Barbara Gibbons

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.

The use of DNA probes to monitor minimal residual disease in childhood acute lymphoblastic leukaemia

DNA probes to both the joining region (JH) of the immunoglobulin heavy chain gene (IgH) and to the β chain of the T‐cell antigen receptor complex (TCR) have been used as tumour‐specific markers to monitor the rearrangements of the IgH chain gene and the TCR β gene in the blast cells of children presenting with acute lymphoblastic leukaemia (ALL) of B or T cell origin. Blast cells from 68 children with early B cell ALL and eight with T‐ALL were examined at presentation, at day 28 after commencement of therapy and at varying times thereafter. An additional 43 patients (42 with B cell ALL, one with T‐ALL) were studied both at presentation, at completion of their 2‐year treatment course and 3 months later. Twelve patients, drawn from both these groups, were studied at relapse as were a further eight patients in whom an extramedullary relapse had occurred. Persistence of clonally‐derived cells as a predictor of early relapse was seen in the day 28 bone marrows of 11/76 newly‐diagnosed children (nine early B and two T‐ALL) followed by rapid, overt relapse in four of the early B ALL cases. No minimal residual disease (MRD) was detected in bone marrows from any of the 43 patients completing their 2‐year treatment course, but six of these subsequently relapsed at varying time periods thereafter. Identical patterns of rearrangement at both presentation and relapse were seen in most cases. Oligoclonality, or multiple IgH chain gene rearrangements was seen in the blast cells of 15% of patients with early B cell ALL. No correlation between oligoclonality, high white count, unfavourable phenotype, or abnormal karyotype could, however, be ascertained.

Childhood monosomy 7 revisited

Summary. Monosomy 7 is found in acute myeloid leukaemia (AML) and myelodysplasia and is characteristic of a rare chronic myeloproliferative disease (MPD) of young children. We have seen 16 children with monosomy 7. Their clinical features and response to treatment are discussed.

Possible evidence for genomic imprinting in childhood acute myeloblastic leukaemia associated with monosomy for chromosome 7

Monosomy or deletion of chromosome 7 is a frequent finding in both de novo and secondary acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). Based on analysis of deletions of chromosome 7 in such patients, it has been suggested that there is a critical region of the chromosome lying within bands q21‐q31. We have examined bone marrow and peripheral blood samples from 10 patients with MDS, AML and biphenotypic acute leukaemia who had monosomy for or rearrangement of chromosome 7, seeking evidence of non‐random allele loss that might suggest the presence of imprinted genes on the chromosome.

Infant acute lymphoblastic leukaemia with t(11;19)

Seven cases of infant acute lymphoblastic leukaemia with t(11:19) (q23;p13) are described. They are characterized by a high white cell count, organomegaly, early central nervous system (CNS) disease, and a poor prognosis. Blasts are usually of an immature early B‐cell lineage although monocytoid features are present in some cases. The characteristics of infant acute leukaemia with t(11;19) are very similar to those found with t(4;11), and the presence of t(11;19) may indicate the same poor prognosis.

Analysis of ras gene mutations in childhood myeloid leukaemia

Previous studies have shown that approximately 30% of adult acute myeloid leukaemias and 20% of adult acute lymphoid leukaemias contain point mutated ras oncogenes. In order to assess whether ras oncogenes are also involved in childhood leukaemias, we have used polymerase chain reaction (PCR) amplification and synthetic oligonucleotide probes to study the nature and frequency of ras gene mutations in childhood leukaemias, concentrating largely on the acute myeloid leukaemias (AML). Thirty‐four childhood presentation AML DNAs were screened for mutations in and around codons 12, 61 and 117 of N‐. K‐ and H‐ras. Eight of these samples (24%) contained ras mutations. As in the adult disease, the gene predominantly involved was N‐ras (6/8), with occasional activation of K‐ras (2/6). The most common base change was a G→A transition at codon 12 or 13 (4/8). Of the patients with mutant ras. 4/8 were diagnosed as AML FAB subtype M5. Five of the 34 childhood AMLs analysed displayed abnormalities of chromosome 7. However, none of these cases contained a mutant ras gene.

