Fine-needle aspiration diagnosis of clonal extramedullary hematopoiesis in a case of myeloproliferative neoplasm.

Extramedullary hematopoiesis (EMH)-the proliferation of hematopoietic progenitors outside of the bone marrow (BM) is a well-known phenomenon in myeloproliferative neoplasms (MPN). Abundant literature describes EMH at various body sites in cases of MPN, and some studies showed the presence of cytogenetic changes associated with MPN in the EMH tissues. We present a case of an 80-year-old female, with a history of MPN, presenting with mediastinal adenopathy. The transbronchial fine-needle aspiration (FNA) of the mediastinal lymph node showed EMH with atypical megakaryocytes and del(13q) demonstrated by fluorescence in situ hybridization. The subsequent BM biopsy demonstrated myelofibrosis with atypical megakaryocytes harboring the same cytogenetic abnormality. Our case highlights the capability of FNA cytology for providing accurate morphologic, immunohistochemical, and cytogenetic diagnosis of clonal EMH.

LN-Derived Fibroblastic Reticular Cells and Their Impact on T Cell Response-A Systematic Review.

Fibroblastic reticular cells (FRCs), usually found and isolated from the T cell zone of lymph nodes, have recently been described as much more than simple structural cells. Originally, these cells were described to form a conduit system called the "reticular fiber network" and for being responsible for transferring the lymph fluid drained from tissues through afferent lymphatic vessels to the T cell zone. However, nowadays, these cells are described as being capable of secreting several cytokines and chemokines and possessing the ability to interfere with the immune response, improving it, and also controlling lymphocyte proliferation. Here, we performed a systematic review of the several methods employed to investigate the mechanisms used by fibroblastic reticular cells to control the immune response, as well as their ability in determining the fate of T cells. We searched articles indexed and published in the last five years, between 2016 and 2020, in PubMed, Scopus, and Cochrane, following the PRISMA guidelines. We found 175 articles published in the literature using our searching strategies, but only 24 articles fulfilled our inclusion criteria and are discussed here. Other articles important in the built knowledge of FRCs were included in the introduction and discussion. The studies selected for this review used different strategies in order to access the contribution of FRCs to different mechanisms involved in the immune response: 21% evaluated viral infection in this context, 13% used a model of autoimmunity, 8% used a model of GvHD or cancer, 4% used a model of Ischemic-reperfusion injury (IRI). Another four studies just targeted a particular signaling pathway, such as MHC II expression, FRC microvesicles, FRC secretion of IL-15, FRC network, or ablation of the lysophosphatidic acid (LPA)-producing ectoenzyme autotaxin. In conclusion, our review shows the strategies used by several studies to isolate and culture fibroblastic reticular cells, the models chosen by each one, and dissects their main findings and implications in homeostasis and disease.

Reticulin staining pattern in the differential diagnosis of benign parathyroid lesions.

AIM: A reticulin staining pattern (RSP) can be used for the differential diagnosis of endocrine gland lesions, as in the adrenal and hypophysis glands. We aimed to use RSP for the differential diagnosis of parathyroid gland lesions. MATERIALS AND METHODS: In this study, we evaluated 97 parathyroid lesions in 85 patients, as well as 29 normal parathyroid glands. All sections were stained with a silver impregnation-based kit for the reticulin stain. The RSPs were classified as short thick fiber-, anastomosing- and nodular/alveolar-pattern. The dominant pattern was accepted as being greater than 50% in each section. RESULTS: Short thick fibers and anastomosing and nodular RSPs were seen in adenomas, but there was no alveolar pattern. Although nodular/alveolar patterns were seen in focal areas in hyperplasia, they never became the dominant pattern. Nodular dominant RSPs were seen in adenomas; however, nodular RSPs were not seen in hyperplasia in a dominant pattern (p = 0.049). While short thick fibers were not seen in normal glands, they could be seen in adenomas (p < 0.001) and in hyperplasia (p < 0.001). CONCLUSION: RSPs can be used in the differential diagnosis of parathyroid lesions. While short thick reticular fibers support adenomas and hyperplasia rather than normal tissue, a nodular dominant pattern supports adenomas rather than hyperplasia.

