Thomas Buhr

Classification and staging of Ph-negative myeloproliferative disorders by histopathology from bone marrow biopsies

The present study illustrates characteristic features of histopathology in the 3 non-leukemic, Ph-negative groups of chronic myeloproliferative diseases (CMPD). Attention is paid to the final outcome of CMPD, especially its transformation into acute leukemias and the occurrence of myelofibrosis from bone marrow biopsies (BMB) in a total of 1,716 CMPD patients. Essential thrombocythemia (ET), polycythemia vera (P. vera), and chronic megakaryocytic granulocytic myelosis (CMGM) can readily be distinguished by histopathology from BMB in the great majority of patients without regarding laboratory data, leaving a compartment of about 12% unclassifiable cases. Histologic patterns of staging are the increase in number and pleomorphism of megakaryocytes (MK), increase in number and density of reticulin fibers and collagen fibrosis, and excess of blasts. These 3 criteria are each graded from 0 to 3 in every biopsy. From these, a staging results by means of the histology of BMB in each of the Ph-negative CMPD. This staging provides a classification by defined criteria which permits comparative studies, the possibility of monitoring the individual patients by follow-up histology, and offers a baseline for reliable evaluation of results from therapy protocols.

European Bone Marrow Working Group trial on reproducibility of World Health Organization criteria to discriminate essential thrombocythemia from prefibrotic primary myelofibrosis.

Background The World Health Organization classification of myeloproliferative neoplasms discriminates between essential thrombocythemia and the prefibrotic phase of primary myelofibrosis. This discrimination is clinically relevant because essential thrombocythemia is associated with a favorable prognosis whereas patients with primary myelofibrosis have a higher risk of progression to myelofibrosis or blast crisis. Design and Methods To assess the reproducibility of the classification, six hematopathologists from five European countries re-classified 102 non-fibrotic bone marrow trephines, obtained because of sustained thrombocytosis. Results Consensus on histological classification defined as at least four identical diagnoses occurred for 63% of the samples. Inter-observer agreement showed low to moderate kappa values (0.28 to 0.57, average 0.41). The percentage of unclassifiable myeloproliferative neoplasms rose from 2% to 23% when minor criteria for primary myelofibrosis were taken into account. In contrast, the frequency of primary myelofibrosis dropped from 23% to 7%, indicating that the majority of patients with a histological diagnosis of primary myelofibrosis did not fulfill the complete criteria for this disease. Thus, over 50% of cases in this series either could not be reproducibly classified or fell into the category of unclassifiable myeloproliferative neoplasms. Conclusions World Health Organization criteria for discrimination of essential thrombocythemia from prefibrotic primary myelofibrosis are poorly to only moderately reproducible and lead to a higher proportion of non-classifiable myeloproliferative neoplasms than histology alone.

JAK2V617F allele burden discriminates essential thrombocythemia from a subset of prefibrotic-stage primary myelofibrosis

OBJECTIVE Among Philadelphia chromosome-negative myeloproliferative neoplasms (Ph(-) MPN), essential thrombocythemia (ET) and the prefibrotic phase of primary myelofibrosis (PMF) represent two subtypes with considerable overlap. MATERIALS AND METHODS In this study, histopathological classification of 490 MPN cases was correlated with the allelic burden of JAK2(V617F) and MPL(W515L). RESULTS Ph(-) MPN entities largely overlap with regard to JAK2(V617F) and MPL(W515L) allele burden, but ET displayed mutant allele burden <50%. PMF with different stages of myelofibrosis all yielded similar JAK2(V617F) allele burden. At initial presentation one-quarter of prefibrotic PMF cases exhibited an allele burden exceeding 50% (38% median JAK2(V617F) alleles, n=102). In ET, its main differential diagnosis, not a single case was found with >40% JAK2(V617F) alleles (median, 24% JAK2(V617F) alleles; n=90; p<0.001). Increase in JAK2(V617F) alleles during follow-up could not be linked to fibrosis or blastic progression but was related to polycythemic transformation in ET. MPL(W515L) was found in 3% of ET and 8% of PMF, with a significantly higher percentage of mutated alleles in fibrotic than prefibrotic PMF (median, 78% MPL(W515L) alleles; p<0.05). CONCLUSION Histopathological categories ET and prefibrotic PMF correlate with significant differences in mutant allelic burden of JAK2(V617F), but not of MPL(W515L) which, by contrast to JAK2(V617F), shows a higher percentage of mutated alleles in fibrotic than in prefibrotic cases. Thus, for Ph(-) MPN in which ET and prefibrotic PMF represent the most probable diagnoses, a JAK2(V617F) allele burden >50% favors a diagnosis of prefibrotic PMF.

