John T. Reilly

Characterization of an acute micromegakaryocytic leukaemia: evidence for the pathogenesis of myelofibrosis

Summary The current hypothesis for the pathogenesis of myelofibrosis involves the intramedullary release of growth factors from defective or abnormal megakaryocytes. We describe a case of an acute micromegakaryocytic leukaemia, in a patient with chronic myelofibrosis, that provides additional evidence for this concept. The micromegakaryocytes, which reached 223 × 109/1, were characterized morphologically by both light and electron microscopy, immunocytochemically and by platelet peroxidase activity. The cells were shown to have a mature cytoplasm, containing alpha granules and the associated proteins: vWF:Ag, fibrinogen, fibronectin and protein S. DNA analysis, by both a Seescan Solitaire Plus image analysis system and flow cytometry, revealed nuclear immaturity, with 92% of cells being diploid.

Evaluation of a Novel Stable Whole Blood Quality Control Material for Lymphocyte Subset Analysis: Results From the UK NEQAS Immune Monitoring Scheme

The UK NEQAS immune Monitoring Scheme (UK NEQAS) evaluates the performance of laboratories routinely performing T-lymphocyte subset analysis on HIV-infected individuals. The scheme originally issued fresh whole blood, but a significant problem was that of analyte stability, especially 36 h postphlebotomy. To circumvent this problem, we have developed a novel stabilisation procedure that ensures retention of leucocyte light scatter and immunological staining characteristics for up to 300 days. In addition, the stabilised whole blood preparation is fully compatible with flow cytometer technology, incorporating either whole blood lysis or no wash, no lyse techniques. The ranges of interlaboratory coefficient of variation for the stabilised material are now tighter than those previously obtained with fresh whole blood. Development of this novel material has enabled overseas laboratories to participate in the UK NEQAS immune Monitoring Scheme and could in the future lead to the production of reference and/or calibration reagents for leucocyte immunophenotyping.

Pathogenesis of idiopathic myelofibrosis: present status and future directions.

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia. is a chronic neoplastic disorder characterized by bone marrow fibrosis, extramedullary haemopoiesis, splenomegaly and a leucoerythroblastic blood picture (Ward & Block, 1971). The differential diagnosis encompasses the many causes of secondary bone marrow fibrosis (McCarthy, 1985), as well as the recently recognized entities of myelodysplastic syndrome with fibrosis (Pagliuca et al, 1989; Lambertenghi-Deliliers et al, 199 1) and transitional myelodysplasiamyelofibrosis (Reilly & Dolan, 1991). Failure to distinguish these conditions may account, in part, for the discrepancies in the literature, especially with regard to karyotypic patterns and prognosis. Since Heuck’s (1879) fist description and Dameshek’s (1951) hypothesis that the disease be classified as a myeloproliferative disorder, considerable progress has been made in the elucidation of its pathogenesis, especially during the last decade. Nevertheless its prognosis remains poor when compared to other chronic myeloproliferative disorders (Rozman et al, 1991) and has not changed significantly during the past 20 years. The aim of this annotation is to address these new insights and to envisage future directions of research in the next few years.

Efficacy of recombinant interferon‐alpha (rIFN‐α) in polycythaemia vera: a study of 17 patients and an analysis of published data

The efficacy and tolerability of rIFN‐α has been evaluated in 17 selected patients with symptomatic polycythaemia vera, diagnosed according to the PRV Study Group criteria. Complete disease control (CR) was achieved, after 1–12 months, in nine patients, with partial control in a further five cases. Three patients failed to respond. Pruritus significantly improved in 83% (10/12) of cases, following 1–28 weeks of treatment. Six patients (35%), however, were unable to tolerate rIFN‐α, on account of weight loss, myalgia and mental changes. Overall, α‐interferon therapy significantly improved venesection requirements, MCV and PCV values, platelet counts, pruritus scores and the degree of splenomegaly. Analysis of pooled published data (100 evaluable patients, including the present study) revealed an overall CR of 60%, a PR of 27%, and a failure rate of 13%. Significant pruritus control (>50% improvement) occurred in 77% of cases. rIFN‐α appears to be an effective therapy for PV‐associated myeloproliferation and/or pruritus, although side‐effects remain a concern. Long‐term studies are now indicated to determine if the natural history of the disease is altered, in particular whether the incidence of myelofibrosis and/or leukaemic transformation is reduced.