Association of mast cells and bone marrow reticulin fibrosis in patients with bcr-abl negative chronic myeloproliferative neoplasms.

BACKGROUND: We aimed to investigate the association of bone marrow mast cell numbers (MCN) and the degree of reticulin fibrosis in patients with chronic myeloproliferative neoplasms (MPN). METHODS: This was a case-control study that recruited 47 patients who were diagnosed with bcr-abl negative MPN. Thirty patients with lymphoma served as controls. JAK2 mutation was studied and all subjects underwent bone marrow biopsy at the time of diagnosis. Mast and CD34+ cells were counted. Marrow reticulin fiber was graded. RESULTS: Thirty-four patients had essential thrombocythemia (ET), 8 patients had primary myelofibrosis (PMF) and 5 patients had polycythemia vera (PV). Fourteen MPN patients had JAK2, whereas the controls had not. MCN was higher in patients than controls (p = 0.001). There was no significant difference regarding CD34. Reticulin fibrosis was present in 57.4% of MPN patients, whereas there was any in controls. PMF patients had more CD34 + cells than PV and ET. PMF patients had more reticulin fibers compared with other subgroups (p < 0.001). MCN, but not CD34+ cell counts, was significantly higher in JAK2(+) patients than JAK2(-) patients. CONCLUSION: MCN and reticulin fibrosis were significantly increased in MPN patients. JAK2 positivity had significantly increased MCN compared to patients without JAK2. JAK2 was associated with increased reticulin fibrosis.

Human Mesenchymal Stromal Cells Engineered to Express Collagen VII Can Restore Anchoring Fibrils in Recessive Dystrophic Epidermolysis Bullosa Skin Graft Chimeras.

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genodermatosis caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils at the dermal-epidermal junction. With no curative treatments presently available, retrovirally transduced autologous epidermal grafts and intradermal lentivirally engineered fibroblast injections are being investigated. Alternative approaches aim to infuse allogeneic mesenchymal stromal cells (MSCs) to provide a more generalized treatment for RDEB. We investigated whether healthy human MSCs could be engineered to overexpress C7 and correct RDEB in a human:murine chimeric model. Initially, engineered MSCs incorporated ex vivo into RDEB grafts, their presence confirmed by fluorescence in situ hybridization, revealed recovery of function of the dermal-epidermal junction with no signs of blister formation. Importantly, the detection of anchoring fibrils by transmission electron microscopy corroborated structural recovery. Next, MSCs cotransduced to express C7 and luciferase were delivered intradermally into grafted RDEB skin, resulting in localized MSC persistence with deposition of de novo C7 at the site. Notably, C7 expression was sufficient to restore anchoring fibril density to normal levels. In contrast, intravenously injected engineered MSCs were undetectable within grafts and lacked anchoring fibril reconstitution. Our data suggest that although localized correction may be achievable using engineered MSCs, strategies for systemic administration require further modeling.

TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity.

Fibroblastic reticular cells (FRCs), a subpopulation of stromal cells in lymphoid organs and fat-associated lymphoid clusters (FALCs) in adipose tissue, play immune-regulatory roles in the host response to infection and may be useful as a form of cell therapy in sepsis. Here, we found an unexpected major role of TLR9 in controlling peritoneal immune cell recruitment and FALC formation at baseline and after sepsis induced by cecal ligation and puncture (CLP). TLR9 regulated peritoneal immunity via suppression of chemokine production by FRCs. Adoptive transfer of TLR9-deficient FRCs more effectively decreased mortality, bacterial load, and systemic inflammation after CLP than WT FRCs. Importantly, we found that activation of TLR9 signaling suppressed chemokine production by human adipose tissue-derived FRCs. Together, our results indicate that TLR9 plays critical roles in regulating peritoneal immunity via suppression of chemokine production by FRCs. These data form a knowledge basis upon which to design new therapeutic strategies to improve the therapeutic efficacy of FRC-based treatments for sepsis and immune dysregulation diseases.

The sympathomimetic agonist mirabegron did not lower JAK2-V617F allele burden, but restored nestin-positive cells and reduced reticulin fibrosis in patients with myeloproliferative neoplasms: results of phase II study SAKK 33/14.

