Limitations of the use of single base changes in the p53 gene to detect minimal residual disease of breast cancer.

BACKGROUND/AIMS: Peripheral blood progenitor cell (PBPC) transplantation is frequently used in the treatment of malignant diseases, but contamination of the graft by tumour cells is a real concern and may lead to disease relapse. The feasibility of applying heterogeneous single base genetic changes as tumour specific markers to detect minimal residual disease in PBPC harvests was studied, using the p53 gene and breast cancer as models. METHODS: Tumour tissues from 51 patients with cellular aliquots from PBPC harvests available were studied. Thirty eight patients had metastatic disease or were at high risk of metastasis, and 13 had high risk stage II/III disease with four or more involved axillary lymph nodes. Tumour DNA was screened for p53 mutations in exons 5 to 9, using denaturing gradient gel electrophoresis, followed by sequencing. Based on sequence information, allele specific primers were designed for each mutation and the non-radioisotopic, amplification refractory mutation system (ARMS) was used to screen DNA from PBPC harvests for minimal residual disease. Attempts were made to optimise each system, based on parameters determined using the T47D breast cancer cell line with a confirmed point mutation in codon 194. RESULTS: Twelve different somatic mutations were found, two of which could not be sequenced. The remainder were point mutations. Only five of the 10 ARMS systems were successfully optimised, and minimal residual disease detection sensitivities ranged from one copy of tumour DNA in 10(2) to 10(3) copies of wild-type DNA. Using ARMS, three of five patients and eight of 12 of their PBPC harvests showed minimal residual disease. CONCLUSIONS: These results suggest that the use of single base genetic changes in minimal residual disease detection is relatively insensitive and is limited to a small number of patients and to certain mutations. In addition, it is labourious and therefore unlikely to play an important role in clinical practice.

A novel 8-bp insertion in codon 281 of p53 in a patient with acute lymphoblastic leukaemia and 2 separate leukaemic clones. Mutations in brief no. 219. Online.

We report a novel p53 insertion in a case of aggressive acute lymphoblastic leukaemia in a 16 year old male, in which 2 separate leukaemic clones were previously identified by T-cell receptor sigma and immunoglobulin heavy chain gene rearrangement studies. Initial p53 mutation screening of blast cells from 29 patients with acute leukaemia by PCR-denaturing gradient gel electrophoresis showed that 2 had a silent codon 213 polymorphism and only the index case had a somatic mutation identified to be an 8 bp insertion in codon 281 (5'CCGGGGGG-3'). This insertion was associated with the second, more aggressive clone which underwent clonal evolution and became resistant to cytotoxic chemotherapy. With an allele-specific primer in PCR, we were able to demonstrate the presence of this clone as a minority at disease presentation, and in 2 of 3 collections of peripheral blood progenitor cells.

Fas antigen (CD95) expression in peripheral blood progenitor cells from patients with leukaemia and lymphoma.

Fas antigen (CD95) is a cell surface receptor belonging to the tumour necrosis factor/nerve growth factor superfamily and is able to induce apoptosis when triggered by its' natural ligand or an anti-Fas antibody. Fas expression is low on CD34+ bone marrow (BM) progenitor cells, but is increased by various cytokines in vitro. We investigated Fas expression on CD34+ cells from 39 peripheral blood progenitor cell (PBPC) harvests and from 5 normal BM harvests by dual colour flow cytometry to determine if Fas expression was altered during mobilisation. By including calibrated microbeads during flow cytometry, we quantified the number of Fas antigen molecules per cell. A low percentage of PBPC (22%) and normal BM (23%) CD34+ cells expressed Fas antigen. Fas expression varied on CD34+ cells from different diseases and the highest expression was found in ALL (52%). There was a significant three fold increase in the number of Fas molecules/cell expressed on CD34+ cells (PBPC 6,230 molecules/cell, BM 2,236; p = 0.0003). This level of expression was considerably less than that for CD3/CD19 lymphocytes (33,095 molecules/cell) and CD14 monocytes (47,467 molecules/cell) in the PBPC harvest. In conclusion, mobilisation including the use of growth of factors, has minimal effect on CD34 progenitor cell Fas expression.

