Significance of chromosome abnormalities in leukemia.

The review deals with technical considerations in relation to culturing and studying the chromosomes of leukemic cells. Specific and non-random chromosomal changes are described by the historic approach. Then follows a description of the application of chromosomal studies to diagnosis, progress and followup in the chronic and acute leukemias. A section is devoted to the application of chromosomal studies in bone-marrow transplantation. Finally, lines of research for leukemia using a genetic approach are suggested.

Lymphocytotoxic antibodies in leukemic patients treated with bone marrow transplantation.

Lymphocytotoxic antibodies were studied sequentially in a series of 42 patients with leukemia who received a bone marrow graft. Of these patients, 38% had cytotoxic antibodies before bone marrow transplantation (BMT). After BMT the antibody status changed with time, but 62% of the patients had antibodies at some time after BMT. During the first 10 weeks after BMT, 40% of the patients had antibodies. Thereafter the frequency rose to 50% and remained at that level beyond one year after BMT. In successful grafts the gamma globulins are of donor origin six months after BMT; thus donor B cells are capable of forming lymphocytotoxic antibodies even when the immune system is suppressed by cyclosporine. The antibodies had recognizable HLA specificity in about half the cases before and after BMT. When donor and patient were HLA-identical, HLA specificity did not correspond to donor/recipient antigens. In two cases in which the donor was matched for only one haplotype, antibodies formed by recipient cells, active against donor HLA antigens, were found.

Chimerism in skin of bone marrow transplant recipients.

Skin biopsies from 3 patients receiving one-haplotype-matched bone marrow grafts have provided a unique opportunity to demonstrate the presence of donor cells in situ using immunohistological techniques and a monoclonal antibody directed against an epitope common to HLA-A2 and HLA-A28 antigens. The infiltrating cells were also analyzed in consecutive tissue sections with a panel of monoclonal antibodies to human leukocyte antigens, T cells, and epidermal Langerhans cells. Most of the infiltrating cells were shown to be T lymphocytes of donor origin, regardless of whether the histological changes were consistant with graft-versus-host disease (GVHD) or were eczematous. Donor T cells were also shown to colonize histologically normal skin soon after transplantation. Epidermal keratinocytes, dermal endothelium, and adnexal structures did not express the donor HLA type (i.e., were host derived) but the origin of the epidermal Langerhans cells could not clearly be established. The data show that donor cells preferentially migrate to certain sites in skin after transplantation and are not always associated with GVHD.

HLA-DR7 association with African Burkitt's lymphoma.

Association between HLA-DR7 and Burkitt's lymphoma previously reported has been confirmed by a second study. Analysis of additional data from a second study of 33 Ghanaian patients with African Burkitt's lymphoma and 54 Ghanaian controls matched for age and ethnic origin showed that 39.4% of cases, but only 14.8% of controls, had HLA-DR7 (p less than 0.01). The relative risk of 3.7 is similar to that observed in the earlier study (3.3). Combining the earlier and present studies, analysis of clinical data from 94 patients with Burkitt's lymphoma and 116 controls shows the relative risk of Burkitt's lymphoma among individuals with HLA-DR7 was 3.4 (p less than 0.001). There was an increased relative risk of the disease associated with HLA-DR7 in: patients under 10 years of age; and patients with advanced stages of disease (Stage III or IV). However, comparison of relative risks by sequential analysis of 2 X 2 tables showed that these differences by age and stage were not statistically significant.

Study of the HLA system in Burkitt's lymphoma.

Seventy-eight patients with Burkitt's lymphoma and seventy controls from Ghana were typed for HLA-A, B, C and DR antigens, to determine whether there is an association between the HLA system and Burkitt's lymphoma. Increased relative risk was observed in Burkitt's lymphoma patients with DR7, HLA-A1 and B12(BW44).

Immunogenicity of hydatidiform mole.

The HLA and ABO systems were studied in patients with complete hydatidiform mole (CHM). The study confirmed that CHM can immunize the patient against paternally derived HLA-ABC antigens. In patients who required subsequent treatment for trophoblastic tumours the frequency of antibody detection was much higher (10/14) at the time of treatment than it had been at the time of evacuation (2/14). The source of immunogen was considered to be the stroma of the placental villi. It was demonstrated that in a series of 82 CHMs none of the 25 patients mated to a husband of the same ABO group as her own required treatment for postmole trophoblastic tumours. Among unlike matings 17 out of 57 of the patients required treatment. Thus it appears that trophoblastic proliferation after evacuation of mole is favoured by an ABO difference between maternal environment and the molar tissue.

The expression of major histocompatibility antigens in the chorionic villi of molar placentae.

Frozen sections of chorionic villi from molar placentae, ranging in menstrual age from 9 to 19 weeks, were studied by an immunoperoxidase technique using the monoclonal antibody to HLA-A,-B,-C antigens W6/32. The distribution of class I HLA antigens in molar placentae was compared with that in normal placental tissue of comparable menstrual age. The results showed that the distribution of these antigens in molar placentae is similar to that seen in normal placentae. In both tissues, class I HLA antigens were absent from the syncytiotrophoblast and the cytotrophoblast cells of the chorionic villi. However, these antigens were found to be present on cells in the mesenchymal stroma of both normal and molar chorionic villi.