The ß-3 sympathomimetic agonist BRL37344 restored nestin-positive cells within the stem cell niche, and thereby normalized blood counts and improved myelofibrosis in a mouse model of JAK2-V617F-positive myeloproliferative neoplasms. We therefore tested the effectiveness of mirabegron, a ß-3 sympathomimetic agonist, in a phase II trial including 39 JAK2-V617F-positive patients with myeloproliferative neoplasms and a mutant allele burden more than 20%. Treatment consisted of mirabegron 50 mg daily for 24 weeks. The primary end point was reduction of JAK2-V617F allele burden of 50% or over, but this was not reached in any of the patients. One patient achieved a 25% reduction in JAK2-V617F allele burden by 24 weeks. A small subgroup of patients showed hematologic improvement. As a side study, bone marrow biopsies were evaluated in 20 patients. We found an increase in the nestin+ cells from a median of 1.09 (interquartile range 0.38-3.27)/mm2 to 3.95 (interquartile range 1.98-8.79)/mm2 (P<0.0001) and a slight decrease of reticulin fibrosis from a median grade of 1.0 (interquartile range 0-3) to 0.5 (interquartile range 0-2) (P=0.01) between start and end of mirabegron treatment. Despite the fact that the primary end point of reducing JAK2-V617F allele burden was not reached, the observed effects on nestin+ mesenchymal stem cells and reticulin fibrosis is encouraging, and shows that mirabegron can modify the microenvironment where the JAK2-mutant stem cells are maintained. (Registered at clinicaltrials.gov identifier: 02311569).

Histological dating of subdural hematoma in infants.

BACKGROUND: After infant deaths due to non-accidental head injury (NAHI) with subdural hematoma (SDH), the magistrates ask experts to date the traumatic event. To do so, the expert only has tools based on adult series of NAHI. We aimed to develop an SDH dating system applicable to infants aged under 3 years. METHODS AND RESULTS: We studied a retrospective multicenter collection of 235 infants who died between the ages of 0 and 36 months, diagnosed with SDH by forensic pathological examination and with known posttraumatic interval (PTI). Two pathologists assessed blindly and independently 12 histomorphological criteria relating to the clot and 14 relating to the dura mater in 73 victims (31 girls, 42 boys) whose median age was 3.8 months. Histopathological changes were significantly correlated with PTI for the appearance of red blood cells (RBCs) and the presence or absence of siderophages, and regarding the dura mater, the quantity of lymphocytes, macrophages, and siderophages; presence or absence of hematoidin deposits; collagen and fibroblast formation; neomembrane thickness; and presence or absence of neovascularization. Dating systems for SDH in adults are not applicable to infants. Notably, neomembrane of organized connective tissue is formed earlier in infants than in adults. CONCLUSION: Our dating system improves the precision and reliability of forensic pathological expert examination of NAHI, particularly for age estimation of SDH in infants. However, the expert can only define a time interval. Histopathology is indispensable to detect repetitive trauma.

Ethanol extract of Curcuma longa rhizome mitigates potassium bromate-induced liver changes in the Wistar rat: Histological, histochemical and immunohistochemical assessments.

The effects of Curcuma longa rhizome on hepatic cells, glycogen, connective tissue fibres and filamentous cytoskeleton were evaluated following KBrO3-induced liver injury in Wistar rats. Thirty-five male rats were randomly divided into seven groups (n=5). Group 1 were normal saline treated rats. Hepatic injury was induced in groups 2 to 7 by oral administration of 100mg/kg KBrO3 for 2 weeks. Following induction, rats in group 2 were sacrificed while groups 3, 4, 5 were given oral dose of EECLOR at 100, 200, 400mg/kg respectively. Group 6 rats were treated with silymarine while group 7 rats were left untreated. The rats were sacrificed and the liver sections were stained with H&E, Masson trichrome, Gordon and Sweets, PAS, Feulgen reaction, anti-vimentin antibody for demonstration of general histoarchitecture, elastic fibre, collagen fibre; glycogen, nuclear DNA and filamentous cytoskeleton respectively. Groups 2, 3, 7 developed intranuclear vacuolation, plasma coagulation, plamolysis, karyopyknosis, karyorrhexis and karyolysis, hyperchromatism, DNA fading and pleomorphism. Immunohistochemical study revealed near negative immunoreaction for vimentin. These pathological changes were ameliorated in EECLOR-treated groups in a manner comparable to silymarine-treated group. The study concluded that ameliorative effects of EECLOR in KBrO3-induced liver injury could be due to its vimentin stabilization property.

