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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3943-3951
CD95 (APO-1/Fas) Mutations in Childhood T-Lineage Acute
Lymphoblastic Leukemia
By
Christian Beltinger,
Elke Kurz,
Thomas Böhler,
Martin Schrappe,
Wolf-Dieter Ludwig, and
Klaus-Michael Debatin
From the Sektion Hämatologie/Onkologie,
Universitäts-Kinderklinik and Abteilung Molekulare Onkologie,
Deutsches Krebsforschungszentrum (German Cancer Research Center),
Heidelberg, Germany; the Abteilung Pädiatrische Hämatologie
und Onkologie, Medizinische Hochschule Hannover, Germany; and the
Abteilung für Hämatologie, Onkologie, und Tumorimmunologie,
Robert-Rössle-Klinik, Humboldt Universität Berlin, Germany.
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ABSTRACT |
CD95 (APO-1/Fas)-mediated apoptosis is pivotal in normal lymphocyte
homeostasis and mutations of CD95 cause a benign autoimmune lymphoproliferation syndrome (ALPS) in humans and mice. However, tumors
only rarely develop in these patients, and no CD95 mutations have yet
been directly implicated in tumorigenesis. We therefore examined 81 de
novo childhood T-lineage acute lymphoblastic leukemias (T-ALL)
including 54 steroid-poor responders, 10 relapsed T-ALL, and 10 leukemic T-cell lines, for the presence of CD95 mutations using
single-strand confirmation polymorphism and sequence analysis. In
leukemic blasts and normal T cells of one patient, a heterozygous mutation in exon 3 of CD95 causing a 68Pro  68Leu change
associated with decreased CD95-mediated apoptosis was found. In
leukemic blasts and normal T cells of a second patient, a homozygous
mutation in the promoter of CD95 causing disruption of a consensus
sequence for AP-2 binding without decreasing constitutive CD95
expression was detected. No large intragenic alterations of CD95 were
found, no homozygous loss was detected in the cell lines, and no CD95 mutations were detected in the relapses. The data presented here show
that CD95 mutations occur in some T-ALL and may be of biological importance.
| |
INTRODUCTION |
APO-1 (CD95/FAS)-MEDIATED apoptosis is a
major mechanism of growth control, at least in lymphoid cells and
perhaps in other cells. CD95 is expressed in many normal
and malignant human cells,1 including T-cell
leukemias.2-5 CD95 expression has been associated with a
better prognosis in B-cell lymphomas6 and with
responsiveness to chemotherapy in acute myeloid leukemia,7
whereas most primary T-cell leukemias are constitutively resistant
against CD95-induced apoptosis.4,5,8
The role of the CD95 system in tumorigenesis in animal models is
complex. Production of CD95 ligand in tissue compartments inducing
apoptosis in T cells9 may explain the existence of immunologically privileged sites like the central nervous system (CNS),
long known as a sanctuary site for acute lymphoblastic leukemia (ALL).
A similar effect is exerted by CD95 ligand producing tumor cells like
melanoma,10 hepatocellular carcinoma,11 colon carcinoma,12,13 and lung carcinoma,14 which may
explain the phenomenon of tumors as immunologically privileged tissues.
