Adoptive immunotherapy for relapse of chronic myeloid leukemia after allogeneic bone marrow transplant: equal efficacy of lymphocytes from sibling and matched unrelated donors.

Lymphocyte transfusion from the marrow donor (DLT) is well established as an effective therapy for relapse of CML post allogeneic BMT. Reports thus far have been mostly limited to patients who received DLT from a matched sibling donor. We compared the efficacy and toxicity of DLT in 30 patients who were treated with cells from their HLA-identical sibling (n = 18) or from their phenotypically HLA-matched unrelated marrow donor (n = 12). The overall probability of obtaining a cytogenetic remission was 69% (95%CI: 51-83%) and was not significantly different between the two groups. The disease stage at the time of DLT was the only factor associated with cytogenetic remission by multivariate analysis; patients treated in cytogenetic or molecular relapse (n = 11) were seven times more likely (RR = 7.4, 95%CI: 2.4-22.4, P = 0.0005) to respond compared to patients treated for hematologic relapse (n = 19). There was a trend towards more acute GVHD II-IV in the unrelated donor group (58 vs 39%, P = 0.09), but the probability of developing extensive chronic GVHD was not significantly different (56 vs 39%, P = 0.4). We conclude that transfusion of donor cells from HLA-matched volunteer donors does not appreciably increase the risk of GVHD compared with transfusion of cells from HLA-identical siblings in patients with CML who relapse following allogeneic BMT. Conversely, there is no evidence for an increased graft-versus-leukemia effect after DLT from volunteer donors.

Molecular analysis of transient cytogenetic relapse after allogeneic bone marrow transplantation for chronic myeloid leukaemia.

Serial quantification of residual disease in CML patients after allogeneic BMT is useful for early detection of relapse. However, the fact that some cytogenetic relapses appear to be transient may complicate protocols for early therapeutic intervention based on molecular analysis and could result in the unnecessary treatment of some patients. To determine the frequency and significance of transient cytogenetic relapse, we have studied serial samples from 98 CML patients after allogeneic BMT by conventional cytogenetics and competitive RT-PCR for BCR-ABL mRNA. During the period of study, 26 patients had cytogenetic or haematologic evidence or relapse. In four cases (15% of those who relapsed; 4% of all patients) relapse appeared to be transient; i.e., subsequent marrow samples were completely Ph chromosome-negative despite the fact that there had been no change in treatment, including the level of immunosuppression. BCR-ABL mRNA levels broadly paralleled the cytogenetic findings. Of these four patients, two subsequently progressed to frank haematologic relapse and two remained strongly positive for BCR-ABL transcripts and are therefore presumably still at risk of relapse. Analysis of B cell-enriched, T cell-enriched and lymphoid-depleted fractions for three patients demonstrated that transient relapse was not due to the proliferations of BCR-ABL-positive lymphoid cells. In contrast, BCR-ABL-positive myeloid precursor cells were detected in two of three patients tested. We conclude that transient cytogenetic relapse followed by sustained remission is a relatively infrequent occurrence after current allogeneic transplant regimens.

Correlation between the proportion of Philadelphia chromosome-positive metaphase cells and levels of BCR-ABL mRNA in chronic myeloid leukaemia.

We have sought to define the relationship between the proportion of marrow metaphases showing the Philadelphia chromosome (Ph) and levels of BCR-ABL mRNA assessed by quantitative polymerase chain reaction (PCR) in patients with chronic myeloid leukaemia (CML). From a total of 141 patients, 164 PCR assays were performed on peripheral blood samples taken within 2 weeks of a bone marrow specimen analysed by cytogenetics. BCR-ABL mRNA was quantified in all 106 PCR-positive samples by competitive PCR; results ranged from < 10 to 3.4 x 10(6) transcripts/micrograms RNA. Twenty-one chronic-phase patients had a median of 5.0 x 10(5) BCR-ABL transcripts/micrograms RNA; no difference in levels of the fusion mRNA was found between 15 Ph-positive and six Ph-negative, BCR-ABL-positive patients. Ph-positive metaphases were not detected in any individual who was PCR negative (n = 58) and in only a single patient who was PCR positive with < 10(3) BCR-ABL transcripts/micrograms RNA (n = 44). Conversely, of 41 samples from patients in haematological remission who had > 10(3) BCR-ABL transcripts/micrograms RNA, 30 had at least one Ph-positive metaphase. The highest level of BCR-ABL transcripts at which Ph-positive metaphases were not detected was 1.5 x 10(4). For the 46 patients who had at least one Ph-positive metaphase, a good correlation (Spearman coefficient = 0.83, P < 0.0001) was found between the percentage of Ph-positive metaphases and BCR-ABL transcript levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Relapse of chronic myeloid leukemia after allogeneic bone marrow transplant: the case for giving donor leukocyte transfusions before the onset of hematologic relapse.