Genetic studies in hydatidiform mole with clinical correlations.

In an elective study of 163 hydatidiform moles 38 were classified as partial mole (PHM) and 125 as complete mole (CHM) on the basis of pathology. Genetic studies showed the PHM to be triploid with one maternal and two paternal chromosome sets. In all cases of PHM the molar pregnancy resolved spontaneously after evacuation. On the basis of genetic studies CHM which were diploid could be subdivided into two entities: homozygous androgenetic CHMs that were 46,XX, and heterozygous CHMs which were androgenetic and usually 46,XY. In informative cases in this series the frequency of heterozygous CHM was 10 per cent. Twenty-two (17.6 per cent) of all the patients with CHM required subsequent chemotherapy for post-mole trophoblastic tumour. Where patients with CHM could be classified as having homozygous or heterozygous CHM the requirement for treatment (17.8 per cent and 25 per cent, respectively) was not found to be significantly different in the two groups.

Flow cytometry used to distinguish between complete and partial hydatidiform moles.

Cells prepared from fresh tissue from a normal conception, and three complete hydatidiform moles (CHMs), were shown to be diploid by flow cytometry, while triploid populations were identified in tissue from two partial hydatidiform moles (PHMs). Similar results were found in cells from formalin-fixed, paraffin-embedded material. In nine cases where cytogenetic analysis was carried out this correlated with the results from flow cytometry. Using flow cytometry we were able to show that two cases which were pathologically complete were in fact diploid, despite the finding of trisomy at one locus in the molar tissue using biochemical markers. The most likely explanation of apparent trisomy of the enzymes in the molar tissue was contamination by maternal tissue.

Cytogenetics of acute promyelocytic leukaemia: incidence of t(15;17) at the Royal Marsden Hospital, London.

This is the first report from a laboratory in the U.K. of the incidence of t(15;17) in acute promyelocytic leukaemia. One of the cases has a variant translocation, t(15;17)(q22;q25). A t(15;17) translocation was present in all cases considered to be adequately studied.

One haplotype matched bone marrow grafts: an investigation using the HLA system as a marker of cellular origin.

HLA antigens were used as markers to study the lymphocyte population in 31 patients with leukaemia, treated with a one-haplotype matched bone marrow transplant (BMT). In 24 patients substained engraftment was achieved and the recipient was repopulated with B and T lymphocytes of donor HLA type. Repopulation occurred at the same rate for lymphocytes of the B and T cell classes, usually within 2 weeks of grafting. In two additional cases bone marrow engraftment was successful but the lymphocyte population was chimeric and cells of both donor and host HLA type were present in the recipient for many weeks. Three patients relapsed after engraftment and peripheral blood lymphocytes were exclusively of host or donor HLA type, or a chimeric population was present. In one chimeric case, peripheral blood T lymphocytes were of donor origin, and B lymphocytes were of host origin. Mononuclear cells in the bone marrow were of host HLA type. The use of the HLA system as a marker is a useful additional approach to determine engraftment or chimerism following an allogeneic one haplotype matched bone marrow transplant.

ABO genotyping of complete hydatidiform moles.

It has been suggested that the ABO blood group of a patient and her partner influence the clinical outcome for patients having a pregnancy with a complete hydatidiform mole (CHM). Since CHM lack red blood cells, it has not previously been possible to type CHM serologically and investigate the relationship between the blood group of the CHM and that of the patient. In the present study we have demonstrated the feasibility of using molecular genotyping to determine the ABO genotype of CHM, the ABO genotype being consistent with the androgenetic origin of CHM in all cases. In the series of 48 cases of CHM, the requirement for chemotherapy was not significantly different in those patients with a CHM of like blood group compared with those with a CHM of unlike blood group.

The human chorionic gonadotrophin alpha subunit gene in gestational trophoblastic disease. 1. Restriction fragment length polymorphisms in hydatidiform moles.

The distribution of EcoR1 and HindIII restriction fragment length polymorphisms in the region of the gene coding for the alpha subunit of human chorionic gonadotrophin have been examined in 71 normal controls, six families, and 65 cases of hydatidiform mole, all of European origin. In control samples the genotypes generated by the presence or absence of the restriction sites, fitted a Hardy Weinberg distribution while in family studies the two polymorphisms showed Mendelian inheritance. The distribution of the genotypes in hydatidiform moles differed from that in control samples. However, the frequency of the alternative alleles R+, R- and H+, H- was not significantly different in the two populations and differences in the distribution of genotypes could be explained in terms of the unique origin of hydatidiform moles. The frequency of the rare homozygous genotype R-R-,H+H+, which has previously been suggested to be associated with the development of choriocarcinoma, was similar in those complete moles which resolved spontaneously (35 per cent) and those which progressed to trophoblastic tumours (27 per cent). No association was found between the genotype of the hydatidiform mole and the subsequent clinical outcome of the patient. Thus the pattern of restriction fragment length polymorphisms associated with the human chorionic gonadotrophin alpha subunit gene would not appear to be an appropriate marker of clinical outcome in patients with hydatidiform moles.

