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Keywords:

  • PNH;
  • dual pathogenesis;
  • CAMPATH-1H;
  • clonal selection

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Paroxysmal nocturnal haemoglobinuria (PNH) cells are deficient in glycosylphosphatidylinositol (GPI) linked antigens due to a somatic mutation of the PIG-A gene in a haemopoietic stem cell. It appears that a PNH clone reaches detectable proportions only when there is selection in its favour. GPI-deficient T lymphocytes have been identified in patients treated with CAMPATH-1H, a monoclonal antibody against the GPI-linked CD52 molecule. CAMPATH-1H selects for cells that are deficient in CD52 (such as PNH-like cells) promoting the development of a PNH-like clone (analogous to PNH). We report that 10/15 patients with chronic lymphocytic leukaemia developed PNH-like lymphocytes after therapy with CAMPATH-1H. The remaining five patients developed no PNH-like cells at any stage, including one patient who received 12 weeks of therapy. The inactivating PIG-A mutation has been identified in one patient. This mutation was detectable by an extremely sensitive mutation-specific PCR-based analysis in the patient's mononuclear cells prior to CAMPATH-1H therapy. The frequency and phenotype of GPI-deficient lymphocytes after CAMPATH-1H and the detection of a PIG-A mutation in the lymphocytes prior to CAMPATH-1H therapy indicated that such mutations were present in a very small proportion of cells prior to selection in their favour by CAMPATH-1H. This suggests that a large proportion of individuals have cells with PIG-A mutations that are not detectable by flow cytometry and thus may have the potential to develop PNH.

Paroxysmal nocturnal haemoglobinuria (PNH) is a chronic disorder characterized by episodes of intravascular haemolysis often associated with abdominal pain, recurrent life-threatening venous thrombosis and an intimate association with aplastic anaemia (AA) ( Hillmen et al, 1995 ; Socie et al, 1996 ). PNH is a clonal disorder arising due to a somatic mutation in a pluripotent haemopoietic stem cell. The resulting PNH clone is deficient in all surface antigens that are normally anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. In 1993 the PIG-A gene was identified by complementation of a GPI-deficient mutant cell line ( Miyata et al, 1993 ) thereby demonstrating that it is an essential step in the biosynthetic pathway of GPI structures. PIG-A mutations have been identified in all cases of PNH reported to date ( Takeda et al, 1993 ; Rosse & Ware, 1995, Luzzatto & Bessler 1996). Thus a PIG-A mutation is necessary for the development of PNH.

But does a PIG-A mutation in a pluripotent haemopoietic stem cell inevitably lead to the development of PNH? The answer to this intriguing question lies in the relationship between AA and PNH. A significant proportion (up to 52%) of patients with AA will eventually develop a PNH clone ( Schrezenmeier et al, 1995 ). In addition the majority of patients with PNH have significant cytopenias at some stage in their illness ( Hillmen et al, 1995 ). Considering that both AA and PNH are rare disorders this association cannot be a chance finding. Thus PNH appears to occur only in individuals with bone marrow failure (AA). There is now overwhelming evidence that AA results, at least in part, from an immune-mediated attack against the haemopoietic stem cell ( Young & Barrett, 1995). It has been postulated that the PNH clone, presumably because of the lack of one or more GPI-linked surface proteins, is able to avoid the aplastic process and therefore has a relative growth and/or survival advantage over the residual normal haemopoiesis ( Rotoli & Luzzatto, 1989). This theory indicates that in order to develop PNH a patient must have both a PIG-A mutation in a haemopoietic stem cell and an underlying aplastic process. This hypothesis will be referred to as the dual pathogenesis of PNH. The finding that approximately half of patients with AA develop a detectable PNH clone at some stage of their disease suggests that PIG-A mutations are reasonably frequent occurrences but do not usually result in PNH because the PNH clone has no selective advantage over normal haemopoiesis.