B-CLL developing in a patient with PV is not affected by V617F mutation of the Janus kinase 2

To the Editor: We present the case of a 79-year-old Caucasian male with polycythemia vera (PV) and the rare concomitant occurrence of B-cell chronic lymphocytic leukaemia (B-CLL). A few cases of PV and BCLL were reported in the 1980s (1–7), but with the exception of chronic myeloid leukaemia (CML), which is characterised by the Philadelphia chromosome, it has not been studied before whether chronic myeloproliferative disorders (CMPD) and lymphoproliferative malignancies are clonally related (8). The recently discovered Janus kinase 2 (JAK2) gain-of-function point mutation (1849G>T/V617F) in CMPDs enables a lineageinvolvement analysis (9–11), in order to elucidate a common origin from an aberrant haematopoietic stem cell (HSC). Indeed, a minority of PV cases showed the JAK2 clonality not only in HSC and myeloid cells but also in lymphoid cells (12). Whether these findings have any implication on the development of lymphoproliferative disorders is a matter of debate, because B-CLL and other lymphoid neoplasms were reported not to harbour the mutation (10). Clinically, the patient’s PV was known for 6 yr, before examination of the bone marrow confirmed the diagnosis according to WHO criteria, but additionally B-CLL was histologically identified, which revealed a CD23 immunophenotype (Fig. 1A, B). Flow cytometry of peripheral blood led to the detection of 12.9% leukaemic B cells characterised by kappa, CD19, CD5, CD20, CD22, CD23, human leucocyte antigen (HLA)-DR, CD25 and CD38, but only 0.1% regular differentiated B lymphocytes. B-CLLtypical trisomy 12, deletion of 11q22.3, 13q14 and 17p13 (13) were not detectable by fluorescent in situ hybridisation (FISH) of the isolated B-cell fraction. Physical examination revealed spleen enlargement andpalmarerythemaonbothsides.Laboratoryexaminations showed low erythropoietin level (2.5 U/L), elevated erythrocyte count (6.7 · 10/L), high haemoglobin (16.3 g/dL) and haematocrit (50%) with lowered levels of Mean Corpuscular Haemoglobin (MCH) (24.2 pg),MeanCorpuscularVolume (MCV) (75.7 fL) and Mean Corpuscular Haemoglobin Concentration (MCHC) (32.0 g/dL), leucocytosis (18.7 · 10/L) with 9% lymphocytes and 1% myelocytes and thrombocytosis (621 · 10/L). Therapy was based on intermittent phlebotomy, anticoagulants and analgetics. Quantification of the mutated T allele by pyrosequencing (11) was performed on mononuclear blood cells (28.3%) and formalin-fixed paraffinembedded (FFPE) bone marrow cells (25.7%). Purified granulocytes (51.1%) and laser-microdissected megakaryocytes (52.5%) represented the PV clone, whereas 100% wild-type JAK2 was detected in CD19/CD5 leukaemic B cells, CD19/CD5 B and CD3 T lymphocytes. The mutation status was further confirmed by BsaXI restriction analysis (14) (Fig. 1B, C). As a result of the lack of typical B-CLL aberrations and the unfeasibility of an X-chromosome inactivation assay in this case, we cannot rule out that both malignancies share a JAK2 aberrant HSC and that JAK2 occurred on the background of clonal haematopoiesis (15). However, although lymphocytes were reported to display JAK2 in PV patients (12), in our case the development of B-CLL was not affected by the underlying PV JAK2 clone. Presumably, the mutation is an advantage for CMPD development, but an unfavourable factor for lymphoproliferative tumorigenesis. Therefore, we conclude that the occurrence of PV and B-CLL in this patient represents two separate neoplasms of the myeloid and lymphoid lineage, which do not share a common JAK2 precursor cell. Eur J Haematol 2006: 77: 539–541 doi:10.1111/j.0902-4441.2006.t01-1-EJH2940.x All rights reserved 2006 The Authors Journal compilation 2006 Blackwell Munksgaard