Interestingly, CD95-deficient lpr/lpr mice do not develop
malignancy unless they exhibit a concomitant T-cell defect, which
favors the development of B-cell lymphomas, suggesting a tumor
suppressor function of CD95.15 In a similar way,
lpr/lpr mice show accelerated development of T-cell lymphomas only
in the presence of additional oncogenic events such as overexpression
of L-MYC.16
Progression of tumors may be facilitated by production of soluble (s)
CD95 in the tumor via alternative splicing. sCD95 secreted by this
mechanism neutralizes Fas ligand, thus causing resistance against
CD95-mediated apoptosis.17 sCD95 was detected in a human T-cell leukemia cell line18 and found to be increased in
the serum of patients with lymphoid but not myeloid
malignancies.19,20
Mutations in CD95 with defective CD95-mediated T-cell apoptosis have
been described in patients with autoimmune lymphoproliferative syndrome
(ALPS).21-25 Their phenotype with massive benign
lymphadenopathy, autoimmunity, and increased number of
CD3+CD4-CD8- lymphocytes is similar
to the lpr syndrome described in mice.26 Most of
the mutations, of which the majority were point mutations in the death
domain, were heterozygous, showed a dominant negative phenotype, or
required additional genetic or other factors to result in ALPS. Of
note, only 3 patients developed malignancies: hepatitis-associated
hepatocellular carcinoma, multiple neoplasia, and
osteosarcoma.23,24 A CD95 germline mutation, causing an apoptosis defect and associated with ALPS and Hodgkin's disease, has
been described in 1 family.25
In addition to its role in negative physiological growth control,
considerable evidence from different laboratories suggests that
apoptosis induced by anticancer drugs involves activation of the CD95
system,27-32 whereas others found chemotherapy-induced apoptosis to be CD95-independent.33
Because the incidence of CD95 mutations in malignancies has not been
investigated yet with sensitive methods, the relevance of CD95
mutations for tumorigenesis and tumor progression is unknown. We
therefore investigated childhood T-lineage ALLs (T-ALL) for CD95
mutations. We show in this report that CD95 is mutated in some T-ALL.
These mutations, which differ from those causing ALPS, may be of
biological significance.
| |
MATERIALS AND METHODS |
Patients and case reports.
Samples from 81 randomly chosen children with de novo T-ALL were
investigated. In addition 10 children with relapsed T-ALL were
examined, in 5 of which the initial disease was investigated as well.
From 60 children entered on the German ALL-BFM 90 trial34 complete clinical data were available
(Table 1). Immunologic marker analysis was
performed at the central reference laboratory of the ALL-BFM trials
(Robert-Rössle-Klinik, Humboldt Universität, Berlin,
Germany). Cell surface and intracytoplasmic (cy)/intranuclear antigens
were detected by standard direct or indirect immunofluorescence assays
as previously described.35,36 Immunophenotypic subgroups of
T-ALL were defined as follows: pro-T-ALL: cyCD3+, CD7+; pre-T-ALL: cyCD3+, CD7+, CD2+ and/or CD5+ and/or CD8+; cortical
T-ALL: cy or membrane CD3+, CD7+, CD1a+; and mature T-ALL: membrane
CD3+, CD1a-.37 Because more samples were available from
children with initially high leukocyte counts the selection was biased
towards high-risk patients, which included a high number of
steroid-poor responders.38
Patient 7 was a boy found to be microcephalic and micrognathic in
infancy who at the age of 4 years developed a pharyngeal epithelial
carcinoma for which he was treated with radiation therapy, methotrexate, and a single dose of cyclophosphamide. At the age of 14 years, he presented with lymphadenopathy, marked hepatosplenomegaly, a
white blood cell count (WBC) of 98200/µL with 84% lymphoid blasts, hemoglobin 4.5 g/dL, platelet count of 127000/uL, and CNS disease. Immunophenotyping showed an early T-ALL. No cytogenetic and molecular tests were performed. He responded poorly to steroid induction and died
of pneumonia after 11 months of palliative therapy. The maternal
grandfather had died of Hodgkin's disease.
Patient 14, a girl, has previously been partially
described.39 She presented at the age of 18 months with
massive hepatosplenomegaly, a WBC of 743000/µL with 94% lymphoid
blasts, hemoglobin 7.6 g/dL, platelet count of 52000/µL, and CNS
disease. The immunophenotype showed a T-cell ALL with an intermediate
thymocyte phenotype. Karyotype was 45, XX,  11, 14, and a
marker chromosome, most likely der11. The T-cell receptor (TCR) showed
a germline configuration of the  -gene by Southern blot analysis.
Molecular analysis showed a heterozygous deletion of the ALL-1
gene, eliminating exon 8.39 She responded poorly to
steroids, blasts persisted in her bone marrow at day 15 but the
deletion was undetectable in her bone marrow at day 33. Fourteen months
after initial diagnosis she developed T-ALL, whose immunophenotypic and
molecular features differed from her initial disease. Immunological
markers had changed to a mature T-cell phenotype. The blasts now
expressed TCR  and proteins as determined by flow cytometry. No
ALL-1 deletion was found. Despite intensive chemotherapy, she
did not achieve remission and died of sepsis. There was no familiy
history of malignant disease. The parents were not consanguinous.
