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Bone marrow (BM) fibrosis is a potential side effect of thrombopoietin receptor agonist (TPO-RA) treatment. We aimed to investigate stromal seromarker profiles and growth factors in order to elucidate pathogenic and dynamic aspects of immune thrombocytopenia (ITP)-related BM fibrosis before and during TPO-RA treatment. Connective tissue metabolites [procollagen I and III peptides (PINP/PIIINP); hyaluronan (HYA), C-terminal-telopeptide (ICTP), and fibrosis-related growth factors (transforming growth factor-beta (TGF-beta), HGF, basic fibroblast growth factor)] were measured in blood samples acquired before initiation of TPO-RA and subsequently at 6-month intervals for up to 2 years. BM fibrosis was graded MF-0 in 8 (18%), MF-1 30 (65%), and MF-2 8 (18%) in the last available BM biopsy. In the 21 patients having more than one biopsy, the grade of fibrosis from the first to the last available biopsy decreased in 2 (10%), remained unchanged in 15 (71%), and increased in 4 (19%). Pretreatment levels of PIIINP, PINP, ICTP, and HYA were significantly increased in ITP versus controls. PINP, PIIINP, and HYA decreased on TPO-RA; ICTP remained unchanged. PINP:ICTP was lower before and during treatment compared to controls. Pretreatment, TGF-beta was lower than in controls; HGF exhibited the opposite pattern. HYA, ICTP, and TGF-beta tended to increase while PINP and platelet-derived growth factor tended to decrease with increasing fibrosis grade. In conclusion, ITP is associated with deranged patterns of extracellular matrix seromarkers and growth factors, indicating that BM stromal remodeling is enhanced. During TPO-RA treatment for up to 2 years, this profile was partially reversed while mild BM reticulin fibrosis was still present in the majority of patients. These observations likely reflect a BM injury by autoimmunity that is modified by TPO-RA.

Monocytosis can develop during disease course in primary myelofibrosis simulating that seen in chronic myelomonocytic leukemia, and should not lead to disease reclassification. In contrast, at presentation, rare cases have clinical, morphologic, and molecular genetic features truly intermediate between primary myelofibrosis and chronic myelomonocytic leukemia. The taxonomy and natural history of these diseases are unclear. We identified cases which either: (1) fulfilled the 2008 World Health Organization criteria for primary myelofibrosis but had absolute monocytosis and, when available, chronic myelomonocytic leukemia-related mutations (ASXL1, SRSF2, TET2) or (2) fulfilled criteria of chronic myelomonocytic leukemia but had megakaryocytic proliferation and atypia, marrow fibrosis, and myeloproliferative-type driver mutations (JAK2, MPL, CALR). Patients with established primary myelofibrosis who developed monocytosis and those with chronic myelomonocytic leukemia with marrow fibrosis were excluded. By combining the pathology databases of two large institutions, six eligible cases were identified. Patients were predominantly male and elderly with monocytosis at diagnosis (average 17.5%/2.3 × 10 3 /μl), organomegaly, primary myelofibrosis-like atypical megakaryocytes admixed with a variable number of chronic myelomonocytic leukemia-like hypolobated forms, variable myelodysplasia, marrow fibrosis and osteosclerosis. All had a normal karyotype and no myelodysplasia-associated cytogenetic abnormalities. Five of the patients in whom a more extensive molecular characterization was performed showed co-mutations involving JAK2 or MPL and ASXL1, SRSF2, TET2, NRAS, and/or KRAS. Disease progression has occurred in all and two have died. Rare patients present with features that overlap between primary myelofibrosis and chronic myelomonocytic leukemia and are thus difficult to classify based on current World Health Organization criteria. Biologically, these cases likely represent primary myelofibrosis with monocytosis, dysplasia, and secondary (non-driver) mutations at presentation. Alternatively, they may represent a true gray zone of neoplasms. Their clinical behavior appears aggressive and innovative therapeutic approaches may be beneficial in this particular subset.

