularity and fiber content. These are important to assess the dynamics of disease processes with a significant impact on risk stratification, survival patterns and, especially, therapy-related changes. A panel of experienced European pathologists and a foreign expert evaluated, at a multi-headed microscope, a large number of representative slides of trephine biopsies from patients with myelofibrosis in an attempt to reach a consensus on how to grade cellularity and fibrosis. This included a critical evaluation of previously described scoring systems. During the microscopic analysis and subsequent discussion and voting, the importance of age-dependent decrease in cellularity was recognized. Grading of myelofibrosis was simplified by using four easily reproducible categories including differentiation between reticulin and collagen. A consensus was reached that the density of fibers must be assessed in relation to the hematopoietic tissue. This feature is especially important in order to avoid a false impression of a reduced fiber content in fatty and/or edematous bone marrow samples after treatment. The consensus for measuring myelofibrosis by clear and reproducible guidelines achieved by our group should allow for precise grading during the disease process and after therapy.

Assessment of peripheral blood lymphocyte subsets in idiopathic myelofibrosis.

The objective of this study was to contribute to a better characterization of the immunological profile of idiopathic myelofibrosis (IM) at presentation by analysing the blood lymphocyte subsets and their possible correlations with other disease features. Absolute blood lymphocytes and lymphocyte subsets were assessed in 31 IM patients, compared with those from 34 healthy individuals, and correlated with the patients' main clinical, hematological and bone marrow histologic features. The mean lymphocyte count of the IM patients was 1.1 (SD 0.6) x 10(9)/L, versus 1.6 (SD 0.49) x 10(9)/L in controls (p = 0.0006), with 24 of the 31 patients (77.4%) showing lymphocytopenia (< 1.5 x 10(9)/L). IM patients had significantly lower counts of CD3, CD4, CD8, and CD3 -/ CD56+ cells, and significantly higher CD3 +/CD56 + lymphocyte counts. Although no significant differences were found between patients and controls with regard to CD19+/CD5+ cell counts, increased CD5 + B-cell lymphocytes were observed in three IM patients. In one of the latter patients, Ig gene rearrangement analysis of the heavy chain gene demonstrated such a subpopulation to be clonal, but the patient did not develop features of chronic lymphoid leukemia during a 5-yr follow-up. No correlation was found between the patients' blood lymphocyte counts and other disease features. We conclude that most IM patients have absolute lymphopenia, decreased T cells and increased cytotoxic T cells at diagnosis, and 10% of them show an increased CD5 + B-cell subpopulation.

[Quantitative evaluation of juxtatrabecular fibrosis in myelodysplastic syndromes]. / Valoración cuantitativa de la fibrosis yuxtatrabecular en los síndromes mielodisplásicos.

PURPOSE: Myelofibrosis is a common, poor-prognosis feature of myelodysplastic syndromes (MDS). The aim of this work was to evaluate quantitatively the extent of the juxtatrabecular fibrosis in primary MDS and in secondary myelodysplasias, along with the presence of immature precursor cells in anomalous position, that is, the displacement of granulopoiesis from the paratrabecular area to central positions. PATIENTS AND METHODS: Twenty-seven bone marrow samples were examined: 9 from primary MDS, 9 from secondary myelodysplasias, and 9 normal. The percentage of myeloblasts and promyelocytes with nucleoli located in the central areas was estimated, in an attempt to correlate such feature with the degree of juxtatrabecular fibrosis. The analysis of data was performed with the Kruskal-Wallis test, values of p < 0.01 being significant. RESULTS: Sectorial juxtatrabecular fibrosis was present in all the myelodysplastic samples, ranging from 6% to 55% of the trabecular surface; the highest values found in the controls were about 3.6% (p < 0.01). Although the juxtatrabecular fibrosis figures are higher in secondary MDS, the difference is not significant with regard to the primary MDS in the number of patients studied here. The count of myeloblasts and promyelocytes with nucleoli present in the MDS was significantly greater than that of the control group. The number of promyelocytes with nucleoli was significantly higher in the primary MDS with respect to the secondary ones, whereas no significant difference was seen between the two types of MDS regarding the myeloblast count. CONCLUSIONS: The increased number of central immature precursor cells in MDS was not directly correlated with the extent of the juxtatrabecular fibrosis. Although the number of cases is small, the fact that the juxtatrabecular fibrosis was higher in the two deceased patients in the series (49% and 56%, respectively) suggests a poor-risk prognosis for such finding.

Morphometric assessment of bone marrow fiber content in acute nonlymphatic leukemia at presentation.

The number of intersections of reticulin fibers per sq mm of fat cell-free marrow parenchyma with the lines of a grid ocular (i/sq mm) represents an objective measure of the bone marrow reticulin fiber content. This method was used to assess the reticulin fiber content of bone marrow biopsies from 50 cases of acute nonlymphatic leukemia (ANLL) at presentation and 20 controls. Seventeen (34%) of the 50 patients with ANLL showed fibrosis, i.e., had a reticulin fiber score above the upper 99% confidence limit of the mean of 20 normal control biopsies. The frequencies of marrow fibrosis, as defined above, were 47% (16 of 34) in the combined subtypes of undifferentiated (M0), myeloid (M1), myelomonocytic (M4) and monocytic (M5) acute leukemia and 7% (1 of 15) in the combined subtypes of acute myeloid leukemia with partial maturation (M2) and acute promyelocytic leukemia (M3) (P less than .01). The fibrosis scores of M0/M1/M4/M5 patients were significantly higher than those of M2/M3 patients (P less than .05) and of controls (P less than .005). Finally, the survival of patients with and without fibrosis was not different.