Simultaneous application of immunolabelling and in situ hybridization to detect the origin of B and T lymphocytes in a case of acute lymphocytic leukaemia after bone marrow transplantation.

Previous cytogenetic studies, using selective mitogens, on a patient with B cell acute lymphocytic leukaemia during the 6 years of remission after bone marrow transplantation from an HLA-identical sister indicated persistence of recipient B lymphocytes in the peripheral blood. Such studies are necessarily limited to dividing cells at metaphase, which represent only a small proportion of the total cell population. We have now combined the techniques of immunolabelling and in situ hybridization on the patient's peripheral blood lymphocytes in order to define accurately their individual lineage and gender. A clear difference in the proportion of the persisting recipient lymphocytes was found between B and T lymphocyte lineages.

Radiation damage in patients treated by total-body irradiation, bone marrow grafting, and cyclosporin.

The bone marrow (BM) and peripheral blood (PB) from 63 patients were assessed for the presence of chromosomal aberrations after bone marrow transplantation (BMT) following total body irradiation (TBI) for leukemia. Forty-one patients showed no abnormalities in either BM or PB, and 22 had aberrations in either BM or PB or both. Only stable aberrations were found in the BM, but both stable and unstable abnormalities were present in the PB, the majority showing only unstable aberrations. Among the 25 patients who had a leukemic relapse, clonal chromosomal abnormalities were found in the BM of 12 out of the 16 cases for whom marrow was studied at the time of the relapse. A statistically significant negative correlation between leukemic relapse and graft versus host disease (GvHD) was found, but the relationships between chromosome damage and leukemic relapse, GvHD, and the pretransplant radiation dose and between the radiation dose and both leukemic relapse and GvHD were not significant.

Factors influencning mitotic yields from suspension cultures of normal and acute leukaemic bone marrows.

Cytogenetic studies of bone marrow are often hampered by a paucity of dividing cells. In an attempt to improve mitotic yields we have tried to promote mitotic activity in suspension cultures of bone marrow from both normal and leukaemic subjects. Enrichment of the media always increases yield and the addition of bone marrow stimulating factor (BMSF) may do so. The best prospect for improving the yield of mitoses in acute myeloid leukaemia is to culture the marrow in an enriched medium with BMSF.

Marrow dysplasia with C trisomy and anomalies of the granulocyte nuclei.

An unusual case of bone marrow dysplasia is reported. The features of particular interest are the very high incidence of nuclear drumsticks on the polymorphs, a curious appearance of these same nuclei on electron microscopy, and C trisomy of the bone marrow cells; it is possible that the condition resulted from gold and/or x-ray treatment.

Karyotypic analysis and chromosome polymorphisms in four choriocarcinoma cell lines.

Four choriocarcinoma cell lines were karyotyped and examined for genetic homozygosity or heterozygosity using chromosomal polymorphisms. The BeWo, Jar, and ElFa lines had modal chromosomes in the hypertriploid range, while the DoSmi line was hypotetraploid. A number of chromosomal rearrangements were seen in all lines but there was no common rearrangement. The BeWo and Jar cell lines, derived from tumors following term births, were shown to be heterozygous by the presence of both X and Y chromosomes. The ElFa and DoSmi lines, established following molar pregnancies, were shown to be heterozygous; the ElFa line by the presence of both X and Y chromosomes and the DoSmi line by the examination of Q-band polymorphisms. Thus, all four lines, whatever their origin, were shown to be genetically heterozygous.

Cytogenetic studies in patients previously treated for Hodgkin's disease.

A cytogenetic study was made of bone marrow cells and lymphocytes from patients who had been successfully treated with various regimens for Hodgkin's disease. Most of the patients had been off treatment for at least 3 years before the study began. They were divided into three groups according to the intensity of the therapy received. The frequency of gaps and breaks in the chromosomes of lymphocytes was above normal limits and similar in the three treatment groups. In contrast, the frequency of both lymphocytes and bone marrow cells with rearranged karyotypes was correlated with the intensity of treatment. Clones of cells with an abnormal karyotype were found in only two patients, both of whom were in the group receiving the most intensive therapy, i.e., chemotherapy and total nodal irradiation.

Cytogenetic studies of leukemic recurrence in recipients of bone marrow allografts.

Cytogenetic studies were made in 20 leukemic patients who relapsed after treatment by allogeneic bone marrow transplantation (BMT). Seven of the eight patients in whom no chromosomal abnormalities were detected in leukemic cells before BMT developed clonal abnormalities after BMT, and in two of these patients two independent clones were observed. Most patients in whom clones were detected before BMT showed evidence of clonal evolution after BMT. Nonclonal abnormalities were also observed in clonal cells. These additional abnormalities, both clonal and nonclonal, were attributed to the effects of total body irradiation received by the patient before BMT. There was no evidence of recurrence of leukemia in donor cells among these patients. We concluded that the cells found in leukemic recurrence were derived from the original leukemic clone.