Christopher A. Juttner

Circulating autologous stem cells collected in very early remission from acute non-lymphoblastic leukaemia produce prompt but incomplete haemopoietic reconstitution after high dose melphalan or supralethal chemoradiotherapy.

Summary. Haemopoietic reconstitution (HR) using autologous peripheral blood stem cells (PBSC) was attempted after intensive chemotherapy or chemoradiotherapy in two patients with relapsed acute non‐lymphoblastic leukaemia (ANLL). The PBSC were collected by leukapheresis very early in first remission and cryopreserved in liquid nitrogen. Both patients demonstrated early evidence of trilineage engraftment. The first patient received melphalan 200 mg/m2 followed by rescue with 1·3 × 108 mononuclear cells/kg body weight containing 29 × 104 granulocytemacrophage progenitor cells (CFU‐GM)/kg, and HR was evident by Day 14. The second patient was treated with supralethal chemoradiotherapy followed by rescue with 3·0 × 108 mononuclear cells/kg containing 23 × 104 CFU‐GM/kg. He demonstrated early engraftment with near normal peripheral blood counts by Day 16. There was a subsequent fall in both bone marrow cellularity and peripheral blood counts to a level of low but persistent activity. There was a further phase of haematological recovery from 8 weeks following transplantation with an increase in peripheral blood counts and bone marrow cellularity until final relapse at 13 weeks. This study demonstrates that circulating stem cells have haemopoietic reconstitutive capacity, previously only shown with buffy coat cells from chronic granulocytic leukaemia. The minimum number of PBSC required for satisfactory engraftment remains unknown, although it seems probable that the ratio of pluripotent stem cells to committed progenitor cells is lower in very early remission peripheral blood than in either allogeneic normal bone marrow or autologous bone marrow collected laterin stable remission. The question of leukaemic contamination of the PBSC remains to be answered.

High levels of circulating haemopoietic stem cells in very early remission from acute non-lymphoblastic leukaemia and their collection and cryopreservation

Summary. Circulating myeloid progenitor cells (PB CFU‐GM) were measured in the peripheral blood of 13 patients with acute non‐lymphoblastic leukaemia (ANLL) as they entered first remission. The mean of the recorded peak levels was 2796 × 105 CFU‐GM/1, representing a 2 5‐fold increase above the mean value in normal subjects. These elevated levels of PB CFU‐GM occurred regularly during the very early remission phase when platelet counts rose rapidly. Five of the patients had PB mononuclear cells collected by continuous‐flow leukapheresis during this early recovery phase. CFU‐GM were assayed as a measure of the number of haemopoietic stem cells in each collection. The cells were concentrated and then cryopreserved in liquid nitrogen. Leukapheresis was also performed on five normal subjects for comparison. Low numbers of CFU‐GM were harvested from normal subjects, mean 033 ± 0‐06 × 104 CFU‐GM/kg body weight for each leukapheresis. In ANLL patients entering remission, however, very large numbers of CFU‐GM were regularly harvested. A mean of 11 ± 2 × 104 CFU‐GM/kg body weight were cryopreserved after each leukapheresis, representing 5 times the number of CFU‐GM considered necessary for successful autologous haemopoietic reconstitution. Haemopoietic stem cell viability was assessed after varying periods of cryopreservation. There was no significant stem cell loss after up to 24 months storage. Thus, it is possible to collect and cryopreserve large numbers of CFU‐GM and by inference pluripotent haemopoietic stem cells from the peripheral blood of patients with ANLL during very early remission. The possible biological and therapeutic implications are discussed.

Expression of a 150-kD cell surface antigen identified by monoclonal antibody YB5.B8 is associated with poor prognosis in acute non-lymphoblastic leukaemia.

Peripheral blood specimens, obtained from 71 patients with newly-diagnosed acute non-lymphoblastic leukaemia (ANLL) prior to the initiation of therapy, were assayed for the presence of a myeloid leukaemia-associated cell surface antigen identified by monoclonal antibody YB5.B8. The antibody bound to cells from 22 patients, and these patients had a poorer overall survival rate than those whose cells failed to bind the antibody (p less than 0.025). Fifty patients were treated with daunorubicin/cytosine arabinoside/6-thioguanine (DAT) according to a standard protocol and survived at least to the end of the induction phase (7 days). Of the 34 patients whose cells were YB5.B8 negative, 28 obtained a complete remission. In contrast, only four of the 16 patients whose cells expressed YB5.B8 antigen obtained complete remission (p less than 0.001). Expression of the YB5.B8 antigen in ANLL appears to be a strong prognostic indicator which is independent of other known prognostic factors such as patient age, leucocyte count and pre-existing hematopoietic abnormality.