Flow cytometry using annexin V can detect early apoptosis in peripheral blood stem cell harvests from patients with leukaemia and lymphoma.

Quantifying progenitor cells in peripheral blood stem cell (PBSC) harvests by flow cytometric enumeration of CD34+ cells does not account for cell viability. Cell membrane asymmetry in early apoptosis exposes phosphatidylserine on the cell surface. This can be detected by staining with annexin V FITC. Apoptosis in 30 autologous PBSC harvests mobilised by cyclophosphamide + G-CSF or standard chemotherapy + G-CSF was analysed immediately after collection by dual-colour flow cytometry with CD34 PE and annexin V FITC. Harvests contained a median of 3.4 x 10(6)/kg (range 0.3-91.8) CD34+ cells. Of these 87.6% (range 30-96.5) were annexin V-. In 10% of harvests more than 50% of CD34+ cells were apoptotic. Differences in PBSC mobilisation or collection could not explain the variation in annexin V binding. Cyclophosphamide + G-CSF significantly increased the yield of CD34+ cells but did not increase apoptosis. Comparison of consecutive harvests showed no difference in the numbers of CD34+ cells collected but found a significant decrease in apoptotic CD34+ cells through multiple collections. Analysis of annexin V binding in PBSC harvests is a simple flow cytometry technique which gives additional information on the status of CD34+ progenitor cells.

Differential expression of cell adhesion molecules by human hematopoietic progenitor cells from bone marrow and mobilized adult peripheral blood.

The mechanisms responsible for mobilization of hematopoietic progenitor cells (HPC) from the bone marrow into the circulation are unknown. One possibility is that HPC downregulate cell adhesion molecule expression. We studied normal human bone marrow and adult peripheral blood following 4 g/m2 cyclophosphamide and recombinant human granulocyte colony-stimulating factor (rHuG-CSF). Each sample was studied for the coexpression of CD34 and a panel of cell adhesion molecules by dual immunocytometry. Bone marrow HPC express the immunoglobulin gene superfamily members of ICAM-1 (CD54), PECAM-1 (CD31) and LFA-3 (CD58), the integrins VLA-4 (CD49d/CD29), VLA-5 (CD49e/CD29) and LFA-1 (CD11a/CD18), L-Selectin (CD62L), HCAM (CD44) and CD36. Mobilized peripheral blood HPC display less expression of LFA-3 (CD54) and VLA-5 (CD49e). Significant differences in cell adhesion molecule expression do exist between sessile and circulating HPC, but the biological relevance of these observations is currently unclear.

Molecular detection of tumor contamination in peripheral blood stem cell harvests.

A total of 96 peripheral blood stem cell harvests (PBSCH) were collected following standard chemotherapy from 27 patients with leukemia, lymphoma, and myeloma. Tumor molecular markers were analyzed in diagnostic samples by Southern blot [immunoglobulin heavy chain (IgHJ), T cell receptor (TcR) delta chain, TcR beta chain] and polymerase chain reaction (PCR) amplification [IgH, TcR delta, t(14;18) translocation] and found in 11 of 22 and 13 of 27 patients, respectively. At a sensitivity of 2 to 5%, Southern blot analysis failed to detect tumor in PBSCH. Using PCR with sensitivities of 10(-4) (IgH, TcR delta) to 10(-6) [t(14;18)] tumor was present in 34 PBSCH collected from five patients with acute lymphoblastic leukemia and two with lymphoma. In these patients, PBSCH collected after successive courses of chemotherapy remained consistently positive. However, no tumor was detected in 28 PBSCH from five patients with lymphoma and one with myeloma. Of eight patients who had bone marrow examined (six concurrently) within 3 weeks of PBSCH, one had tumor in the PBSCH but not in the bone marrow, and three had tumor in the bone marrow but not in the PBSCH, indicating a possible advantage in using PBSC for autologous transplantation in these patients. Although PBSC are an alternative source of stem cells to bone marrow and are considered to have a lower incidence of tumor contamination, the majority of PBSC in this study were positive by PCR analysis. PCR analysis was unable to determine if a positive result represents clonogenic cells capable of initiating relapse following transplant.