Fourteen patients with chronic myeloid leukemia (CML) relapsing after allogeneic bone marrow transplant (BMT) were treated with leukocyte transfusions from the original marrow donor (sibling, n = 9; volunteer unrelated, n = 5). The relapse was defined at the molecular level in two cases, cytogenetically in five cases and hematologically in seven cases. Ten patients responded, seven of seven patients with cytogenetic/molecular relapse compared with three of seven with hematologic relapse (P < .03). All five recipients of cells from unrelated donors responded. Eight of the 10 responders have achieved polymerase chain reaction-negative status and this persisted in three patients for more than 2 years; no responder has shown sign of relapse. Reversible marrow aplasia occurred in two patients, both treated in hematologic relapse. Severe graft-versus-host disease occurred in four patients and was fatal in one. We confirm previous reports that donor leukocyte transfusions are effective in the management of CML in relapse after BMT. In this series, therapeutic intervention before the onset of hematologic relapse was associated with an increased likelihood of response and no marrow aplasia.

Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation.

We have developed a competitive polymerase chain reaction (PCR) titration assay that estimates the number of BCR-ABL transcripts in chronic myeloid leukemia patients to monitor minimal residual disease after bone marrow transplantation (BMT). The assay gave reproducible results and allowed differences in BCR-ABL message levels of half an order of magnitude to be distinguished. Of 91 patients studied by nonquantitative PCR, 28 who had a positive PCR result on at least one occasion posttransplant were analyzed by competitive PCR. Seventeen patients had no evidence in their marrow of cytogenetic relapse during the period of observation; BCR-ABL transcript numbers in these cases ranged from approximately 10 to 800/micrograms RNA. Ten of the 11 patients who relapsed cytogenetically were studied when Philadelphia-positive metaphases were first detected in their marrow; transcript numbers ranged from 1,600 to 7 x 10(5)/micrograms RNA. Patients in hematologic relapse had between 9 x 10(4) and 10(6) BCR-ABL transcripts/micrograms RNA. Patients who progressed from cytogenetic remission to cytogenetic relapse and then to hematologic relapse had increasing numbers of BCR-ABL transcripts in their blood. Three patients had clear evidence of rising numbers of BCR-ABL transcripts before routine detection of cytogenetic relapse. Conversely patients without cytogenetic relapse generally had low or falling numbers of transcripts. We conclude that serial monitoring of residual disease post-BMT by estimating the number of BCR-ABL transcripts provides more information than conventional cytogenetics or nonquantitative PCR and may identify patients in need of therapeutic intervention before the onset of overt relapse.

Phenotypic characterization of normal and CML CD34-positive cells: only the most primitive CML progenitors include Ph-neg cells.

We studied the sequence of acquisition of CD33, CD38 and HLA-DR antigens on CD34+ cells from marrow and blood of Ph-chromosome positive CML patients and normal marrow. We examined the Ph status of the various CML cell populations. The mean proportions of normal and CML CD34+ cells expressing CD33 and CD38 were not significantly different. However, a significantly greater proportion of CML CD34+ cells expressed HLA-DR antigens compared with normal CD34+ cells and the level of HLA-DR expression per CML cell was abnormally high. When the sequence of acquisition of these antigens on normal and CML CD34+ cells was evaluated using 3-colour fluorescence analysis, the results suggested that HLA-DR was expressed earlier than CD38 or CD33 and these findings were confirmed by following the acquisition of CD38 and CD34+/DR+/CD38-subpopulation during liquid culture. We performed cytogenetic studies on CD34+ subpopulations in 6 cases. In 4 cases there were some Ph-negative metaphases detectable in the CD34+/DR-subpopulation (range 12.5 to 60%). In the CD34+/DR+ fractions, however, all 6 patients had only Ph-positive metaphases and only 1/5 patients had detectable Ph-negative metaphases in the CD34+/CD38-subpopulation. We conclude that expression of HLA-DR antigens may precede the expression of CD38 on CD34+ cells during normal stem cell differentiation. In CML DR may be expressed aberrantly and Ph-negative cells are found predominantly in the DR negative subpopulation.

Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia in first chronic phase: correlations with acute graft-versus-host disease and relapse.

We have studied 61 patients who underwent allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML) in first chronic phase. Minimal residual disease was detected by the amplification of the leukaemia-specific BCR-ABL fusion mRNA with the polymerase chain reaction (PCR) using a highly sensitive nested primer strategy. As a general pattern, patients often had detectable BCR-ABL (PCR positive) for up to 6 or 9 months post BMT after which time BCR-ABL became undetectable (PCR negative). The conversion from PCR positive to PCR negative was not associated with the time at which cyclosporin A treatment was stopped. Six patients (10%) have relapsed during the period of this study, two within 1 year and four more than 1 year after transplant. The relationship between PCR positivity more than 1 year post transplant and relapse was significant (P = 0.036) but 15 patients who were PCR positive beyond 1 year remain in complete clinical and cytogenetic remission. Thus late positivity identifies a group of patients at increased risk of relapse but is of little predictive value for individual patients. Of the four late relapses, two had been persistently PCR positive and two were initially PCR positive, converted to negative and subsequently to positive again. Although all relapses were preceded by PCR positivity, relapse may occur only 12 months after a PCR negative result. The proportion of patients PCR negative at 3/4 months after BMT was found to increase significantly with the severity of acute GVHD (P = 0.002) but no relationship was found between acute GVHD and subsequent PCR results. There was no clear association between severity of chronic GVHD and PCR result.

Analysis of the tumorigenic potential of common marmoset lymphoblastoid cells expressing a constitutively activated c-myc gene.

The respective roles of Epstein-Barr virus (EBV) and c-myc in the pathogenesis of endemic Burkitt's lymphoma (BL) are unclear. In order to help resolve the question whether constitutive expression of the c-myc gene in an EBV-immortalised B cell is sufficient to induce a tumorigenic phenotype, B cells from a common marmoset (Callithrix jacchus) were immortalised with EBV, transfected with a constitutively activated c-myc gene and inoculated into the host animals. Despite the cell line transfected with c-myc displaying enhanced growth characteristics, in vitro and in vivo experiments demonstrated that this was not sufficient to induce a tumorigenic phenotype. This supports our previous findings with EBV-immortalised human B cells transfected with an activated c-myc gene (Hotchin et al., 1990).

Paroxysmal nocturnal hemoglobinuria: correction of abnormal phenotype by somatic cell hybridization.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disorder thought to result from a somatic mutation in a hemopoietic stem cell. PNH may evolve to aplastic anemia or to acute leukemia. PNH cells are deficient in proteins attached to the cell membrane via a glycosylphosphatidylinositol structure, called the GPI anchor, and the primary lesion in PNH is thought to be a defect in the biosynthesis of the GPI anchor. We have recently established permanent lymphoblastoid cell lines that have the PNH phenotype and we report now the isolation of human-human somatic cell hybrid clones obtained by fusing them with normal lymphoblastoid cells. In all of 21 hybrid clones, obtained from five different patients, the expression of three different GPI-linked proteins on the hybrid cells was normal. These findings indicate that the PNH mutant gene is recessive with respect to the normal allele and that a recessive mutation can cause a clonal preneoplastic disorder.

Minimal residual disease after bone marrow transplant for chronic myeloid leukaemia detected by the polymerase chain reaction.

We describe the methodology and application of the polymerase chain reaction to detect BCR-ABL mRNA as a marker for CML cells. The technique is highly sensitive enabling the routine detection of 1 leukaemic cell in 10(5) or 10(6) normal cells and is therefore the most sensitive method available for detecting minimal residual disease. Analysis of marrow or blood from 80 patients after bone marrow transplantation for CML shows that residual leukemia is often detectable for several months but that most subsequently become PCR negative. Patients who relapsed were all PCR positive before the detection of Philadelphia positive metaphases in bone marrow aspirates.