Captopril mitigates splenomegaly and myelofibrosis in the Gata1low murine model of myelofibrosis.

Allogeneic stem cell transplantation is currently the only curative therapy for primary myelofibrosis (MF), while the JAK2 inhibitor, ruxolitinib. Has been approved only for palliation. Other therapies are desperately needed to reverse life-threatening MF. However, the cell(s) and cytokine(s) that promote MF remain unclear. Several reports have demonstrated that captopril, an inhibitor of angiotensin-converting enzyme that blocks the production of angiotensin II (Ang II), mitigates fibrosis in heart, lung, skin and kidney. Here, we show that captopril can mitigate the development of MF in the Gata1low mouse model of primary MF. Gata1low mice were treated with 79 mg/kg/d captopril in the drinking water from 10 to 12 months of age. At 13 months of age, bone marrows were examined for fibrosis, megakaryocytosis and collagen expression; spleens were examined for megakaryocytosis, splenomegaly and collagen expression. Treatment of Gata1low mice with captopril in the drinking water was associated with normalization of the bone marrow cellularity; reduced reticulin fibres, splenomegaly and megakaryocytosis; and decreased collagen expression. Our findings suggest that treating with the ACE inhibitors captopril has a significant benefit in overcoming pathological changes associated with MF.

Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis.

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

Defective WNT signaling associates with bone marrow fibrosis-a cross-sectional cohort study in a family with WNT1 osteoporosis.

This study explores bone marrow function in patients with defective WNT1 signaling. Bone marrow samples showed increased reticulin and altered granulopoiesis while overall hematopoiesis was normal. Findings did not associate with severity of osteoporosis. These observations provide new insight into the role of WNT signaling in bone marrow homeostasis. INTRODUCTION: WNT signaling regulates bone homeostasis and survival and self-renewal of hematopoietic stem cells. Aberrant activation may lead to osteoporosis and bone marrow pathology. We aimed to explore bone marrow findings in a large family with early-onset osteoporosis due to a heterozygous WNT1 mutation. METHODS: We analyzed peripheral blood samples, and bone marrow aspirates and biopsies from 10 subjects with WNT1 mutation p.C218G. One subject was previously diagnosed with idiopathic myelofibrosis and others had no previously diagnosed hematologic disorders. The findings were correlated with the skeletal phenotype, as evaluated by number of peripheral and spinal fractures and bone mineral density. RESULTS: Peripheral blood samples showed no abnormalities in cell counts, morphology or distributions but mild increase in platelet count. Bone marrow aspirates (from 8/10 subjects) showed mild decrease in bone marrow iron storages in 6 and variation in cell distributions in 5 subjects. Bone marrow biopsies (from 6/10 subjects) showed increased bone marrow reticulin (grade MF-2 in the myelofibrosis subject and grade MF-1 in 4 others), and an increase in overall, and a shift towards early-phase, granulopoiesis. The bone marrow findings did not associate with the severity of skeletal phenotype. CONCLUSIONS: Defective WNT signaling associates with a mild increase in bone marrow reticulin and may predispose to myelofibrosis, while overall hematopoiesis and peripheral blood values are unaltered in individuals with a WNT1 mutation. In this family with WNT1 osteoporosis, bone marrow findings were not related to the severity of osteoporosis.

Prognostic significance of a comprehensive histological evaluation of reticulin fibrosis, collagen deposition and osteosclerosis in primary myelofibrosis patients.

