Xlinked clonality assays

Table 9.2 Summary of WHO diagnostic criteria for myeloproliferative disorders.

(a) Diagnostic criteria for polycythemia vera

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

or Hb >18.5 g/dl (men) or >16.5 g/dl (women) B2 Neutrophil leukocytosis (neutrophil count >12 x 109/L)

A2 Absence of cause of secondary polycythemia B3 Bone marrow biopsy showing panmyelosis with prominent

A3 Splenomegaly erythroid and megakaryocyte proliferation

A4 Acquired clonal genetic abnormality other than B4 Low serum erythropoietin

Philadelphia chromosome or BCR-ABL

A5 Endogenous erythroid colony formation in vitro

A1 + A2 + one of A3, A4 or A5 establishes PV. A1 + A2 + two of B establishes PV.

(b) Diagnostic criteria for essential thrombocythemia

1 Platelet count >600 x 109/L

2 Bone marrow biopsy showing megakaryocytic lineage proliferation with an increased number of enlarged mature megakaryocytes

3 No evidence for polycythemia vera

Normal red cell mass or Hb <18.5 g/dl (men), 16.5 g/dl (women)

Stainable iron in marrow, normal serum ferritin or normal MCV. If this condition is not met (iron deficiency) PV cannot be excluded unless a

trial of iron fails to increase red cell mass or Hb levels to the PV range.

4 No evidence for CML

No Philadelphia chromosome or BCR-ABL gene rearrangement

5 No evidence for IMF

Collagen fibrosis is absent

Reticulin fibrosis is minimal or absent

6 No evidence for a myelodysplastic syndrome

No del(5q), t(3;3)(q21;q26), inv (3)(q21;q26)

No significant granulocytic dysplasia; few if any micromegakaryocytes

7 No cause for reactive thrombocytosis due to

Underlying inflammation or infection

Underlying neoplasia

Prior splenectomy

All seven criteria need to be met to confirm a diagnosis of ET.

(c) Characteristic features of idiopathic myelofibrosis


Clinical/laboratory findings

Prefibrotic IMF Minimal or absent reticulin fibrosis

Mild anemia

Hypercellular BM with increased number of neutrophils

Mild to moderate leukocytosis

Increased numbers and clustering of atypical megakaryocytes

Mild to marked thrombocytosis

No or mild leukoerythroblastosis

No or mild splenomegaly or hepatomegaly

No or minimal red cell poikilocytosis

Fibrotic IMF Reticulin and/or collagen fibrosis

Splenomegaly and hepatomegaly

Decreased BM cellularity

Moderate to marked anemia

Diluted marrow sinuses containing pockets of hematopoietic precursors

WBC and platelet count variable

Increased numbers and clustering of atypical megakaryocytes

New bone formation


Prominent red cell poikilocytosis (dacrocytes)

also clonally derived. Similar patterns were also observed for ET and IMF.

Subsequent studies using methylation of X-linked genes as a marker of gene inactivity have demonstrated an unbalanced pattern of methylation consistent with clonality in white blood cells or bone marrow cells from a high proportion of patients with ET, PV or IMF. However, in most of the early studies no control was performed to exclude the possibility of

Myeloproliferative disorders 93








Clonal mat




Digestion with Hhal pat

Digestion with Hhal pat

PCR amplification with 32P-labeled primer. Polyacrylamide gel electrophoresis of products

PCR amplification with 32P-labeled primer. Polyacrylamide gel electrophoresis of products

mat mat

(a) First exon of the human androgen receptor locus (HUMARA). The first exon contains two recognition sites for the methylation-sensitive restriction enzyme Hhal. Methylation of these sites correlates with X-chromosome inactivation. The sites lie very close to a highly polymorphic (CAG)n repeat. PCR primers can therefore be designed which flank both the Hhal site and the polymorphic (CAG)n repeat.

(b) Determination of clonality of a population of cells using the HUMARA. *Methylated restriction site. Hhal sites on the active X chromosome are unmethylated and are therefore digested with the enzyme, whereas methylated sites will not be cleaved. If the maternally and paternally derived alleles contain different numbers of the (CAG) repeat, their size can be distinguished following amplification by PCR and separation by polyacrylamide gel electrophoresis. Adapted from Allen etal. (1992).

skewed Lyonization, a situation which can mimic true clonality. The most appropriate tissue to use for such a control is T cells, and the demonstration of a polyclonal pattern in T cells together with a skewed pattern in granulocytes (or other appropriate lineage) in female patients (Figure 9.3) was generally considered as evidence for the presence of a clonal myeloid malignancy.

Interestingly, a small number of well-characterized patients with PV and ET possess polyclonal T cells and polyclonal granulocytes (Figure 9.3). These results suggest that, in some patients, only a small proportion of granulocytes are part of the neoplastic clone or that the granulocytic lineage is not involved at all. Alternatively, polyclonal hematopoiesis may still be present in some patients fulfilling current criteria for an MPD. These results have been confirmed in patients with ET by analysis of polymorphisms at the level of mRNA and hence do not merely reflect alterations in the methylation status of these patients' granulocytes. The use of RNA-based methods has also shown that a small number of ET patients possess clonal platelets but polyclonal granulocytes and T


- + w -

- + ^


- + w -

. + ^


- + 1



(a) Clonal

Fig. 9.3 HUMARA assay results

Examples of results obtained with three MPD patient samples are shown.

G, granulocytes; T, T cells; + and - refer to the presence and absence of predigestion with the methylation-sensitive restriction enzyme Hha\. (a) Clonal pattern observed in most MPD patients. T cells exhibit balanced X-inactivation whereas granulocytes display a skewed pattern. (b) A small number of patients have polyclonal T cells and polyclonal granulocytes. (c) This result is ambiguous since both the granulocytes and T cells give a skewed pattern. This is consistent with either excessive Lyonization or with T cells arising from the malignant clone.

cells. These various observations have contributed to a growing realization that heterogeneity exists within each diagnostic category and especially for ET. It has also been suggested that ET patients demonstrating polyclonal hemopoiesis may be at a lower risk of suffering a thrombotic event.

Although a minority of MPD patients possess polyclonal granulocytes, the finding of a skewed pattern in granulocytes with polyclonal T cells was thought to be potentially useful as a positive diagnostic test. Initial studies suggested that this pattern was rare in normal women. However, these control women were usually young and the MPDs are mainly observed in the elderly. When a large number of normal elderly women were studied, a significant number showed a clonal pattern in unfractionated blood cells. It was subsequently found that a skewed pattern in granulocytes with polyclonal T cells, identical to that found in MPD patients, is present in 25-50% of normal elderly women.

A number of mechanisms, not necessarily mutually exclusive, could account for this age-related skewed X inactivation. Firstly, an acquired mutation could lead to a proliferative advantage. Secondly, stem cell depletion could result in stochastic predominance of one cell type. However, several lines of evidence suggest that the predominant mechanism contributing to this phenomenon is selection for polymorphic X-linked differences. Twin studies in humans have suggested the presence of X-linked genes which regulate stem cell kinetics; elderly cats develop skewing towards one parental G6PD allele; and strain-dependent differences in the response of mouse stem cells to cytokines have been linked to a number of genetic loci, including some on the X chromosome.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook

Post a comment