Patient cells and cell lines.
Leukemic blasts were obtained by Ficoll-Hypopaque separation of blood
or bone marrow at the time of diagnosis. Mycoplasma-free T-cell lines
Jurkat 16, Molt 4, CEM, CEMDOXOR (a doxorubicine-resistant
CEM derivative cross-resistant to APO-1) and CEMCD95R (an
APO-1-resistant CEM derivative cross-resistant to
doxorubicine),27 HPB, HUT 78, Walser, SKW 3, and H9 are
routinely maintained in our laboratory and were kept in continuous
suspension culture as previously described.40
DNA and RNA isolation.
Genomic DNA and total RNA were extracted using Quiagen Genomic-tips and
RNEasy kit, respectively, according to the manufacturer's directions
(Quiagen, Hilden, Germany). DNA from 50 unrelated donors without
hematologic or immunologic disease was provided by Dr Bartram
(Heidelberg, Germany).
Long-distance polymerase chain reaction (PCR) and restriction enzyme
digestion.
CD95 was amplified by long-distance PCR using genomic DNA from patient
cells. Two overlapping primer pairs were constructed according to the
published sequence of CD9541-43: CD95LoFlank,
5 -ATTAGATGCTCAGAGTGTGTGCACAAGGCTGG-3 with
CD95LoInt2A, 5-ACATACCTGGAGGACAGGAGTTGATGTCAGTC-3,
spanning the 5 flanking region to intron 2 and CD95LoInt2B;
5-CTGAGATCCAAACTGCTATACAAGTGACCTGC-3 with CD95LoEx9,
5-GGCTGTGCTCATTGACATGGGAGAAAGTCATG-3, spanning intron 2 with the untranslated region of exon 9. Primers CD95LoFlank and
CD95LoInt2A were used with the Expand Long Template PCR System (Boehringer Mannheim, Mannheim, Germany). Two hundred fifty ng of DNA
were amplified according to the manufacturer's directions using buffer
#3 and 0.75 µL of enzyme mix. Primers CD95LoInt2B and CD95LoEx9 were
used with the Advantage-GC Genomic PCR kit (Clontech, Heidelberg,
Germany) according to the manufacturer's directions, with a final
GC-Melt concentration of 1 mol/L. PCR conditions for both primer pairs
were as follows: denaturing for 2 minutes at 94°C; 10 cycles of
94°C for 10 seconds, 65°C for 30 seconds, and 68°C for 12 minutes; followed by 26 cycles of 94°C for 10 seconds, 65°C for
30 seconds, and 68°C for 12 minutes, with an increment of 20 seconds per cycle; and finished by a final extension at 68°C for 7 minutes. PCR was performed with PTC 200 thermocyclers (MJ Research,
Watertown, MA). PCR products were digested with BamHI and
HindIII (Promega, Madison, WI), separated on 0.7% agarose
gels, stained with ethidium bromide, and visualized under ultraviolet
light.
PCR and single-strand conformation polymorphism analysis (SSCP).