Different involvement of the megakaryocytic lineage by the JAK2 V617F mutation in Polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis.

Atypical megakaryocytes provide the histomorphological hallmark of all Philadelphia-chromosome negative chronic myeloproliferative disorder (Ph− CMPD) subtypes and have not been studied so far for the JAK2V617F mutation. The mutant gene dosage was determined in isolated megakaryocytes from 68 cases of JAK2+/Ph− CMPD by a pyrosequencing assay. Megakaryocytes from essential thrombocythemia (ET) showed significantly lower levels of mutated JAK2 alleles compared to patients with chronic idiopathic myelofibrosis (cIMF) with manifest fibrosis and polycythemia vera (PV) but not to prefibrotic cIMF. Solely, ET JAK2V617F in megakaryocytes is associated with a PV-like phenotype, and at least in one patient, the JAK2 mutation was exclusively acquired within the megakaryocytic lineage. The overt differences between prefibrotic and fibrotic cIMF suggested a causative role of the gene dosage of mutant JAK2 in fibrotic progression. Megakaryocyte analysis of a follow-up of eight individual cases with sequential biopsies, however, showed that progression to homozygosity of V617F mutated JAK2 and onset of manifest fibrosis appeared to be independent events. We conclude that megakaryocytes might be the predominant or even the exclusive lineage that acquires the JAK2V617F mutation in ET and that the JAK2V617F evolution to higher gene dosages represents a dynamic and complex process substantially involving megakaryocytes.

Opposite expression pattern of Src kinase Lyn in acute and chronic haematological malignancies

Lck/yes-related novel (Lyn) tyrosine kinase overexpression has been suggested to be important for leukaemic cell growth making it an attractive target for therapy. By contrast, Lyn deficiency was shown to be responsible for a phenotype resembling myeloproliferative neoplasm (MPN) in mice. We aimed to shed more light on Lyns role in haematological neoplasm and systematically investigated Lyn expression in MPN, acute and chronic leukaemia subtypes (n = 236). On top, B-cell chronic lymphocytic leukaemia (B-CLL) and chronic myeloid leukaemia significantly overexpressed Lyn when compared to de novo acute lymphoblastic leukaemia, de novo acute myeloid leukaemia (AML) and Philadelphia-chromosome-negative myeloproliferative neoplasms (p < 0.001). Most of acute leukaemia subtypes showed a notable down-regulation of Lyn mRNA but anyhow individual cases were labelled for the active form of Lyn protein. Intriguingly, secondary AML evolved in myelodysplastic syndromes revealed almost undetectable Lyn. Overexpression of Lyn in B-CLL was associated with a significant down-regulation of microRNA-337-5p suggesting that aberrant expression of this particular microRNA could be involved in post-transcriptional control of Lyn mRNA fate. We conclude that tyrosine kinase Lyn contributes to the malignant phenotype in certain leukaemia subtypes and therefore attracts targeted therapy.

