Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia.

Donor lymphocyte infusions (DLI) have been shown to enhance the graft-versus-leukaemia (GVL) effect and induce haematological and molecular remission in patients with relapsed CML following allogeneic bone marrow transplantation (BMT). The potent donor cell-mediated cytolysis following DLI may lead to a short period of aplasia before the re-establishment of donor haematopoiesis. The absence of detectable donor cells in patients prior to DLI infusion may result in permanent aplasia in certain patients. We report on four patients who relapsed 1, 3, 6.5 and 7 years post-BMT for chronic phase CML and were treated with DLI from their original BMT donor. Polymorphic short tandem repeats (STRs) were used to assess haematological chimaerism both prior to and following DLI. At the time of relapse, STR-PCR indicated the presence of donor cells in all four patients, at levels ranging from 1-40%. A clinical and molecular response was seen in 4/4 patients following a short period of cytopenia and all patients remain in clinical remission with a follow-up of 2 months-3 years post-DLI. STR-PCR indicated that a response was occurring during the period of pancytopenia when metaphase analysis was unsuccessful. Lineage-specific analysis of the cellular response to DLI was monitored using STR-PCR of peripheral blood (PB) and bone marrow (BM) lymphocyte-enriched fractions and CD2-positive and -negative T cell fractions. In one patient BM and PB CD34-positive and -negative fractions were also assessed. A change in the ratio of donor:recipient cells in the PB lymphocyte fraction was the earliest molecular indication of an anti-leukaemic response. Subsequent conversion to donor chimaerism occurred in the other lineages and the granulocyte fraction was the last lineage to convert. In conclusion, lineage-specific STR-PCR permits detailed monitoring of subtle changes in donor/recipient cell dynamics in specific lineages following DLI during the crucial pancytopenic phase and may be a useful predictor of haematological response to DLI therapy.

Comparative Study of FISH, PCR and Cytogenetics on 25 Patients Submitted to Bone Marrow Transplantation (BMT) for Chronic Myelogenous Leukemia (CML); Which Tests can we Use in Routine Analysis Post BMT?

Chronic Myeloid Leukaemia (CML) shows an excellent response to allogeneic bone marrow transplantation (BMT) with a 60-80% long term disease free survival in recipients of unmanipulate marrow. The most frequent cause of treatment failure is leukaemic relapse, due to the re-emergence of malignant recipient clones. Clinical and haematological relapse is usually preceded by molecular evidence of relapse. Early detection of molecular relapse may allow intervention with immunotherapy such as donor lymphocyte infusions (DLI). This study was undertaken to compare results from two centres who employ either Fluorescent In Situ Hybridisation (FISH) or polymerase chain reaction (PCR) analysis of DNA polymorphisms as their routine method of detecting residual host cells following BMT for CML in order to establish (1) if these methods are equivalent for routine laboratory use in reporting of chimaerism results to the referring clinician, and (2) if these methods are beneficial for indicating new and early therapeutic strategies. FISH analyses for the X and Y chromosomes (in sex mismatched patients) and/or FISH for BCR and ABL loci were compared with short tandem repeat PCR (STR-PCR) and conventional karyotyping in serial analyses in 25 patients submitted to BMT for Philadelphia positive (Ph) CML. Comparison of all results on samples assessed between 1 and 13 years post BMT indicated that FISH and PCR, performed on the same bone marrow samples displayed similar results in more than 90% of patients in first 3 years after BMT which increased to a concordance rate of 100% in long term survivors. In contrast, comparison of FISH or PCR versus cytogenetic analysis indicated a low concordance rate, with less than 50% of samples showing similar results during all the follow-up period. Eighty percent of recipients (22 patients) had evidence of mixed chimaerism following BMT (initial level of positivity 1-6% recipient cells) during the follow-up period. This low percentage of recipient cells remained stable in 7 patients, while 9 patients reverted to a donor profile. All 16 patients are in haematological remission. In addition the 3 patients with complete donor chimaerism remain in remission. In the remaining 6 patients, a progressive increase in recipient cells occurred (progressive mixed chimaerism, PMC), and was followed by haematological relapse. We conclude that FISH and PCR can be used to monitor CML patients post BMT and transient or stable low level mixed chimaerism is not associated with leukaemia relapse, but PMC is predictive of imminent relapse and its detection may help to illucidate the timing of early intervention with donor lymphocyte infusion.

Donor chimaerism is a strong indicator of disease free survival following bone marrow transplantation for chronic myeloid leukaemia.

