David Swirsky

Diagnosis of acute promyelocytic leukaemia by RT-PCR: detection of PML-RARA and RARA-PML fusion transcripts

Summary. Acute promyelocytic leukaemia (APL; AML M3) is identified by a unique t(15;17) translocation which fuses the PML gene to the retinoic acid receptor alpha gene (RARA). Reverse transcription coupled with the polymerase chain reaction (RT‐PCR) has been used to develop a diagnostic test for APL based on the PML‐RARA fusion message. Separate PCR assays were designed to amplify either PML‐RARA (15q+ derived) or RARA‐PML (17q‐ derived) chimaeric transcripts. PML‐RARA transcripts were detected in every case from a series of 18 APL patients with cytogenetically confirmed t(15;17) translocations, whereas RARA‐PML messages were detected in only 67% (12/18) of these patients. This suggests that it is the 15q + derivative which mediates leukaemogenesis. Furthermore the PCR approach (or Southern analysis) may be used to identify in which of the alternative PML introns the breakpoint occurs; 52% of cases (15/29 patients) utilize a 5′ PML intron and 48% the 3′ intron (14/29 cases). Neither the choice of PML intron nor the expression of the 17q‐derivative could be correlated with the microgranular variant of APL (M3V), overall survival rate, age, sex or presence of coagulopathy. Finally, the fusion message is undetectable in five remission samples. This indicates a possible use for RT‐PCR in monitoring remission patients for evidence of relapse.

Guidelines on the management of acute myeloid leukaemia in adults

1 Bone marrow aspirate and trephine biopsy unless the peripheral blast count is high. 2 Immunophenotyping [CD3, CD7, CD13, CD14, CD33, CD34, CD64, CD117 and cytoplasmic myeloperoxidase (MPO)]. 3 Cytochemistry (MPO or Sudan Black, combined esterase). Can be omitted if four-colour flow cytometry is available. 4 Cytogenetics [with reverse-transcription polymerase chain reaction (RT-PCR) for AML 1-ETO and CBFBMYH11 in non-acute promyelocytic leukaemia (APL) and promyelocytic leukaemia (PML) and retinoic acid receptor-alpha (RARA) in suspected APL; fluorescent in situ hybridisation (FISH) in selected cases].

Incidence of AML1/ETO fusion transcripts in patients entered into the MRC AML trials

Acute myeloid leukaemia (AML) with the t(8;21)(q22;q22) is deemed to be a ‘good‐risk’ disease. 396 patients with AML at diagnosis were screened for the presence of t(8;21) and AML1/ETO fusion transcripts by cytogenetic and RT‐PCR techniques respectively. 32 cases of t(8;21) were detected, all of which were also PCR positive. A further 19 cases were detected at the molecular level, predominantly but not exclusively in M1 and M2 FAB types. Approximately 12% of all new cases of AML are estimated to have AML1/ETO fusion transcripts and it is suggested that molecular screening should be performed in all cases with the possible exception of the M3 FAB type.

Cytogenetic and morphological abnormalities in paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by the expansion of a haematopoietic stem cell clone with a PIG‐A mutation (the PNH clone) in an environment in which normal stem cells are lost or failing: it has been hypothesized that this abnormal marrow environment provides a relative advantage to the PNH clone. In patients with PNH, generally, the karyotype of bone marrow cells has been reported to be normal, unlike in myelodysplastic syndrome (MDS), another clonal condition in which cytogenetic abnormalities are regarded as diagnostic. In a retrospective review of 46 patients with a PNH clone, we found a karyotypic abnormality in 11 (24%). Upon follow‐up, the proportion of cells with abnormal karyotype decreased significantly in seven of these 11 patients. Abnormal morphological bone marrow features reminiscent of MDS were common in PNH, regardless of the karyotype. However, none of our patients developed excess blasts or leukaemia. We conclude that in patients with PNH cytogenetically abnormal clones are not necessarily malignant and may not be predictive of evolution to leukaemia.