Because of the presence of various overlapping findings, the discrimination of polycythemia vera (PV) from prefibrotic/fibrotic primary myelofibrosis (PF/F-PMF) and essential thrombocythemia (ET) may be challenging, particularly in suboptimal bone marrow biopsy specimens. In this study, we assessed whether differences in the expression of nuclear factor-erythroid 2 (NF-E2), nerve growth factor receptor (NGFR; CD271), CD34, CD68, p53, CD3, CD20, and CD138 by immunohistochemistry could be useful in separating among them. Higher frequencies of nuclear positive erythroblasts with NF-E2 were observed in ET and PV cases (50% ± 13.3% and 41.5% ± 9.4%, respectively) when compared with both PF-PMF (21% ± 11.7%) and F-PMF (28.5% ± 10.8%). We found that with a cutoff level of at least 30% nuclear staining for NF-E2 in erythroblasts, we could reliably exclude the possibility of PMF. Conversely, NGFR+ stromal cells per high-power field (HPF) was significantly increased in F-PMF (53.5 ± 19.1/HPF) and PF-PMF (13.5 ± 3.8/HPF) compared with ET (4.4 ± 2.2/HPF) and PV (6.6 ± 3.3/HPF). Similarly, differences in CD34-microvessel density was remarkable in F-PMF and PF-PMF cases in comparison with PV and ET (49.9 ± 12.1/HPF, 29.3 ± 12.4/HPF, 13.7 ± 4.6/HPF, and 11.9 ± 5.1/HPF, respectively). Thus, the assessment of NF-E2 and NGFR expression and the evaluation of CD34-microvessel density may provide additional support in reaching a correct diagnosis in these cases of myeloproliferative neoplasms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Thrombopoietin-receptor agonists increase platelet counts by stimulating the thrombopoietin receptor. Bone marrow fibrosis has been reported in patients receiving thrombopoietin-receptor agonists. This study determined the extent of myelofibrosis, its clinical relevance, and incidence of phenotypic or karyotypic abnormalities in patients with immune thrombocytopenia treated with thrombopoietin-receptor agonists. The grade of myelofibrosis was assessed before (n=15), during (n=117) and after (n=9) treatment in bone marrow biopsies from 66 patients. The proportion of bone marrow biopsies showing no fibrosis (myelofibrosis grade 0) decreased from 67% pre-treatment to 22% at last biopsy, of which 59% had grade 1 myelofibrosis and 18% had grade 2 myelofibrosis. The median duration of treatment with thrombopoietin-receptor agonists to last bone marrow biopsies was 29 months; patients who had two or more biopsies significantly more frequently had myelofibrosis grades 2/3 in the last bone marrow biopsies as compared to the first. Older age was associated with higher grades of fibrosis. No differences in blood counts or lactate dehydrogenase levels were found between patients with myelofibrosis grades 0/1 and those with grade 2. No clonal karyotypic or immunophenotypic abnormalities emerged. This study found that thrombopoietin-receptor agonists induce myelofibrosis grades 2/3 in approximately one-fifth of patients with immune thrombocytopenia, increasingly with >2 years of treatment with thrombopoietin-receptor agonists. Annual/biannual follow-up with bone marrow biopsies is, therefore, recommended in patients being treated with thrombopoietin-receptor agonists in order to enable prompt discontinuation of these drugs should grades 2/3 myelofibrosis develop. Discontinuation of thrombopoietin-receptor agonists may prevent development of clinical manifestations by stopping progression of fibrosis in grade 2/3.