Dyskeratosis congenita fibroblasts are abnormal and have unbalanced chromosomal rearrangements.

Dyskeratosis congenita (DC) is a rare inherited disorder characterized by bone marrow failure, dystrophic changes in the skin and mucous membranes, and a predisposition to malignancy. The DC locus has been mapped to Xq28. The primary defect responsible for this disease remains unknown. We have studied four patients with this disease, three from one family and one from another. In all four patients, primary skin fibroblast cultures were abnormal both in morphology (polygonal cell shape, ballooning, and dendritic-like projections) and in growth rate (doubling time about twice normal). Fibroblast survival studies using four clastogens (bleomycin, diepoxybutane, mitomycin-c, and 4-nitroquinoline-1-oxide) and gamma radiation showed no significant difference between DC and normal fibroblasts. Cytogenetic studies performed on peripheral blood lymphocytes showed no difference between DC and normal lymphocytes with or without prior incubation with clastogens. However, bone marrow metaphases from one of three patients and fibroblasts from two of four patients (who were the eldest of the 4) showed numerous unbalanced chromosomal rearrangements (dicentrics, tricentrics, and translocations) in the absence of any clastogenic agents. Cell-specific differences and a higher rate of chromosomal rearrangements in the older patients appear to correlate with the clinical evolution of the disease. These findings suggest that the DC defect predisposes DC cells to developing chromosomal rearrangements.

p53 in chronic myeloid leukemia cell lines.

Four human chronic myeloid leukemia (CML) cell lines, BV173, K562, KCL-22, and KYO-1, were studied for inactivation of human tumor suppressor gene p53. Southern blotting showed allele deletion in KCL-22 and cytogenetic studies showed a chromosome 17 deletion in KYO-1 but no gross structural abnormalities in the other two lines. Northern blotting showed increased amounts of normal size p53 mRNA in BV-173 and KYO-1, trace amounts in KCL-22, and none in K562. Direct sequencing of p53 cDNA revealed a missense point mutation in KYO-1 and a single base pair deletion consistent with a coding frame shift in KCL-22. Both abnormalities in these myeloid cell lines were located in the highly conserved region of p53. Studies with two monoclonal antibodies showed that the three cell lines with p53 mRNA had readily detectable p53 proteins. In KYO-1 and BV173 cells the p53 protein was located mainly in the nuclei but KCL-22 cells had weak staining in the cytoplasm. Our data support the assumption that inactivation of p53 tumor suppressor function in myeloid blast transformation of CML may result from point mutations or deletions that produce mutant proteins.

Inactivation of the retinoblastoma susceptibility gene in a human high grade non-Hodgkin's lymphoma cell line.

We studied the expression of the retinoblastoma (RB) gene product (p105) in a B-cell line established from a patient with non-Hodgkin's lymphoma (large cell type). The karyotype of this cell line, named Ri-1, showed amongst other changes an apparent deletion of one chromosome 13 on band q14. No p105 could be detected by immunoprecipitation analysis and Western blotting in Ri-1 cells. Northern blotting revealed that RB mRNA is not expressed in Ri-1. Southern blotting confirmed the loss of one RB allele but showed a normal gross structure of the remaining allele. This suggests that the inactivation of the RB gene in Ri-1 cells is due to deletion of one allele and point mutations or small deletions in the other, as is often the case in retinoblastomas. Our findings imply that inactivation of the RB gene may play a role in the pathogenesis of high grade malignant lymphomas and that studies of RB in primary lymphoma samples would be of interest.

Acute myeloid leukaemia relapsing following interleukin-2 treatment expresses the alpha chain of the interleukin-2 receptor.