KARYOTYPIC AND RAS GENE MUTATIONAL ANALYSIS IN IDIOPATHIC MYELOFIBROSIS

Summary. Karyotypic analysis was performed in a total of 69 patients with well‐characterized idiopathic myelofibrosis. Karyotypic abnormalities were detected in 46% of cases examined during the chronic phase (29/63); with three abnormalities, del (13q), del(20q) and partial trisomy 1q, accounting for 75% of all abnormalities at diagnosis. The absence of del(5q), trisomy 8 and 21, as well as the rarity of monosomy 7, contrasts with pooled published data and may reflect our exclusion of closely related disorders, in particular MDS with fibrosis. Chromosomal aberrations increased to approximately 90% (8/9) in patients analysed during acute transformation. Mutational activation of codons 12, 13 and 61 of N‐, Ha‐ and Ki‐ras genes were assessed by polymerase chain reaction and hydridization with synthetic non‐radioactive digoxigenin‐labelled probes. Three mutations were detected in samples of peripheral blood DNA taken from 50 patients during the chronic phase of their disease: one N12 Asp (GGT GAT) and two N12 Ser (GGT AGT) mutations. The results from this study indicate that karyotypic abnormalities are present in at least 29% of cases at diagnosis and that del(13q), del(20q) and partial trisomy 1q are the most frequent findings. Ras mutations were relatively infrequent (6%) and appeared restricted to the N‐ras gene. Karyotypic analysis at diagnosis was found to be of prognostic significance.

Investigation of calmodulin and basic fibroblast growth factor (bFGF) in idiopathic myelofibrosis: Evidence for a role of extracellular calmodulin in fibroblast proliferation

The urinary concentration of calmodulin and basic fibroblast growth factor (bFGF) was determined in a total of 53 patients with various chronic myeloproliferative disorders (CMPD), including 22 patients with idiopathic myelofibrosis (IMF). Calmodulin excretion was significantly elevated in IMF (0.29u2003±u20030.04u2003μg/mmol creatinine) (P<0.001), when compared to polycythaemia vera (PV) (0.14u2003±u20030.02), essential thrombocythaemia (ET) (0.13u2003±u20030.04), chronic myeloid leukaemia (CML) (0.16u2003±u20030.02), unclassified myeloproliferative disorders (UMPD) (0.11u2003±u20030.02) and age‐matched controls (0.1u2003±u20030.02) (P<0.001). In contrast, bFGF was slightly elevated in all CMPD conditions when compared to age‐matched controls. A neutralizing antibody to calmodulin was demonstrated to significantly influence the in vitro proliferation of normal human fibroblasts, an effect dependent on both cell density and the presence of fetal calf serum (FCS). Essentially, the antibody reduced FCS‐induced proliferation of low‐density fibroblasts but had little or no inhibitory effect on high‐density fibroblasts in the absence of FCS. In addition, extracellular calmodulin was shown not to interact with known fibroblast mitogens, namely IFG‐1, EGF, bFGF and PDGF. We conclude that extracellular calmodulin should be considered, in addition to PDGF, TFG‐β and EGF, as a potential mitogen involved in the stromal reaction of idiopathic myelofibrosis.

Differential effect of G-CSF and GM-CSF in acquired chronic neutropenia.

Acquired chronic neutropenia is a condition of uncertain aetiology that may be complicated by severe bacterial infections. Advances in molecular biology have resulted in the availability of recombinant myeloid regulatory growth factors suitable for the treatment of isolated neutropenias. Recombinant human granulocyte colony-stimulating factor (G-CSF) increases circulating neutrophil numbers in congenital idiopathic neutropenia (Welte et al, 1990). familial cyclic neutropenia (Hammond et al, 1989) and acquired chronic neutropenia (Jakubowski et al. 1989). In contrast, recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) has been unsuccessful in the treatment of both congenital and cyclic neutropenia (Welte et al, 1990; Freund et al. 1990) and may result instead in an eosinophilia. The use of GM-CSF has not, to our knowledge, been previously reported in acquired chronic neutropenia. We describe a patient with chronic idiopathic neutropenia. complicated by refractory perianal sepsis, whose neutrophil count corrected with clinical improvement during G-CSF therapy, but whose treatment had to be discontinued due to cutaneous vasculitis. Subsequent treatment with GM-CSF, although resulting in an increased total leucocyte count due to an eosinophilia, failed to correct the neutropenia. A 5 5-year-old man presented with a perianal abscess and fistula. which despite treatment by surgical debridement and prolonged broad-spectrum antibiotics, failed to heal. Total white cell count was 0.8 x 10/l with neutrophils 0 .2 x loy/ 1. Bone marrow aspirate and trephine biopsies revealed a hypercellular marrow with myeloid hyperplasia but maturation arrest at the myelocyte stage. The patient had taken no recent medication and had no previous history of bacterial infections. Autoantibodies and antibodies to neutrophilspecific antigens were not detected. Karyotypic analysis was normal. A diagnosis of chronic idiopathic neutropenia was made and because of the presence of advancing infection, treatment with G-CSF at a dose of 4 pg/kg/d subcutaneously was commenced. The neutrophil count rose from 0.2 x 109/1 to 1.0 x 1OY/1 by day 10 (Fig 1) and coincided with the