AIMS: To evaluate whether a comprehensive histological evaluation of reticulin fibrosis, collagen deposition and osteosclerosis in bone marrow trephine biopsies (BMBs) of primary myelofibrosis (PMF) patients may have prognostic implications. METHODS AND RESULTS: Reticulin fibrosis, collagen deposition and osteosclerosis were graded from 0 to 3 in a series of 122 baseline BMBs. Then, we assigned to each case a comprehensive score [reticulin, collagen, osteosclerosis (RCO) score, ranging from 0 to 9] that allowed us to distinguish two groups of patients, with low-grade (RCO score 0-4) and high-grade (RCO score 5-9) stromal changes. Of 122 patients, 88 displayed a low-grade and 34 a high-grade RCO score. The latter was associated more frequently with anaemia, thrombocytopenia, peripheral blood blasts and increased lactate dehydrogenase levels. The RCO score was correlated strictly with overall mortality (P = 0.013) and International Prognostic Scoring System (IPSS) risk categories, and was able to discriminate the overall survival of both low- and high-grade patients (log-rank test: P < 0.001). Moreover, it proved to be more accurate than the European Consensus on Grading of Bone Marrow Fibrosis (ECGMF grade) in identifying high-risk patients with poor prognosis. Finally, a combined analysis of RCO scores and IPSS risk categories in an integrated clinical-pathological evaluation was able to increase the positive predictive value (PPV) for mortality in high-risk patients. CONCLUSION: The comprehensive RCO score, obtained by histological evaluation of reticulin fibrosis, collagen deposition and osteosclerosis was prognostically significant and more accurate than ECGMF grade in identifying high-risk patients and improved PPV when applied in addition to IPSS.

Rethinking the usefulness of bone marrow biopsy on treatment decision in CLL patients at diagnosis.

We aimed to investigate the role of bone marrow infiltration pattern (BMIP) and bone marrow reticulin fibrosis (BMRF) in determining treatment demand in patients with diagnosis of chronic lymphocytic leukemia (CLL). We retrospectively evaluated the data of 65 patients, who were followed with the diagnosis of CLL at Istanbul Training and Research Hospital, Department of Hematology, between July 2007 and June 2016. The median age of the patients was 64 years (range, 32-83). Twenty-three (35.4%) patients were female, and 42 (64.6%) were male. Early/mild grade BMRF was observed in 46 (70.8%) patients and advanced grade BMRF in 19 (29.2%) patients. Eleven (23.9%) of 46 patients with early/mild grade BMRF and 10 (52.9%) of 19 patients with advanced grade BMRF required treatment during follow-up (p = 0.04). According to the BMIP, 14 (21.5%) patients had diffuse and 51 (78.5%) patients had non-diffuse BMIP. Eleven (78.6%) of 14 patients with diffuse BMIP and 10 (19.6%) of 51 patients with non-diffuse BMIP required treatment during follow-up (p < 0.001). In univariate analysis, both advanced grade BMRF and diffuse BMIP had an impact on occurrence of treatment demand (p = 0.028, HR = 3.535 vs. p < 0.01 HR = 15.033). Multivariate analysis also revealed diffuse BMIP to be effective (p < 0.001, HR 13.089), while advanced grade BMRF failed to significantly influence treatment demand (p = 0.140, HR 2.664). In conclusion, in the light of our findings, it is reasonable to consider that bone marrow biopsy at the time of diagnosis might provide a preliminary information about treatment demand in patients with CLL.

Prognostic impact of bone marrow fibrosis in polycythemia vera: validation of the IWG-MRT study and additional observations.

In 2012, the International Working Group for Myeloproliferative Neoplasms (MPN) Research and Treatment (IWG-MRT) reported an associations between mild bone marrow (BM) fibrosis (⩾grade 1) in polycythemia vera (PV) and a lower incidence of thrombosis during the clinical course and a higher risk of fibrotic progression. The objective in the current study of 262 patients with PV was to validate these observations and also identify other risk factors for myelofibrosis-free survival (MFFS). About 127 (48%) patients displayed ⩾grade 1 reticulin fibrosis at the time of diagnosis; presenting clinical and laboratory features were not significantly different between patients with or without BM fibrosis. In univariate analysis, BM fibrosis had no significant impact on overall, leukemia-free or thrombosis-free survival, whereas a significant association was noted for MFFS (P=0.009, hazard ratio 2.9; 95% confidence interval 1.32-6.78); other risk factors for MFFS included leukocytosis ⩾15 × 109/l, presence of palpable splenomegaly and abnormal karyotype. During multivariable analysis, leukocytosis ⩾15 × 109/l, palpable splenomegaly and ⩾grade 1 BM reticulin fibrosis remained significant. The current study validates the previously observed association between ⩾grade 1 BM reticulin fibrosis in PV and subsequent fibrotic progression, and identifies leukocytosis and palpable splenomegaly as additional risk factors for fibrotic progression; additional studies are required to clarify the impact of BM fibrosis on thrombosis and that of abnormal karyotype on MFFS.