Comparison of myeloma cell contamination of bone marrow and peripheral blood stem cell harvests

It could be speculated for patients with myeloma and other lymphoproliferative disorders that peripheral blood stem cells may be preferable to bone marrow for autologous transplantation because they may be less contaminated by neoplastic cells. To test this possibility, the immunoglobulin heavy chain gene rearrangement and limiting dilution polymerase chain reaction were used to sensitively quantify myeloma cells in bone marrow and peripheral blood stem cell collections, taken at a similar time, from eight patients with multiple myeloma. Levels of residual disease in the peripheral blood stem cell harvests were variable and did not reflect the tumour burden in the marrow. Peripheral blood stem cells contained 1.7 to 23 700‐fold fewer myeloma cells compared with the bone marrow and would have resulted in reinfusion of 0.08 to 59 480‐fold fewer myeloma cells based on total reinfused CFU‐GM and 0.24 to 24 700‐fold fewer myeloma cells based on total reinfused nucleated cells. Assuming that the proportion of clonogenic myeloma cells is equivalent, peripheral blood stem cells may be better than bone marrow as a source of haemopoietic stem cells for transplantation in multiple myeloma. The clinical follow‐up suggested that patients transplanted with peripheral blood stem cells containing a low number of myeloma cells had better disease control than those transplanted with peripheral blood stem cells containing a high number.

Residual leukemia cannot be detected in very early remission peripheral blood stem cell collections in acute non-lymphoblastic leukemia

We have used a combined cell culture and cytogenetic approach to study the level of residual leukemia during the very early remission (VER) phase of acute non-lymphoblastic leukemia. Clonogenic leukemic cells were induced to proliferate by phytohemagglutinin-stimulated leucocyte conditioned medium and identified by a leukemia-associated karyotype t(8;21) and a morphological marker (Auer rod). When leukemic blasts were cultured, the leukemic karyotype and Auer rods were most readily detected after 3-9 days. When VER blood cells were cultured, no leukemia-associated karyotype or Auer rods could be detected. Based on the number of VER blood cell derived metaphases analysed, the incidence of leukemic blasts among dividing cells is less than 2%.

Long-term effects of cancer treatment and consequences of cure: Cancer survivors enjoy quality of life similar to their neighbours

To assess the long-term effects of cancer treatment and consequences of cure, 102 index cancer cases were compared with 95 neighbourhood controls of similar age and sex and with 78 cardiac controls. The quality of life experienced by these three groups was examined using multiple instruments with proven psychometric properties. All the major quality of life domains (physical, psychological and social) were covered. The findings revealed that the index cases were similar to their neighbours in areas of subjective well-being. However, the index cases exhibited more sexual dysfunction, were more conscientious, determined and emotionally disciplined, and applied the defence mechanisms of displacement and reaction formation more often than the neighbourhood controls. The cardiac controls were older, more anxious, more conventional/less imaginative and used suppression as a defence mechanism to a greater degree than the index cases. In conclusion, young adult cancer survivors enjoy a quality of life similar to their neighbours, whereas coronary bypass survivors adjust less well psychosocially.

N-RAS MUTATION IN ACUTE MYELOID LEUKEMIA : INCIDENCE, PROGNOSTIC SIGNIFICANCE AND VALUE AS A MARKER OF MINIMAL RESIDUAL DISEASE

Summary The polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridization have been used to investigate the incidence of N‐ras mutation in acute myeloid leukemia (AML). The prognostic significance of these mutations and their value as markers of minimal residual disease have also been assessed. Mutated N‐ras alleles were detected in 9 of 69 AML patients (13%). No significant difference in survival or remission duration was found between those patients with an N‐ras mutation and those without. Four patients with N‐ras mutations at presentation were followed through disease progression. The results showed no consistent pattern of association between the presence of an N‐ras mutation and disease state.