Immunotherapy with recombinant human Interleukin-2 (rhIL-2) was given to nine patients in first complete remission from acute myeloid leukaemia (AML). Five patients relapsed. The median time to relapse after commencing rhIL-2 was 26 weeks (range 2-44). Four patients were studied at relapse. The morphological and cytochemical features at relapse and presentation were similar. Cytogenetic analysis at relapse in patients 1 and 3 showed a normal karyotype. At relapse, patient 4 had the abnormality 46,XY, t(2;3). Patient 2 had the chromosomal abnormality t(8;21) at presentation and relapse. Patients 3 and 4 with M5 AML relapsed rapidly at 2 and 9 weeks after starting rhIL-2 treatment. Relapse leukaemia cells had features normally associated with lymphoid development. Patient 3 was TdT positive, with rearranged immunoglobulin genes, and a proportion of cells expressing the CD7 antigen; patient 4 also expressed the CD7 antigen. Relapse leukaemic cells from three of four patients expressed the alpha chain of the IL-2 receptor as assessed by flow cytometry. After overnight incubation and removal of T-lymphocytes the proportion of cells from these patients expressing the alpha chain increased from 15% to 61% (P less than 0.01). Using tritiated thymidine uptake to assess cell proliferation, two of three patients who expressed the IL-2 receptor alpha chain proliferated in response to 1000 u/ml of rhIL-2 in vitro, with a stimulation index greater than 1.95 (P less than 0.05). Following rhIL-2 immunotherapy for AML, relapse cells may express an inducible form of the alpha chain of the IL-2 receptor, which can mediate a proliferative response. It is possible that rhIL-2 when administered to AML patients in remission, may induce relapse. This may be a particular risk in patients with the M5 subtype.

Origin and function of adherent lymphokine activated killer cells in patients with chronic myeloid leukaemia who relapse following bone marrow transplantation.

Adherent lymphokine activated killer (ALAK) cells are a subpopulation of activated natural killer (NK) cells with MHC unrestricted antitumour activity distinguished by their propensity to adhere to plastic in the presence of interleukin-2 (IL-2). We generated ALAK cells from seven patients with chronic myeloid leukaemia (CML) following Campath-1-depleted bone marrow transplantation (BMT). Five had relapsed and were in chronic phase, one had cytogenetic evidence of relapse and one had prior evidence of cytogenetic relapse but was in complete remission at time of study. Phenotypically the ALAK cells included both CD56+/CD3- NK cells and CD56-/CD3+ T cells. The CD3- subpopulation were studied cytogenetically and their functional activity tested in a 4 h 51Cr release cytotoxicity assay using the pretransplant leukaemia cells as targets. Cytogenetic studies showed that the ALAK cells from six patients were Ph negative, and where donor and recipient were sex mismatched, ALAK cells were exclusively of donor origin. In one patient ALAK cells were Ph positive and of recipient origin in eight of nine metaphases. In the 51Cr release assay the ALAK cells showed significant lysis of the pretransplant leukaemia in five of the seven patients tested. These data indicate that in CML patients who relapse post-BMT the NK cells are usually of donor origin but may be recipient-derived. In most patients these ALAK cells have antileukaemic activity in vitro.

Screening alcohol & drug use in a general practice unit: comparison of computerised and traditional methods.

Systematic screening of patients for areas of health risk in their lifestyle has much potential for primary health care clinicians as a cost-effective and time saving means to identify 'at risk' individuals. In the area of alcohol and drug problems, such early identification increases the likelihood of successful intervention. The present study, conducted at a general practice unit, compared the use of a computer to screen for alcohol and drug use with the two more traditional assessment methods of face-to-face interview and paper and pencil questionnaire. It was found that levels of reported consumption were similar across assessment methods. Although the interview method was strongly preferred overall, patients' preference for the computer increased significantly after use. The computer was also found to be more acceptable to patients reporting non-medical drug use, a potentially threatening and sensitive issue. There was a low refusal rate and most patients were willing to allow their doctor to see the assessment results. This indicates that screening for alcohol and drug use is acceptable to general practice patients, and that the computer can play a useful role as a prevention aid.

Alcohol consumption patterns in South Australia: 1983.

The alcohol consumption patterns of adult South Australians were surveyed by the Australian Bureau of Statistics in October 1983. Results indicate that, since 1977, there has been a significant increase in alcohol consumption by women, while the overall consumption of alcohol, particularly beer, by older men has significantly decreased. Comparisons are made between the drinking behaviour of men and women, and the implications for medical personnel are presented.