Primary myelofibrosis is a type of chronic myeloproliferative neoplasm characterized by progressive bone marrow failure with worsening cytopenia and in a subset of patients, progression to acute leukemia. Published data in patients with myelodysplastic syndromes have shown that the development of monocytosis in the course of myelodysplastic syndromes is associated with a poor prognosis. A similar occurrence has been only sporadically reported in patients with primary myelofibrosis. Over a period of four years we identified 10 out of 237 cases of primary myelofibrosis who developed persistent absolute monocytosis (>1 × 10(9)/l) during the course of disease (5 men and 5 women; median age/range: 68 years/52-82). Monocytosis developed at a median interval of 42 months from diagnosis (range: 1-180) and persisted for a median period of 23 months (range: 2-57). Five patients died after developing monocytosis (range: 20-188 months) and two experienced worsening disease and became transfusion dependent. Monocytosis was associated with increased white blood cells, decreased hemoglobin, decreased platelet count, and the presence of circulating blasts. In three cases, bone marrow biopsies after the onset of monocytosis showed marked myelomonocytic proliferation with morphological shifting from a typical primary myelofibrosis marrow appearance to aspects compatible with an overt 'secondary' chronic myelomonocytic leukemia. Before the development of monocytosis, 5 of 10 patients carried the JAK2V617F mutation; five patients showed karyotypic alterations. No change in JAK2 mutational status or cytogenetic evolution were associated with the development of monocytosis. Four of nine patients analyzed showed KRAS mutation in codon 12 or 13 with low allele burden. This is the first study correlating monocytosis developing in primary myelofibrosis patients with bone marrow morphology, laboratory data, molecular analysis and clinical follow-up. Development of monocytosis in patients with established primary myelofibrosis is associated with rapid disease progression and these patients should be considered as a high-risk group associated with short survival.

Primary immune thrombocytopenia is an acquired autoimmune disorder characterized by platelet count of <100 × 10(9)/l in the absence of other causes of thrombocytopenia. Primary immune thrombocytopenia is defined as 'chronic' when it has been present for more than 12 months without spontaneous remission or maintenance of complete response to therapy. Recently, thrombopoietin receptor agonists became available for treatment of chronic primary immune thrombocytopenia. Anecdotal reports have raised concerns about a possible association between therapy with thrombopoietin receptor agonists and an increase in bone marrow fibrosis. To investigate this association we studied eight patients with primary immune thrombocytopenia in detail comparing fibrosis and other morphological features in pre-therapy and on-therapy bone marrow biopsies, with the longest follow-up reported to date. A slight but significant increase to MF-1 in reticulin fibrosis was observed during therapy, but collagen was never present. On-therapy bone marrows were hypercellular due to panmyelosis with increased trilineage hematopoiesis. Megakaryocytes were increased in number, with acquisition of evident pleomorphism, nuclear hyperlobulation and tendency in some cases to form clusters. The overall picture of the on-therapy marrows was characterized by myeloproliferative neoplasm-like features, resembling essential thrombocythemia or occasionally early primary myelofibrosis. As thrombopoietin receptor agonists are becoming a mainstream treatment for primary immune thrombocytopenia, general pathologists and especially hematopathologists need to be aware of the characteristic morphological changes associated with use of these therapeutic agents, in order to avoid misdiagnosis of a myeloid neoplasm.

This study assessed the grade of bone marrow (BM) fibrosis and its association with a seromarker for collagen-III formation and fibrosis-related cytokines in 25 immune thrombocytopenia (ITP) patients treated with thrombopoietin receptor agonists (Tpo-RA) who had at least one BM biopsy. Assessment of 8 pre- and on-treatment BM biopsies revealed statistically significant increases in reticulin. Reticulin in biopsies performed after a median of 1·4 years of treatment was graded: MF-0 in 3 (12%), MF-1 in 19 (76%), MF-2 in 2 (8%) and MF-3 in 1 (4%). No cytogenetic or flow-cytometric abnormalities were detected. Median pretreatment Procollagen III N-propeptide (PIIINP) (6·6 μg/l) was significantly higher than on-treatment levels (5·6 μg/l); both were higher than controls (3·4 μg/l; P < 0·001). PIIINP was negatively correlated with treatment duration (r = -0·49) suggesting a decelerated reticulin production over time. There was a trend towards an association between grade of reticulin and PIIINP. Transforming growth factor (GF)-beta and basic-Fibroblast GF were not different between patients and controls but Hepatocyte GF (HGF), an anti-fibrotic cytokine, was significantly elevated in patients. In conclusion, low-grade BM reticulin fibrosis is seen in most ITP patients on Tpo-RA. The novel findings of decreasing PIIINP and elevated HGF need further investigation to explore their significance in BM fibrogenesis., (© 2011 Blackwell Publishing Ltd.)