Polycythaemia vera: bone marrow histopathology under treatment with interferon, hydroxyurea and busulphan

Abstract: Little is known about long‐term effects of myelosuppressive therapy on bone marrow of patients with polycythaemia vera, since histopathology from follow‐up biopsies has not been frequently reported. Thus we conducted a retrospective morphometrical analysis of diagnostic and follow‐up biopsies of 62 patients, evaluating fibre content, megakaryocytes and bone marrow cellularity. 8/62 patients were treated with interferon‐α (INF), 11/62 with hydroxyurea (HU) and 11/62 with busulphan (BU). 32/62 served as controls; they were not treated with myelosuppressive drugs but with phlebotomy only. The median observation time was 2.3 yr. Results were compared on the basis of change per time. The bone marrow of the patients with phlebotomies only was characterised by increasing cellularity of haematopoesis, number and volume ratio of megakaryocytes and fibre content. In BU‐ and HU‐treated patients, the haematopoesis was significantly reduced. The IFN patients revealed a reduction of cellularity, which was not significant. The fibre content was reduced by BU only, but not significantly. No correlation between megakaryocytes and fibres was found. – It could be concluded therefore that: 1) fibre proliferation within the bone marrow was not significantly altered by IFN, HU or BU. 2) Cellularity of haematopoesis was reduced significantly by HU and BU but only partly by IFN, corresponding with haematological remission.

Reliability of lymphoma classification in bone marrow trephines

Summary. The aim of this study was to test and establish the accuracy and reliability of lymphoma classification in bone marrow trephines according to the new World Health Organization (WHO) classification by considering predominantly the morphology and immunophenotype. Therefore, we retrospectively compared lymphoma diagnoses, rendered exclusively on bone marrow trephines without knowledge of lymph node diagnosis in 124 patients, with the results of the reference centres that had reviewed lymph node (n = 90) or extranodal biopsies (n = 34). The overall concordance rate was higher than 85% and 91%, respectively, when patients with discordant malignancy grades were excluded. The concordance rate for low‐grade B‐cell lymphomas was 93% and for high‐grade B‐cell lymphomas 84%. The main reasons for discordant diagnoses were divergent immunophenotypes among low‐grade B‐cell lymphomas (6 out of 81, i.e. 7·4%) and discrepant malignancy grades within high‐grade B‐cell lymphomas (6 out of 31, i.e. 19·4%). No relationship between discordant diagnoses and chemotherapy given during the course of the disease with the site of biopsy (i.e. lymph nodes, extranodal sites) was noted. We conclude from our results that bone marrow trephines are a reliable tool, not only for establishing bone marrow infiltration, but also for the subtyping of lymphomas.

Different lineage involvement in myelodysplastic/ myeloproliferative disease with combined MPLW515L and JAK2V617F mutation

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Analysis of Risk Factors of the Evolution of Myelofibrosis in Pre-Fibrotic Chronic Idiopathic Myelofibrosis: A retrospective study based on follow up biopsies of 70 patients by using the RECPAM method

The advanced, fibrotic phase of chronic idiopathic myelofibrosis (CIMF) is preceded by a pre-fibrotic stage. However, the factors which may influence or predict the development of myelofibrosis are not well established. Thus we investigated follow up biopsies of 70 patients with CIMF, diagnosed in stages of no or only scant fiber increase, for the development of myelofibrosis. The influence of histopathological (megakaryocytes, initial fiber content), clinical (age, gender, splenomegaly, chemotherapy) and hematological (Hb, leukocyte- and platelet count) parameters on the development of myelofibrosis was evaluated by using the univariate Log Rank method and the multivariate recursive partition and amalgamation (RECPAM) analysis. Surveying a mean observation period of 47 months we found a development of significant myelofibrosis in 30 of the 70 patients. In the univariate analysis, the development of myelofibrosis was associated with increased megakaryocytes, initial fiber content and age of the patient. In the RECPAM analysis, the patients with a high megakaryocytic count and older age showed the highest risk of developing myelofibrosis (mean 58.3 and 60.1 months). The group with the best prognosis comprised the patients under 60 years which have a low content of megakaryocytes (mean 137 months). We found that the development of myelofibrosis in CIMF was best predicted by an increase of megakaryocytes within the bone marrow, possibly reflecting the release of growth factors by these cells. The next important risk factor was the age of the patients.