Peripheral blood stem cell mobilization using high-dose cyclophosphamide and G-CSF in pretreated patients with lymphoma.

Peripheral blood stem cell (PBSC) mobilization for autologous transplantation is more difficult in treated patients and those with bone marrow involvement. In 17 pretreated lymphoma patients, cyclophosphamide (4 g/m2) and G-CSF mobilized a median circulating peak of 1959 CFU-GM/ml on day 12.5. PBSC harvesting commenced when WBC was 1 x 10(9)/l at day 10.5 collected a median of 21.2 x 10(4)/CFU-GM/kg. 13/17 (76%) patients exceeded the 10 x 10(4) CFU-GM/kg threshold for engraftment. The CFU-GM yield was significantly higher in patients whose WBC recovered from 1-5 x 10(9)/l in less than 3 days and correlated with the maximum WBC pre and post the cyclophosphamide induced nadir. This regime safely mobilized adequate PBSC in the majority of pretreated lymphoma patients.

Direct sequence analysis of TCR V delta 2-D delta 3 rearrangements in common acute lymphoblastic leukaemia and application to detection of minimal residual disease.

T cell receptor delta chain (TCR delta) gene rearrangements were studied by Southern blot analysis in 36 patients with common acute lymphoblastic leukaemia, including 14 adults and 22 children. The majority of patients (68%) had either a rearrangement or deletion of one or more TCR delta genes. The most frequent rearrangement involved a partial recombination of V delta 2 to D delta 3 (55%). D delta 2-D delta 3 rearrangements were present in five patients (14%). To investigate the TCR delta rearrangement as a tumour marker in minimal residual disease studies, presentation samples from 18 patients were amplified by PCR and directly sequenced. Although the size of the V delta 2-D delta 3 junction varied by only 40 bp, sequence analysis showed extensive diversity. This was derived from four factors: deletion of the 5' end of D delta 3 gene (15/18) and 3' end of V delta 2 gene (16/18); the presence of D delta 2 sequences (6/18); insertion of N nucleotides (15/18); association of P nucleotides with intact V delta 2 and D delta 3 genes (5/18). N nucleotides were the major feature, contributing to 75% of the junction. D delta 1 sequences were not involved. Twenty base oligonucleotide probes, constructed from the junctional sequences, were capable of detecting residual tumour cells at the 10(-4) sensitivity level. Cross hybridization studies confirmed the probes to be clone specific. Longitudinal studies on patients undergoing treatment were capable of detecting tumour in remission samples.

Clonal selection in acute lymphoblastic leukaemia demonstrated by polymerase chain reaction analysis of immunoglobulin heavy chain and T-cell receptor delta chain rearrangements.

Immunoglobulin heavy chain (IgH) and T-cell receptor (TcR) genes can be monitored as markers of clonality by polymerase chain reaction (PCR) analysis in acute lymphoblastic leukaemia (ALL). We report the short clinical course of a 16-year-old patient with ALL and a t(4;11) who relapsed early following treatment and subsequently received reinduction chemotherapy followed by peripheral blood stem cell transplantation with interleukin 2 therapy. Despite this, the patient relapsed and died 8 months after presentation. The leukaemic cells were analysed by PCR and showed rearrangements of TcR V delta 2-D delta 3 and IgH CDRIII genes. Direct sequence analysis of the TcR delta and IgH PCR products revealed two leukaemic clones at diagnosis with one present at minimal levels. After initial therapy the major clone was no longer detected even in subsequent relapse samples but the originally minimal clone persisted and increased despite further treatment, indicating drug resistance.