CAMPATH-1H is a humanized monoclonal antibody ( Hale et al, 1990) that is specific for CD52 , an antigen expressed on all lymphocytes and monocytes as well as mature spermatozoa. The function of CD52 is unknown, but CAMPATH-1H was isolated because of its extraordinary ability to kill cells by complement-mediated attack. CAMPATH-1H, when given in vivo, results in a profound prolonged lymphopenia and has been used therapeutically in a range of human diseases, including rheumatoid arthritis, non-Hodgkin's lymphoma and chronic lymphocytic leukaemia ( Osterborg et al, 1997 ). Patients treated with CAMPATH-1H have been reported to develop GPI-deficient T cells and the analysis of the PIG-A messenger RNA from a GPI-deficient T-cell line in one such patient was abnormal, suggesting an underlying PIG-A mutation ( Hertenstein et al, 1995 ). This phenomenon is analogous to PNH with the CAMPATH-1H selecting for a GPI-deficient T-cell clone. GPI-deficient clones are not found in patients who have not experienced a selective process in favour of such a clone (i.e. PNH or CAMPATH-1H therapy). It is not known whether the GPI-deficient clone is present prior to the selection or if it occurs only after the onset of the selective pressure.

We now report the findings in a group of 15 patients with refractory chronic lymphocytic leukaemia (CLL) who were treated with CAMPATH-1H antibody.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Patients

15 patients with CLL whose disease was refractory to conventional therapy, including fludarabine, were treated with CAMPATH-1H (see 1 Table I). Informed consent was obtained from all patients prior to their inclusion in this study. 12 patients completed a full course of CAMPATH-1H (18 × 30 mg doses of CAMPATH-1H over 6 weeks) with three of these patients subsequently receiving a second full course of CAMPATH-1H therapy. The remaining three patients received a median of eight doses (range five to 12) of CAMPATH-1H and stopped because of a satisfactory response and/or neutropenia.

Table 1. Table I. Patient details. Abbreviations: PB, peripheral blood; BM, bone marrow; LN, lymphadenopathy; SP, splenomegaly; ITP, immune thrombocytopenic purpura; Flud, fludarabine; Clb, chlorambucil; Cyclo, cyclophosphamide; DCF, deoxycorformycin; Pred, prednisolone; CHOP, cyclophosphamide, hydroxydoxorubicin, vincristine and prednisolone. Numbers in parentheses indicate number of courses received.Thumbnail image of

Flow cytometry

Leucocytes were prepared from peripheral blood (collected weekly during CAMPATH-1H treatment) and bone marrow (pre-CAMPATH-1H, 3 weeks and post-CAMPATH-1H). by incubation with a 10-fold excess of ammonium chloride (8.6 g/l in distilled H2O) for 5 min, and washed twice in FACSFlow (Becton Dickinson)/0.3% BSA (Sigma Diagnostics). 106 leucocytes were incubated with 10 μl of each antibody per test for 20 min at 4°C. Cells were washed twice in FACSFlow/0.3% BSA, and acquired using a Becton Dickinson FACSort with CELLQuest 3.1 software. 50 000–500 000 total cells were analysed in each test. A sequential gating strategy, using at least three parameters including CD3 for T cells and CD19 for B cells, was used to assess the expression of the GPI-linked proteins CD52, CD55 and CD59. Samples from 13 normal subjects were also studied. Serial dilutions of GPI-deficient lymphocytes into normal lymphocytes were studied in order to ascertain the sensitivity of detection of GPI-deficient lymphocytes. Antibodies to CD3, CD19, CD45RO and CD52 were conjugated in-house; CD55 and CD59 were provided by Cymbus Biosciences.