Although Chronic Myeloid Leukaemia (CML) can be treated successfully with allogeneic bone marrow transplantation (BMT), leukaemia relapse remains a significant clinical problem. Molecular monitoring of the post transplant marrow can be useful in predicting relapse particularly in CML patients where the Philadelphia chromosome or its molecular counterpart, the BCR-ABL fusion messenger RNA can be used as a leukaemia specific marker of minimal residual disease (MRD). We have investigated chimaerism (using polymerase chain reaction of short tandem repeat sequences (STR-PCR)) and MRD status (using reverse transcriptase PCR of the BCR-ABL fusion mRNA) in a serial fashion in 18 patients who were in clinical and haematological remission post allogeneic BMT for chronic phase CML. Eleven patients exhibited complete donor chimaerism with no evidence of minimal residual disease. Five patients had transient or low level stable MC. Late MC and MRD was observed in two patients who relapsed > 6 years after T cell depleted BMT for CML. Thus STR-PCR is an appropriate screening test in the post transplant setting for CML patients, but those patients exhibiting mixed haemopoietic chimaerism should also be monitored using a leukaemia specific sensitive molecular assay.

Prevalence of renal malformation in Turner syndrome.

The presence of renal malformation was evaluated in 43 patients with Turner syndrome (TS) and compared with the karyotype in each case; 28 patients (65%) had a mosaic karyotype and the other 15 (35%) had only 45,X metaphases. Renal malformations characteristic of TS were found in 24% of the complete sample group. Of the 15 cases of pure 45,X karyotype, 8 (53%) had abnormal renal findings, while these were found in only 2 of the 28 mosaic cases (7.1%). The probability of this distribution having occurred by chance is P < 0.05. More than 50% of girls with TS are said to have a renal anomaly. In this study renal malformations were found in 25% of the sample group. A significantly greater association of renal malformation was found with monosomy 45,X than with mosaicism. As mosaicism occurs in up to 60% of all girls with TS, the lower figure reported here represents a truer prevalence than that quoted in older series, where the figures quoted applied only to the 45,X syndrome.

The Irish cystic fibrosis database.

We have found records of 1014 Irish cystic fibrosis patients alive by December 1994, belonging to 883 families. Prevalence in the population is 1/3475 and incidence at birth 1/1461, with a gene frequency of 2.6%. Twenty percent of the patients are aged over 20 years, but at present survival rate falls rapidly after that age. We have identified 85% of the mutations on the CFTR gene in a sample of 29% of the families (506 CF chromosomes). Mutation delta F508 is found in 72% of Irish CF chromosomes, G551D in 6.9%, and R117H in 2%. These are the highest frequencies reported for the latter two mutations world wide. Another seven mutations are found in an additional 4% of CF families. We present new microsatellite haplotype data that could be useful for genetic counselling of CF families bearing some of the 15% of CF mutations still unidentified, and comment on possible uses of our database.

A cluster of cystic fibrosis mutations in exon 17b of the CFTR gene: a site for rare mutations.

Intensive screening has improved our understanding of the profile of mutations in the CFTR gene in which more than 400 mutations have been detected to date. In collaboration with several European laboratories we are involved in such analysis. We have identified 14 new mutations in exon 17b of CFTR, having analysed 780 CF chromosomes, and have compared the frequency of mutations in this exon with that of other regions of the CFTR gene. The results obtained indicate an accumulation of mutations, not only in regions encoding the two nucleotide binding folds, but also in those encoding transmembrane domains of the CFTR gene, in particular exon 17b.

Donor leukemia following allogeneic bone marrow transplantation.

Although allogeneic bone marrow transplantation has been shown to be a highly effective treatment for acute and chronic leukemia, leukemic relapse remains a significant problem. Leukemic relapse occurs in recipient cells in the majority of cases, but the paucity of donor cell leukemias may reflect the sensitivity of the investigative technique. We have developed a highly sensitive technique to identify the origin of all hematopoietic cells in the post transplant state which is based on PCR amplification of microsatellites, polymorphic tandem repetitive elements. We have identified donor leukemia (AML M5) following a sex matched BMT for severe aplastic anemia, verified a previously reported case of donor leukemia following BMT for chronic granulocytic leukemia and recently identified an acquired cytogenetic abnormality(del 11q23) in donor cells four years following an apparently successful BMT for AML. In all cases the donors have remained healthy. Postulated mechanisms include transfer to the transplanted marrow of a dormant oncogene residing in the DNA of either a virus, the chromosomes of degenerating irradiation damaged host leukemic cells or in the marrow stroma which is radioresistant and host in origin following BMT. Using sensitive techniques donor leukemia has been shown to be a more common event than was previously thought and an understanding of its pathogenesis may allow us to elucidate leukemogenic mechanisms in man.