Myelodysplasia in a patient with pre-existing paroxysmal nocturnal haemoglobinuria : a clonal disease originating from within a clonal disease

SUMMARY. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic anaemia, clonal in nature, due to somatic mutation. PNH may evolve to aplastic anaemia; more rarely to a myelodysplastic syndrome (MDS) or to acute myeloid leukaemia (AML). We have studied a patient who suffered from PNH and later developed refractory anaemia with ringed sideroblasts (RARS) associated with trisomy 8. By testing peripheral blood cells with appropriate antibodies we have shown that all of the red cells, neutrophils and monocytes, as well as 20% of the lymphocytes, belonged to the PNH clone; in contrast, only 43% of neutrophils and 22% of monocytes belonged to the MDS clone. We infer that the MDS must have arisen from within the PNS clone.

Identification of PML/RARα rearrangements in suspected acute promyelocytic leukemia using fluorescence in situ hybridization of bone marrow smears: a comparison with cytogenetics and RT-PCR in MRC ATRA trial patients

Identification of PML/RARα rearrangements in suspected acute promyelocytic leukemia using fluorescence in situ hybridization of bone marrow smears: a comparison with cytogenetics and RT-PCR in MRC ATRA trial patients

Paravertebral extramedullary haemopoiesis occurring in a case of congenital dyserythropoietic anaemia type II.

A 63-year-old man, previously diagnosed with hereditary spherocytosis and hereditary haemochromatosis, was referred with a raised ferritin of 1238 lg/l. Haemoglobin and C-reactive protein were normal; a fasting transferrin saturation of 95% confirmed iron overload and HFE mutation studies revealed C282Y heterozygosity. Liver biopsy showed grade IV haemosiderosis and established cirrhosis. The patient was commenced on a venesection programme. At routine follow-up, the patient produced a short history of anorexia, weight loss and abdominal pain. Haemoglobin was 115 g/l with an acute phase response. Peripheral blood morphology showed anisocytosis, occasional nucleated red cells and postsplenectomy changes. A computed tomography scan (top left) revealed paravertebral masses with small volume abdominal lymphadenopathy. Needle biopsy of a paravertebral mass stained with anti-glycophorin-C confirmed extramedullary haemopoiesis (top right). Biopsy of retroperitoneal nodes showed features of an inflammatory, reactive process. On bone marrow aspirate (bottom left) and trephine biopsy (bottom right) there was a predominance of binucleated normoblasts and storage iron was increased. Ham’s acidified serum test was positive with donor serum and patient red cells had elevated i expression, confirming the true diagnosis of congenital dyserythropoietic anaemia type II (HEMPAS) with secondary iron overload. The patient’s clinical condition improved following the discontinuation of venesections. On repeat scanning, the lymphadenopathy had reduced in size and remains under observation. Paravertebral masses consisting of extramedullary haemopoietic tissue are most frequently encountered in cases of thalassaemia intermedia where the stimulus to haemopoiesis is not suppressed by transfusion. It is postulated that in a patient with ineffective erythropoiesis, venesection stimulated significant extramedullary haemopoiesis and was responsible for the patient’s clinical deterioration.

Development and progression of a Philadelphia-chromosome–negative acute myelocytic leukemia clone in a patient with Philadelphia-chromosome–positive chronic myelocytic leukemia

We report a patient with typical Philadelphia-chromosome-positive chronic myelocytic leukemia who developed Philadelphia-chromosome-negative acute myelocytic leukemia following autologous stem cell transplantation. The implications of this observation for disease monitoring and treatment are discussed.

An unusual translocation, t(3;8), and deletion of chromosome 21 in acute myeloid leukemia.

This report describes a case of acute myeloid leukemia (AML) in which there was an unusual karyotypic abnormality involving chromosomes #3, #8, and #21. The clinical and hematologic features are described and cytogenetic findings are discussed in relation to previously reported variants of the translocation t(8;21) in AML. The breakpoint at 8q22 is a consistent feature in these cases.