A 2-Year, Longitudinal, Prospective Study of the Effects of Eltrombopag on Bone Marrow in Patients with Chronic Immune Thrombocytopenia.

BACKGROUND: The long-term effects of eltrombopag on bone marrow (BM) reticulin and/or collagen deposition in previously treated adults with chronic immune thrombocytopenia (ITP) were assessed. METHODS: Three BM biopsies were collected at baseline and after 1 and 2 years of eltrombopag treatment. Specimens were centrally processed, stained for reticulin and collagen, independently reviewed by 2 hematopathologists, and rated according to the European Consensus 0-3 scale of marrow fibrosis (MF). RESULTS: Of 162 patients enrolled, 93 completed all 3 protocol-specified BM biopsies. All patients with a baseline assessment were negative for collagen. Of 159 patients assessed at baseline, 150 (94%) had normal reticulin (MF-0) and 9 (6%) had minimally increased reticulin (MF-1). After 2 years, 83/93 patients (89%) with BM biopsies had MF-0, 10 (11%) had MF-1, and none had MF-2 or MF-3. Five out of 127 patients (4%) at 1 year and 1 out of 93 (1%) at 2 years had collagen deposition. None of the patients had clinical symptoms typical of BM dysfunction or abnormalities of clinical concern based on white blood cell count or peripheral blood smear. CONCLUSION: For most patients with chronic ITP, eltrombopag is not associated with clinically relevant increases in BM reticulin or collagen formation.

Extracellular matrix composition defines an ultra-high-risk group of neuroblastoma within the high-risk patient cohort.

BACKGROUND: Although survival for neuroblastoma patients has dramatically improved in recent years, a substantial number of children in the high-risk subgroup still die. METHODS: We aimed to define a subgroup of ultra-high-risk patients from within the high-risk cohort. We used advanced morphometric approaches to quantify and characterise blood vessels, reticulin fibre networks, collagen type I bundles, elastic fibres and glycosaminoglycans in 102 high-risk neuroblastomas specimens. The Kaplan-Meier method was used to correlate the analysed elements with survival. RESULTS: The organisation of blood vessels and reticulin fibres in neuroblastic tumours defined an ultra-high-risk patient subgroup with 5-year survival rate <15%. Specifically, tumours with irregularly shaped blood vessels, large sinusoid-like vessels, smaller and tortuous venules and arterioles and with large areas of reticulin fibres forming large, crosslinking, branching and haphazardly arranged networks were linked to the ultra-high-risk phenotype. CONCLUSIONS: We demonstrate that quantification of tumour stroma components by morphometric techniques has the potential to improve risk stratification of neuroblastoma patients.

Changes in bone marrow morphology in adults receiving romiplostim for the treatment of thrombocytopenia associated with primary immune thrombocytopenia.

The effects of romiplostim on bone marrow morphology were evaluated in adults with immune thrombocytopenia (ITP). Patients with platelet counts <50 × 10(9)/L, ≥1 prior ITP therapies, and no collagen at baseline received weekly subcutaneous romiplostim starting at 1 µg/kg, adjusted to maintain platelet counts between 50 and 200 × 10(9)/L. Biopsies were scheduled after 1, 2, or 3 years of romiplostim (cohorts 1, 2, and 3, respectively). Irrespective of scheduled time, biopsies were performed earlier if patients discontinued or failed to achieve/maintain a response to romiplostim. Reticulin (silver stain) and collagen (trichrome stain) were graded by two hematopathologists using the modified Bauermeister scale (0-4). Of 169 patients, 131 had evaluable biopsies; 9/131 (6.9 %) had increases of ≥2 grades on the modified Bauermeister scale (cohort 1: 0/34; cohort 2: 2/39; cohort 3: 7/58), including two with collagen. Three of the nine patients had follow-up biopsies, including one patient with collagen; changes were reversible after romiplostim discontinuation. Of the nine patients, one had neutropenia detected by laboratory test and two had adverse events of anemia, both non-serious and not treatment-related. By actual exposure (as some biopsies did not occur as scheduled), the number of patients with grade increases ≥2 were year 1: 3/41, year 2: 1/38, year 3: 5/52. Twenty-four patients sustained platelet counts ≥50 × 10(9)/L for ≥6 months with no ITP medications after discontinuing romiplostim, i.e., they entered clinical remission of their ITP. In conclusion, in patients with ITP receiving romiplostim, bone marrow changes were observed in a small proportion of patients.ClinicalTrials.gov identifier: NCT#00907478.