THE PERIPHERAL BLOOD CFU‐mix: CFU‐GM RATIO DURING VERY EARLY REMISSION FROM ACUTE NON‐LYMPHOBLASTIC LEUKAEMIA

We have reported our initial experience with clinical autografting using very early remission peripheral blood stem cells (PRSC) in two patients with ANLI, (Juttner et al, 198 5) andhave now studied a third patient, who received supralethal chemoradiotherapy at relapse followed by rescue with PBSC containing 24 x 1 O4 CFU-GM/kg. There was a consistent pattern of early tri-lineage engraftment in all three cases, followed by a fall in peripheral blood counts and bone marrow cellularity. This pattern of incomplete recovery despite infusion of 23. 24 and 29 x 1 O4 CFLJ-GM/kg demonstrates that these early remission stem cell collections contain less pluripotent stem cells (PSC) per CFU-GM than does either allogeneic or autologous bone marrow where 2 4 x 1 O4 CFU-GM/kg regularly produce satisfactory engraftment. Thus we would agree with the major postulate of Geissler et al (1986) that early remission PBSC collections have a preferential expansion of the CFU-GM compartment, a situation which appears different from that in animals (Abrams et al, 1981). We are less certain that the CFU-mix is a satisfactory marker for PSC. Purging with 4hydropervxycyclophosphamide eradicates virtually all CFIJ-mix in autologous bone marrow without preventing engraftment (Rowley et al, 198 5 ) . Our initial clinical autografting experience suggests that the PSC:CFU-GM ratio is reduced at least 6-fold during very early remission (Juttner et al, 198 5 ) . If the CFU-mix is a reliable indicator of PSC the CFU-mix: CFUGM ratio should be reduced to the same extent. We have carried out simultaneous assays of CFU-mix and CFU-GM during the very early remission phase in three ANLL patients (To et al, 1985). Our results show that the CFU-mix:CFU-GM ratio in very early remission blood (1 : 14, range 1 : 8-22) is not significantly different from that in normal blood (1 : 14, range 1 :4-23), normal bone marrow (1 : 36, range 1 : 11-84) or ANLL stable remission bone marrow (1 : 2 7). Thus, the changes in CFU-mix levels during very early remission from ANTJ, are more similar to those occurring in CFU-GM than in PSC. This suggests that the CFU-mix is closer in ontogeny to the CFU-GM than it is to the PSC and is little better than the CFU-GM in predicting PSC and repopulating ability. It thus seems that the determination of the haemopoietic reconstitutive capacity of very early remission peripheral blood stem cells remains dependent on clinical autografting studies.

Bone marrow biopsy during induction chemotherapy for acute myeloid leukaemia identifies only 50% of patients with resistant disease

A study was carried out to determine whether bone marrow biopsy performed on day 6 of induction therapy for acute myeloid leukaemia (AML) can identify those patients with resistant disease who would need an intensification of the first course of induction. Bone marrow biopsies were performed on day 6 of induction chemotherapy in 44 patients with AML treated with daunorubicin, cytosine arabinoside and thioguanine. Biopsies were assessed for blast count, trephine cellularity and leukaemic index. Discrimination between patients who went on to achieve remission and those with resistant disease was best achieved using the reduction in bone marrow cellularity from pretreatment marrow to day-6 marrow. However, this discriminator identified only 50% of the patients with resistant disease and included 13% of patients who achieved remission with the first course of chemotherapy. The other parameters of response were even less effective at discriminating between chemotherapy-resistant and chemotherapy-responsive disease.

A differential sensitivity to recombinant human interferon-alpha 2a between normal and chronic myeloid leukaemic peripheral blood granulocyte-macrophage colony-forming units

The sensitivity to recombinant human interferon-alpha 2a (IFN) of peripheral blood granulocyte-macrophage colony-forming units (PB CFU-GM) from patients with chronic myeloid leukaemia (CML) was studied in a semi-solid clonogenic assay, and compared with normal PB CFU-GM. Like normal PB CFU-GM, the growth of CML PB CFU-GM in vitro was found to be dependent on the plating concentration used. The optimal CFU-GM growth occurred when CML PB mononuclear cells (MNC) were plated at low concentrations in the range of 0.01-0.1 x 10(5)/ml, compared to the range of 0.3-3.0 x 10(5)/ml optimal for CFU-GM growth in normal subjects. The optimal plating concentration for CML PB CFU-GM was similar to that observed in PB collected from patients with ovarian carcinoma during haematological recovery following chemotherapy-induced myelosuppression (recovery phase). The recovery phase PB was used as a source of non-leukaemic cells with a higher incidence of CFU-GM similar to that of CML. IFN produced a dose-related inhibition of CFU-GM growth in normal, recovery phase ovarian carcinoma and CML, PB MNC. The IFN concentration required to inhibit 50% of the CFU-GM in culture (LD50) was found to be significantly influenced by the plating concentration. When cells were cultured at 1.0 x 10(5) MNC/ml the mean LD50 for 7 CML patients was similar to that in normal (n = 5) or recovery phase (n = 5) peripheral blood, 273 i.u./ml, 1047 i.u./ml and 795 i.u./ml, respectively. In contrast when CML cells were cultured at 0.03 x 10(5) MNC/ml the concentration for optimal CML CFU-GM growth, the mean LD50 was significantly lower than that in normal PB and recovery phase PB, 4 i.u./ml, 251 i.u./ml and 78 i.u./ml, respectively (p less than 0.05). This is the first report of a differential sensitivity to IFN between CML and non-CML progenitors using an optimized PB CFU-GM assay system and proposes that further study of the in vitro culture of CML progenitors may increase our understanding of the clinical effects of IFN.