PROPOSED CLASSIFICATION FOR THE MYELODYSPLASIA/MYELOFIBROSIS SYNDROMES

We have read with interest the recent article entitled Myelodysplastic syndrome with increased marrow fibrosis: a distinct clinico-pathological entity (Lambertenghi-Deliliers et al, 199 1 ). We would like to present a case that highlights the difficulty that may be encountered in distinguishing this entity from that of idiopathic myelofibrosis and propose the term transitional myelodysplasia-myelofibrosis for patients with the features ofour case. A 72-year-old man presented in November 1990 with a history of lethargy, weight loss and dyspnoea on exertion. Splenomegaly of 6 cm was noted on clinical examination. Haemoglobin concentration was 4.0 g/ dl, white blood count 3.7 x 1OY/1. and platelets 106 x 109/l. The differential WBC was: myeloblasts 3%. myelocytes 7%, neutrophils 52%, eosinophils 2%. basophils 3%. lymphocytes 20%,, monocytes 1 1 /o and circulating micromegakaryocytes (2%). Megakaryocyte fragments were also prominent. Nucleated red blood cells were present and dysmorphic erythrocytic changes included marked anisocytosis, basophilic stippling and tear-drop poikilocytosis. The neutrophils were agranular with many Pelger forms. Karyotypic analysis on peripheral blood was normal. Bone marrow aspirate yielded a dry tap while trephine biopsy showed a marked increase in reticulin and collagen deposition. The patient died 2 months later from bronchopneumonia. Post-mortem examination confirmed significant splenomegaly (9 50 g) with the histological features of extramedullary haematopoiesis. Myelodysplasia associated with bone marrow fibrosis has been recently highlighted by both Pagliuca et a1 (1989) and Takahashi et a1 (1991). The former group stressed the importance of differentiating these patients from those with idiopathic myelofibrosis and emphasized that, in the context of trilineage dysplasia, the absence of hepatosplenomegaly supports the diagnosis of myelodysplasia. In equivocal cases ferrokinetic studies were recommended to exclude extramedullary haematopoiesis. regarded as a sine qua non of idiopathic myelofibrosis. The case of myelodysplasia and bone marrow fibrosis reported by Verhoef et a1 (1990), however. had ferrokinetic evidence of extramedullary erythropoiesis and suggests that intermediate, or transitional,

CLINICO-PATHOLOGICAL FEATURES OF MINIMALLY DIFFERENTIATED ACUTE MYELOID LEUKAEMIA (AML-M0)

