Classification

The diagnosis of the MPDs is made according to a defined set of criteria originally put forward by the Polycythemia Vera Study Group (PVSG) and subsequently modified by Pearson and colleagues to reflect advances in diagnostic practice (Table 9.1). These have led to greater diagnostic uniformity and have helped clinicians make diagnostic decisions in difficult and borderline cases.

A more recent attempt to revise these criteria under the auspices of the World Health Organization has introduced a number of controversial features (Table 9.2). In PV, for example, many feel it is inappropriate to use a raised hemoglobin level (greater than 18.5 g/dl in men; greater than 16.5 g/dl in women) as a specific diagnostic criterion in the absence of a raised red cell mass. In ET and PV, there is concern about the increased reliance on bone marrow histology, the interpretation of which is frequently subjective. This is especially true for ET, in which an abnormal bone marrow biopsy is one of only two positive criteria. In addition, the degree of fibrosis allowable in a diagnosis of ET is vague; the distinction between 'prefibrotic IMF' and ET relies on subtle differences in megakaryocyte morphology and there are no data on interobserver variation when attempting to apply these criteria.

We therefore feel it is most appropriate to use the Pearson modification of the PVSG criteria (Table 9.1). Even with these strict criteria, it can be difficult to differentiate the MPDs from

Table 9.1 Modified PVSG diagnostic criteria for myeloproliferative disorders.

(a) Modified criteria for the diagnosis of polycythemia vera

A1 Raised red cell mass (>25% above normal predicted value) B1 Thrombocytosis (platelet count >400 x 109/L)

A2 Absence of cause of secondary polycythemia B2 Neutrophil leukocytosis (neutrophil count >10 x 109/L)

A3 Palpable splenomegaly B3 Splenomegaly demonstrated on isotope/ultrasound scanning

A4 Acquired cytogenetic abnormality B4 Characteristic BFU-E growth or reduced serum erythropoietin

A1 + A2 + A3 or A4 establishes PV. A1 + A2 + two of B establishes PV.

(b) Diagnostic criteria for essential thrombocythemia

1 Platelet count >600 x 109/L

2 Packed cell volume (PCV) <0.51 for males or <0.48 for females or normal red cell mass in those with a high normal PCV and splenomegaly

3 Stainable iron in marrow or normal serum ferritin or normal red cell mean corpuscular volume (MCV). If measurement suggests iron deficiency then PV cannot be excluded unless a trial of iron therapy fails to increase the red cell mass into the erythrocytotic range.

4 No Philadelphia chromosome or BCR-ABL gene rearrangement

5 Collagen fibrosis of marrow either absent or less than one-third of biopsy area without marked splenomegaly and leukoerythroblastic reaction

6 No cytogenetic or morphological evidence for a myelodysplastic syndrome

7 No cause for reactive thrombocytosis

To make a diagnosis of ET, all criteria need to be met.

(c) Characteristic features of idiopathic myelofibrosis

1 Bone marrow fibrosis

2 Extramedullary hemopoiesis

3 Splenomegaly

4 Leukoerythroblastic blood picture

5 Absence of another chronic myeloproliferative disorder

6 Absence of a condition associated with secondary bone marrow fibrosis

All patients demonstrate points 1, 4, 5 and 6. Most patients would also have points 2 and 3.

Information from Pearson (1998) and Reilly (1998).

reactive causes. Hence, the identification of target genes in these disorders would produce much-needed diagnostic tools for clinicians as well as providing important insights into the regulation of normal hematopoiesis.

The original evidence that MPDs arise as a result of transformation of a hematopoietic stem cell has come from two main sources: analysis of karyotypic abnormalities and analysis of X-chromosome inactivation patterns to determine the clonality of different cell lineages. X-inactivation assays offer a means of assessing the clonality of a population of cells without any acquired cytogenetic or molecular markers. X inactivation occurs in females as a means of dosage compensation so that equal levels of expression of X-linked genes occur in males and females. Early in embryogenesis one X chromosome is inactivated at random in each cell—a process termed 'Lyonization'. The progeny of each cell inherits this inactivation pattern. As a consequence, a normal adult female is a 'mosaic'—some cells carry an active maternal X chromosome, others an active paternal X chromosome.

A population of cells such as a tumor, being clonally derived from a single cell, will therefore all carry either an active maternal X chromosome or an active paternal X chromosome. By contrast, a polyclonal population contains some cells with an active maternal X chromosome and others with an active paternal X chromosome. Assessment of X-inactivation patterns requires the ability to distinguish paternal and maternal X chromosomes and so the various assays are all based on polymorphic X-linked genes. It is also necessary to determine which X chromosome is active. This can be achieved by monitoring expression of an X-linked gene at the protein or RNA level. Alternatively, DNA methylation can be used as a surrogate marker for gene inactivity since, for many genes, meth-ylation status correlates well with transcriptional activity. An example of a DNA-based technique, the human androgen receptor assay (HUMARA), is shown in Figure 9.2.

Initial studies on clonality used a rare polymorphism in the G6PD gene which results in two distinct protein products. In patients with PV, a single isoform was present in erythrocytes, granulocytes, platelets and bone marrow buffy coat, implying that these cells were clonally derived. Both iso-forms were expressed in lymphocytes and skin fibroblasts. The majority of erythroid and granulocyte progenitors were

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