Bone marrow manifestations in multicentric Castleman disease.

This study aimed to document the morphological and immunophenotypic features, and describe the diagnostic features of bone marrow (BM) involvement in human herpes virus 8 Multicentric Castleman disease (HHV8-MCD). BM trephine biopsy (BMTB) specimens from 28 patients were revisited. Samples were evaluated for expression of CD3, CD20, CD138, CD68R, glycophorin C, CD42b, HHV8-latency-associated nuclear antigen (LANA1), Epstein-Barr virus-encoded small RNA and light chains. Presence of significant numbers of HHV8-LANA1(+) lymphoid/plasmacytic cells, noted in 10/28 cases, was indicative of BM involvement and was associated with low CD4 and CD8 counts in peripheral blood. The characteristic morphological appearance of MCD seen in lymph nodes is a rare finding in BMTB. 4/5 cases with lymphoid aggregates were involved by MCD, whereas 6/23 cases without lymphoid aggregates were involved by MCD (P = 0·023). 9/18 cases with hypercellular marrow were involved by MCD, whilst only 1/8 cases with normo/hypocellular marrow showed involvement by MCD (P = 0·070). While 9/21 cases with increased marrow reticulin were involved by MCD, none of the cases with no increase in reticulin were involved by MCD (P = 0·080). Reactive plasmacytosis is a frequent finding. We conclude that bone marrow is involved in a significant proportion of patients with MCD (36%), and involvement can be identified by HHV8-LANA1 immunohistochemistry.

Imatinib reduces bone marrow fibrosis and overwhelms the adverse prognostic impact of reticulin formation in patients with chronic myeloid leukaemia.

AIMS: Before the era of tyrosine kinase inhibitors (TKIs), the presence of bone marrow fibrosis (MF) in patients with chronic myeloid leukaemia (CML) has been established as a poor prognostic factor. The aim of the present study was to evaluate the effects of imatinib treatment on MF and the prognostic significance of MF at this new era of CML therapy. METHODS: The study cohort consisted of 135 patients with CML who were exposed to imatinib. The grades of MF pre and post imatinib together with cytogenetic and molecular responses were evaluated. RESULTS: Severe MF (grade II-III) was observed in 44 (33%) patients prior to imatinib therapy, and in 8 (8%) after 12 months of imatinib treatment (p=0.001). The complete cytogenetic response (CCyR) rates at 12 months did not differ according to the pre-imatinib MF grades, and CCyR rates in patients with grades 0, I, II and III MF were 36/47 (76.5%), 26/33 (78.7%), 12/23 (52.1%) and 7/10 (70%), respectively (p=0.127). There was no significant difference between patients with or without CCyR at 12 months of imatinib regarding grades of MF (p=0.785). The distribution of the major molecular response rates at 18 months according to pre-treatment grades of MF were determined as grade 0 in 38/45 (84.4%), grade I in 21/28 (75%), grade II in 14/21 (66.6%) and grade III in 7/10 (70%) (p=0.112). There was no significant difference in overall survival rates between initial MF mild (grade 0-I) and severe (grade II-III) groups (p=0.278). CONCLUSIONS: According to our findings, MF regresses with imatinib therapy over time, and the MF grades at diagnosis do not have a negative impact on the responses to imatinib treatment. Therefore, the adverse prognostic impact of the MF among patients with CML seems to disappear in the era of the TKIs.