Haemophilia A is an X chromosome linked disorder characterized by a deficiency of coagulation factor VIII. Tens of heterogeneous mutations. mostly point mutations and large deletions, have been identified in factor VIII gene in haemophilia A patients. as recently reviewed by Tuddenham et a1 ( 1 991 ). About one third of point mutations reported to date are recurrent mutations of CpG dinucleotide leading to CT or CA transition. It seems, however, that at different positions the CpG dinucleotides are not affected with the same frequency, In our study of factor VIII mutations in haemophilia A we have identified two patients with loss of TaqI restriction site within the coding region of the factor VIII gene (KFepelova et al. 1992). We have now analysed both mutations using PCR amplification and direct sequencing. In the patient 1 9 Southern blot analysis of TaqI digested DNA indicated loss ofTaqI site in exon 7. Sequence analysis of exon 7 revealed a GAA (Glu) to AAA (Lys) transition in codon 272. Patient 1 9 is a mild haemophiliac (FVIII: C=6%). In his mothers DNA the mutation was not present, and therefore it probably arose de novo in her germ cells. Patient 1 9 is the first reported case with the mutation of CpG dinucleotide in TaqI site in exon 7 (codon 272) of the factor VIII gene. In patient JH 20 with the similar TaqI site loss in exon 7, CpG dinucleotide was not affected (Yossoufian et al. 1988). The mutation is localized near the C-terminal end of the A1 domain. Substitution of negatively charged Glu 272 with positively charged Lys in patient 1 9 may affect properties of this region and cause lower activity and/or stability of altered protein. In the patient 321 Southern blot analysis showed a loss of TaqI site in exon 18. PCR amplification and sequence analysis of exon 18 demonstrated a CGA (Arg) to CAA (Gln) mutation in the codon 1941. This patient suffers from a moderate haemophilia A (FV1II:C = 3%). In the patients sister carriership of haemophilia A was confirmed by demonstration of an abnormal junction fragment using Southern blot analysis. TaqI site mutation in exon 18 has been detected several times (see Tuddenham et al, 199 1, for a review). Eight cases with CGA (Arg) to TGA (Term) mutation are known, all of whom suffer from severe haemophilia A, six of them producing inhibitor. In three patients with a mild to moderate haemophilia a missense mutation CGA (Arg) to CAA (Gln) and in one case a CGA (Arg) to CTA (Leu) have been found. Our patient is probably unrelated to previously reported patients, studied in the U.S.A., Finland and Japan. Both mutations described in our patients probably arose in the CpG dinucleotides in the non-coding DNA strand.

LACK OF CORRELATION BETWEEN CELL SURFACE ACTIVATION ANTIGEN EXPRESSION AND CLINICAL STAGE IN B‐CLL

We read with interest the recent debate regarding CD23 expression in chronic lymphocytic leukaemia and its correlation to clinical stage. Dadmarz & Cawley (1988). in a study of 35 patients, reported low levels of CD23 on peripheral blood lymphocytes of patients with advanced Rai stage disease and suggested that high levels of ‘non-activated’ cells was an unfavourable prognostic sign. In contrast, Gibson et a2 (1989) found no correlation between CD23 antigen expression and clinical stage in 37 patients with B-CLL. although cases requiring chemotherapy had significantly higher expression. It was concluded that clinical disease progression from an indolent form to an active chemotherapy requiring form is manifested by transformation from a nonactivated (CD23 low) to an activated (CD23 high) phenotype. We have evaluated 47 patients with B-CLL (using flow cytometry (FACScan)) correlating Rai stage with the activation markers CD23. CD25. CD71 together with FMC-7 expression and surface membrane immunoglobulin density (SmIg). The mean CD23 antigen expression of all patients was 5 7 f 2 5% similar to the values reported by Gibson et a1 (44&25%)andDadmarz&Cawley (53&25%).However. in contrast to the latter group we found no correlation between CD 23 expression and clinical stage: 5 7 f 2 5% for Rai stage 0-11 (n=35) and 5 8 f 2 6 % for Rai stage 111-IV (n=12) (P<0.01). Furthermore, comparison of CD23 with CD25 also supports the preliminary findings of Gibson et a1 (1 989) in that when CD23 expression is high (>SO%) the CD25 receptor is also increased 49&29%. Whereas low CD23 expression (< 50%) is accompanied by low CD2 5 receptor expression (2 3 f 20%). B-CLL cases can be divided into three distinct groups according to the expression of the two activation antigens, CD25 and CD71, namely, CD25-/CD71CD25+/CD71-’+ and CD25-/CD71+ (Barnett et al, 1989). Morphologically changes in cytoplasm and nucleus are associated with the expression of one or both antigens, including an increase in cell size and the appearance of a nucleolus. Stark et a1 (1986), while reporting a correlation between FMC-7 expression and SmIg density in cases with prolymphocytoid transformation, found no relationship with clinical stage. Our results of FMC7 expression support this finding, stage 0-11 ( n = 3 5 ) 25f22% and stage 111-IV ( n = l l ) 32&25% (P<O.O1). We conclude therefore that the expression of activation antigens in B-CLL, namely CD23. CD25, CD71, FMC-7 and SmIg correlates with differentiation, i.e. prolymphocytoid morphology, and not with clinical stage as defined by the Rai system.