Identification of the PIG-A mutation

DNA was isolated from the post-CAMPATH-1H mononuclear cells of patient 3. The exon 2 of the PIG-A gene was amplified using primers modified from those used by Yamada et al (1995 ). The primers were modified to include a 12 bp extension to facilitate cloning into the Gibco Clone AMP vector system. The cloned exon 2 was amplified using the M13 forward and reverse primers with the ABI PRISMTM dye primer cycle sequencing ready reaction kit with FS DNA polymerase. The product was then analysed with an ABI 373 automatic sequencer.

Mutation-specific polymerase chain reaction for PIG-A mutation

In order to detect an extremely low level of cells containing the PIG-A mutation a highly sensitive nested amplification refractory mutation specific (ARMS) technique was used. Three primers were designed: one complementary to the mutation, a second complementary to the normal sequence or to sequences mutated at an alternative site, and a third downstream of these. The mutation-specific primer was designed to be 6 bp longer than its normal counterpart to enable the two products to be distinguished from each other. These primers were used in a nested PCR using the exon 2 PCR product in order to increase the sensitivity of the assay. The optimum proportion of mutant to normal primer was assessed experimentally to make the assay extremely sensitive for the detection of mutant cells. DNA isolated from the pre-treatment mononuclear cells was used as the template for amplification of exon 2 of PIG-A.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Sensitivity of flow cytometry

Cells from a patient in which the T cells were >95% GPI deficient were serially diluted into normal leucocytes. The percentage of GPI-deficient T cells was then calculated. Fig 2 demonstrates that the analysis could detect as few as a single GPI-deficient cell in 10 000 normal cells.

image

Figure 2.  = 0.9883, slope = 0.93 ± 0.05, P < 0.0001.)

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Flow cytometry, pre-CAMPATH-1H

We have analysed the GPI-linked proteins on T cells and B cells using four-colour flow cytometry from 13 patients prior to CAMPATH-1H treatment and a further 13 normal subjects. There were no detectable GPI-deficient cells in any of the subsets studied in any patient or normal subject (Fig 1b).

image

Figure 2. Fig 1. Flow cytometric plot showing the expression of two GPI-linked antigens on T cells. Three-colour staining (CD3/CD52/CD59) was used and the T cells were identified by their characteristic CD3 versus side scatter plot (a). (b) Pre-CAMPATH; all T cells express both CD52 and CD59. (c) Post-CAMPATH; almost all of the T cells are deficient in both CD52 and CD59. (d) Kinetics of GPI-deficient T-cell expansion.

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Flow cytometry, post-CAMPATH-1H

GPI-deficient T cells developed in 10/15 patients following treatment with CAMPATH-1H. These cells were first detected between 3 and 5 weeks after first the start of CAMPATH-1H therapy. By the end of a full course of therapy the majority of each of these patient's T lymphocytes were GPI deficient (median 90%; see Fig 1c and Table II). The kinetics of emergence of GPI-deficient T cells, as well as depletion of normal T cells, is shown in Fig 1(d) (note logarithmic scale). In all cases assessed, >95% of the GPI-deficient T cells were CD45RO+. In contrast, no GPI-deficient cells were found in any of the remaining five patients at any stage of treatment. No GPI-deficient cells were observed in any of the other cell types, including the CLL cells, monocytes (identified by light scatter characteristics) or stem cells (identified by CD34 expression and side scatter characteristics).

Table 2. Table II. Flow cytometry of patients' cells. Abbreviations: na, not applicable; NT, not tested; nr, not reached.* Bone marrow analysis.Thumbnail image of

PIG-A gene analysis

The PIG-A gene in the GPI-deficient T cells of one of the patients (UPN 5) was studied. A somatic mutation was identified in exon 2 of the PIG-A gene (a single base pair deletion at nucleotide 138; see Fig 3a) which results in a frameshift with a premature stop codon (at nucleotide 293) and no functional gene product. Oligonucleotides were designed in order identify extremely low levels of the mutant gene by the PCR-based ARMS technique (see Fig 3b). The ARMS technique performed on DNA extracted from the mononuclear cells prior to the patient receiving any CAMPATH-1H revealed a faint PCR product (see Fig 3c). This indicates that a very low proportion of cells with this PIG-A mutation were present prior to CAMPATH-1H treatment that were not detectable by flow cytometry.