Retinoblastoma in association with the chromosome breakage syndromes Fanconi's anaemia and Bloom's syndrome: clinical and cytogenetic findings

Two children presenting with sporadic unilateral retinoblastoma and exhibiting a high degree of chromosome breakage were noted to have unusual facies, microcephaly and abnormal skin pigmentation. In the first child the pattern of both spontaneous and mitomycin‐C‐induced chromosome breakage was characteristic of Fanconis anaemia although the degree of breakage was extreme. She also exhibited a striking increase in X‐ray‐induced chromosomal damage in G0 lymphocytes as measured by dicentric formation and increase in chromatid‐type aberrations. She had a number of typical clinical features, including cafe‐au‐lait patches and abnormalities involving the kidney; however, she demonstrated neither the hypoplasia of radius and thumb nor the typical aplastic phase of this disorder. At age 22 months the child became anaemic with trilineage myelo‐dysplasia, which was rapidly followed by the development of acute myeloblastic leukaemia. The early onset (at age 4 months) of retinoblastoma may have been associated with the underlying genomic instability. The second child exhibited a pattern of chromosome breakage characteristic of Blooms syndrome, in addition to a moderate increase in damage induced by mytomycin‐C. She had the typical stunted growth and malar hypoplasia of Blooms syndrome although she did not demonstrate the frequently described erythematous ‘butterfly rash’. Although patients with Fanconis anaemia and Blooms syndrome are recognised to be at an increased risk of cancer, retinoblastoma has not previously been described in patients with either condition. We suggest that underlying recessive chromosome breakage syndromes may be underdiagnosed in paediatric cancer patients, with important implications for prognosis and genetic counselling.

Non-Hodgkin's Lymphoma and Klinefelter Syndrome

Patients with a 47, XXY karyotype (Klinefelter syndrome) appear to have an increased risk of developing a malignancy in adulthood, usually cancer of the breast, extragonadal germ cell tumor, and acute nonlymphoblastic leukemia. There is growing evidence to show that these patients also have an increased risk of developing a malignancy in childhood. There are reports describing the development of acute lymphoblastic leukemia, retinoblastoma, and rhabdomyosarcoma in children with a 47, XXY or mosaic 47, XXY/46, XY karyotype. We report a child with a bone metastasizing, B-cell lineage, non-Hodgkins lymphoma (NHL) who was found to have a 47, XXY karyotype in both the tumor and constitutional cells.

Fluorescence in situ hybridization analysis using cosmid probes to define chromosome 6q abnormalities in ovarian carcinoma cell lines

Deletion of 6q is a frequent finding in ovarian carcinoma, which would suggest that this region contains one or more putative tumor suppressor genes. Chromosome 6q abnormalities in six ovarian carcinoma cell lines were analyzed by G-banding and fluorescence in situ hybridization (FISH). Using a variety of probes, including a chromosome 6 paint, a probe specific for the chromosome 6 centromere, and cosmids that map to q24 (cCI6-115), q25 (cCI6-4), q26 (cCI6-91, cCI6-119), and q27 (cCI6-13, cCI6-24, and cCI6-111), abnormalities of 6q were found in three cell lines. In cell line OAW42 (hypotetraploid), the sequences complementary to cCI6-119, cCI6-91, and cCI6-13 probes were lost in two homologues of chromosome 6, which indicates the deletion of genetic material from bands q26-27. The same bands were translocated in cell line PEO1 (hypertriploid). The probes from this region were absent on two copies of chromosome 6, but hybridized to two or three markers. In cell line 59M (hyperdiploid) an interstitial deletion proximal to q24 was detected in one chromosome 6. We conclude that it is very likely that a gene or genes localized in bands 6q26-27, and perhaps in the region proximal to 6q24, play a critical role in the development or progression of ovarian carcinoma.

Duplication of one of the products of the t(8;21) translocation in a patient with refractory anemia with excess blasts in transformation

A 34-year-old man with refractory anemia with excess blasts in transformation is reported, having a t(8;21) translocation and a duplication of one of the products, der(21). The duplicated product had not been observed at presentation in malignant disease, as far as we are aware; thus, the findings are discussed with regard to this case and other possible smoldering leukemias. The diagnostic use of cytogenetics is also considered.