Deletion delta F508 and clinical expression of cystic fibrosis-related liver disease.

A study of liver function in 108 adult cystic fibrosis patients showed that 20 had established liver disease, and that these had significantly better pulmonary function than the subgroup without liver disease. The relative risk of liver disease for homozygotes vs heterozygotes was 2:1 in our series. Four of the liver patients had a sibling with CF, but three of the sibships were discordant for liver disease. Environmental or genetic factors other than the deletion Delta F508 may influence the development of cystic fibrosis-related liver disease.

Further evidence consistent with Yqh as an indicator of risk of gonadal blastoma in Y-bearing mosaic Turner syndrome.

An 8-year-old girl with some features of Turner syndrome and karyotype 45X/46XY had developed a bilateral gonadoblastoma in her rudimentary ovaries. Her normal Y chromosome showed the characteristic distal fluorescence, as seen in her father's. Another mosaic, this time 45X/46XidicY, and also with some Turner features had rudimentary ovaries, but no gonadoblastoma had developed at age 14. The nature of her idicY, which showed no fluorescent distal Yq and had one of the centromeres inactivated, was confirmed by in situ hybridisation with a Yp-specific probe. Using primers from a human Yp-specific sequence, we amplified DNA extracted from paraffin-embedded ovarian tissue from both cases, and from a normal testicle and a normal ovary as controls. The finding of the expected Y-derived PCR product in the rudimentary gonads from these mosaic patients indicates the presence of their Y chromosome in both. We discuss the validity of the findings, and the possible role of sequences in or near the fluorescent part of Yq in the origin of gonadoblastoma in Y-bearing mosaic Turner syndrome.

Frequency of deletion 508 among Irish cystic fibrosis patients.

We estimate the incidence of cystic fibrosis in Ireland to be at least 1 case per 1838 live births. We have analysed DNA from 44 Irish CF patients for the presence of deletion 508, using the polymerase chain reaction. The deletion was found in 76% of their chromosomes, and approximately 58% of the patients are homozygous for this deletion. Our results are not significantly different from those found in Canadian or UK patient populations, in which frequencies are higher than those found in Southern European countries.

Sequential sampling in clinical cytogenetics: a quality control viewpoint.

We have observed that there is some resemblance between the problems of sampling in industrial quality control and the process of diagnosis of mixed cell populations in cytogenetics. This resemblance enabled us to draw from the methodology of the former science to solve some diagnostic problems in the latter. We considered which of the several sampling procedures available for quality control would be more efficient and more suitable in clinical cytogenetics, concluding that 'sequential sampling' combines both features. We also studied the effect that some abnormalities due to technical factors had on sample size required to reach a diagnosis with a given level of confidence. We give a set of tables for sequential sampling in diagnostic laboratories, illustrating their use in discriminating between mosaicism and pseudomosaicism in prenatal diagnosis and in the diagnosis of the fragile X syndrome. Finally, we discuss the merits of sequential sampling as shown here, comparing it with the current method used in cytogenetics to exclude chromosomal mosaicism.

A computer model for the study of segregation in reciprocal translocation carriers: application to 20 new cases.

We report on a computer program that, given the breakpoints and the chromosomes involved in a translocation, generates all the possible imbalanced gametes, calculates their corresponding imbalances, and arranges them in order of increasing imbalance. When compared to current, more cumbersome criteria from the literature, both methods agreed on 196 cases of 199 (greater than 98%). When compared to observed data from families with aneuploid offspring, both our program and the other reported methods yield a rate of accurate prediction of 87%. The use of the program is illustrated in 20 new translocations from our laboratory. The possible influence of crossing over in meiosis I in altering the gamete that is most likely to be passed to aneuploid live births is discussed.

The fragile X syndrome: the patients and their chromosomes.

We have reviewed recent publications, mostly from 1980 onwards, concerned with the problem of identifying patients with the fragile X chromosome and mental retardation, considering the two practical sides of the problem, that is, identification by their external appearance and by chromosomal studies. We conclude that this condition covers a large range of physical findings which occur in varying degrees in people with the chromosome marker. We have tried to clarify the existent criteria that have to be considered for an accurate cytogenetic diagnosis.

Tables for the cytogenetic study of fragile X chromosomes for diagnostic purposes.

The frequency of expression of the fragile X chromosome varies from patient to patient. Many cases have been reported showing frequencies of less than 2%. With such low frequencies, the risk of erroneous diagnosis is great unless the appropriate number of cells is studied. We present here Tables based on the binomial distribution relating the frequency of expression of the fragile X in a patient with the sample size required to obtain a probability P of correct diagnosis according to different criteria.