image

Figure 3. : DNA from pre-CAMPATH mononuclear cells; Lane 4; DNA from post-CAMPATH cells; Lane 5: DNA from PNH patient with a different mutation, and hence amplifiable with the ‘normal’ primer.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

No GPI-deficient cells can be detected by sensitive four-colour flow cytometry in normal individuals or in patients with CLL. However, GPI-deficient T cells develop in a proportion of patients within a few weeks of starting treatment with CAMPATH-1H, a monoclonal antibody against the GPI-linked antigen CD52. At the end of a 6-week course of CAMPATH-1H GPI-deficient cells usually comprise the majority of the patient's T cells, although absolute numbers are usually low (<0.2 × 109/l). Therefore selection against a single GPI-linked antigen promotes the development of a GPI-deficient population of cells. The finding that these GPI-deficient T cells appear within a very short time after initiation of CAMPATH-1H, and that they do not occur at any stage in a minority of patients, suggests that there are a small number of GPI-deficient T cells in the majority of individuals which only become evident following selection in their favour. This is supported by the fact that these cells are almost exclusively of a memory (CD45RO+) phenotype. The presence of a somatic mutation in the PIG-A gene of one of the patients demonstrated that these cells were monoclonal. The identification of this somatic mutation in a very low proportion of the patient's pre-CAMPATH-1H cells proves that the GPI-deficient clone was present prior to selection in its favour. All of these patients had previously received cytotoxic chemotherapy (see Table I) which may have increased the likelihood of somatic mutations generally and therefore may have increased the probability that these patients have occult PIG-A mutations. However, similar GPI-deficient T cells have been reported in patients receiving CAMPATH-1H for rheumatoid arthritis ( Brett et al, 1996 ) who had not received prior, potentially mutagenic, chemotherapy. The finding that most individuals develop GPI-deficient cells after treatment with CAMPATH-1H and that the PIG-A mutation responsible for such a GPI-deficient clone was present prior to treatment indicates that a significant proportion of individuals have GPI-deficient cells in very low numbers. These findings support the hypothesis that GPI-deficient cells are only capable of expansion to a level where they contribute significantly to haemopoiesis when there is selection in their favour.

Taylor et al (1997 ) have demonstrated that T cells with a PNH phenotype can occur after CAMPATH-1H therapy and that the GPI-deficient phenotype is stable in culture. Follow-up of the CLL patients, some for >2 years, indicated that the PNH phenotype is also irreversible in vivo. Taylor et al (1997 ) were unable to identify a mutation of the PIG-A gene in a single GPI-deficient T-cell line derived from one patient, but stated that they had not excluded disruption of PIG-A as a cause of the phenotype. It is possible that the mutation causing the GPI-deficient phenotype in this model is in another of the genes of the GPI-biosynthetic pathway, but there is still a requirement for selection prior to expansion of any GPI-deficient cells.

The findings presented here are analogous to the current theory of the dual pathogenesis of PNH. In order to develop PNH an individual must have both a haemopoietic stem cell with a PIG-A mutation (GPI-deficient) and a selective pressure to allow the PNH clone to dominate, which in the case of PNH is thought to be an immune-mediated attack against stem cells (aplastic anaemia). These findings strongly support the dual pathogenesis theory of PNH. In view of the finding that many patients with AA will develop a PNH clone it is likely that a PIG-A-deficient haemopoietic stem cell occurs extremely frequently in apparently normal individuals but does not become clinically significant in the absence of aplastic anaemia.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We thank the doctors who entered patients into this study and the patients themselves. We thank Cymbus Bioscience for providing monoclonal antibodies for this study. G.H. is supported by the Medical Research Council and LeukoSite Inc.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
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