Circulating progenitor cells in myelofibrosis: the effect of recombinant alpha 2b interferon in vivo and in vitro.

We have studied circulating progenitor cells in patients with myelofibrosis, the effects of recombinant alpha 2b interferon (IFN) treatment on these cells in vivo and in vitro and the patients' clinical response to IFN treatment. A 75-fold increase in circulating granulocyte macrophage colony forming units (CFU-GM) and a 25-fold increase in multilineage colonies (CFU-GEMM) were seen in the patients compared with controls. Patients who had undergone splenectomy had circulating progenitors within the normal range. IFN treatment of two patients resulted in clinical improvement and reduction in spleen size, but was complicated by a fall in platelet count and persistent malaise. Whilst on treatment the circulating progenitor cells increased up to 5-fold. In contrast, the addition of IFN in vitro to the CFU-GM and CFU-GEMM culture plates resulted in a dose-dependent inhibition of colony growth which was unaffected by the removal of T-cells and monocytes. Thus we confirm that circulating progenitors are raised in patients with myelofibrosis, IFN may be of benefit clinically, reducing spleen size but may increase levels of these cells in vivo. This is in contrast to the inhibitory effect of IFN in vitro on CFU-GM and CFU-GEMM growth.

Assessment of nutritional status and in vivo immune responses in alcoholic liver disease.

Nutritional status and in vivo immune responses were investigated in 30 patients with alcoholic liver disease who were drinking heavily up until emergency hospital admission. Investigations were performed on admission and after 2 wk abstention and adequate hospital diet. No relationship was found between the severity of liver disease revealed histologically and the recent quantity or total duration of alcohol intake, inadequacy of diet, or nutritional status. Skin anergy was more common in those patients with cirrhosis but did not relate to depletion in circulating T lymphocytes, poor nutritional status, or to the direct effect of alcohol toxicity. Acute alcohol toxicity did, however, produce extensive and rapidly reversible metabolic and cellular changes including reduction in serum potassium, magnesium and phosphate and depletion of all circulating lymphocyte subpopulations.

Liver membrane antibodies in alcoholic liver disease. II. Antibodies to ethanol-altered hepatocytes.

Using an indirect immunofluorescence technique, circulating liver membrane antibodies against normal rabbit hepatocytes and ethanol-altered rabbit hepatocytes have been sought in a series of patients with histologically confirmed alcoholic liver disease. Liver membrane antibodies against normal hepatocytes were found in 18 (28%) of the 65 sera examined, but with ethanol-altered hepatocytes as substrate liver membrane antibodies were found in 48 (74%) of the sera. Isolation of F(ab')2 fragments confirmed that the positive results were due to antibody binding. Liver membrane antibodies against ethanol-altered hepatocytes are peculiar to alcoholic liver disease, and there is a similar incidence in the various histological types of alcoholic liver disease. Absorption studies suggest that the liver membrane antibodies are directed against new or altered antigens which are not present in normal hepatocytes. These new or altered antigens may also appear after pretreatment with other primary alcohols and seem likely to be induced by a haptenic effect of the alcohol or a metabolic break-down product. These studies represent a novel approach to the further investigation of the possible role of immunological mechanisms in alcohol-induced liver injury.

Liver membrane antibodies in alcoholic liver disease: 1. prevalence and immunoglobulin class.

Using an indirect immunofluorescence technique liver membrane antibodies of IgG and IgA class have been demonstrated in a statistically significant proportion of sera from patients with alcoholic hepatitis and alcoholic cirrhosis. IgG and IgA class antibodies were found respectively in 23 and 25% of 48 patients with alcoholic hepatitis, in 27 and 33% of 84 with active cirrhosis, and 67 and 58% of 12 with inactive cirrhosis. These results provide evidence of a humoral immune response in alcoholic liver disease which is directed against, as yet undefined, liver-cell membrane antigens.