The CD95 gene consists of nine exons.41 Genomic DNA from
patients and cell lines was amplified with primer pairs covering and
flanking the coding region (Table 2). For
positive SSCP controls, mutant PCR products were made by substituting a
single base within one primer of each primer pair, creating either an
inversion or a transversion. Amplification was done with 100 ng human
DNA; 300 nmol/L of each primer; 50 µmol/L each of dCTP, dGTP, and
dTTP; 40µmol/L dATP; 1µCi [-33P] dATP; 25 or 75 mmol/L KCl; 1.5 or 3.5 mmol/L MgCl2; 10 mmol/L Tris, pH
8.3, 8.8, or 9.2; and 1.2 U Taq DNA polymerase (MBI Fermentas, St
Leon-Rot, Germany). The samples were denatured at 95°C for 3 minutes; amplified for 15 cycles, with each cycle consisting of 45 seconds at 95°C and 1 minute at 70°C, with a decrease of 0.7°C per cycle; followed by 25 cycles, with each cycle consisting of 45 seconds at 95°C, 30 seconds at 50°C or 55°C, and 1 minute at 72°; followed by 5 minutes at 72°C. Two µL of
amplified DNA was diluted with 8 µL of 95% formamide, 10 mmol/L
NaOH, 0.25% bromophenol blue, and 0.25% xylene cyanole; denatured for
5 minutes at 95°C; and flash cooled on ice. Seven µL of the
denatured sample were loaded on a gel either with or without 5%
glycerol. The gels consisted of 0.5 × MDE gel solution (AT
Biochem, Malvern, PA) and 0.6 × TBE (1×= 0.09 mol/L Tris,
0.09 boric acid, 0.002 mol/L EDTA, pH 8.0). Electrophoresis conditions
were 6 to 8 W for 15 to 17 hours at 25°C. Gels were dried and
autoradiography was performed at 70°C. Mutant bands were cut
from the dried gel, the DNA eluated into water overnight, and
reamplified and sequenced using the same primers applied for initial
amplification.
Sequence analysis.
Cycle sequencing was performed with the fmol DNA cycle sequencing
system (Promega, Madison, WI) according to the manufacturer's specifications.
Dinucleotide repeat analysis.
Two polymorphic dinucleotide repeats flanking the CD95 gene, AFM 205xe3
and AFMb362yg5, were chosen from the Genome Data Base and primers
synthesized according to the sequences provided. PCR was performed as
described for SSCP-PCR except that one primer was endlabeled with
 -33P using polynucleotide kinase. Amplimers were
separated using a 8% denaturing polyacrylamide gel run with 60 W. Gels
were dried and autoradiography performed.
Reverse transcriptase (RT)-PCR.
CD95 cDNA synthesis was performed as previously
described.40 A 311 bp fragment of CD95 cDNA was amplified
using primers 5-TCAAGGAATGCACACTCACCAGC-3 and
5-GGCTTCATTGACACCATTCTTTCG-3. To control for RNA
integrity and to quantitate, a 600-bp fragment of GAPDH cDNA was
amplified. Radioactive touchdown PCR was performed as described above,
with 50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L Tris pH
8.3, an annealing temperature of 55°C, and 26 final cycles. Amplification was within the exponential range (data not shown). PCR
products were separated on a 4% polyacrylamide gel. Films were exposed
to the dried gels overnight.
CD95 expression on ALL-blasts.
Cryopreserved ALL-blasts were resuspended in RPMI 1640 supplemented
with 10% fetal calf serum (FCS), penicillin/streptomycin, L-glutamine,
and HEPES buffer at a concentration of 2 × 106
cells/mL. Immunophenotyping was performed with biotinylated anti-APO-1 (IgG3) monoclonal antibody (MoAb) and streptavidin-phycoerythrin, in
conjunction with CD7 fluorescein isothiocyanate (FITC) and CD3 PerCP
mouse MoAb (Becton Dickinson, Heidelberg, Germany).44
Separation of T-ALL blasts from T lymphocytes.
CD7+CD3- T-ALL blasts were separated from
CD3+ T cells by negative selection using an anti-CD3 mouse
MoAb (OKT3) and goat-antimouse antibody-coated magnetic beads
(Dynabeads M-450; Dynal, Hamburg, Germany) as described.45
Apoptosis assay.
Apoptosis of cryopreserved leukemic blasts from six patients and the
Jurkat 16 cell line was measured by assessment of plasma membrane
integrity as determined by uptake of the DNA intercalating dye
propidium iodide (PI; 2.5 µg/µL final concentration) analyzed by
flow cytometry. Percentage of specific cell death following incubation
with anti-CD95 MoAb (anti-APO-1 IgG3, 10 µg/mL) and Protein A (5 ng/ml; Pharmacia, Uppsala, Sweden) was calculated as follows: 100 × (experimental PI uptake [% of cells]) spontanous PI
uptake of cells in medium (% of cells) divided through (100% spontaneous PI uptake [% of cells]). A minimum of 10,000 events were
analyzed in each experiment.
| |
RESULTS |
Lack of large insertions or deletions of the CD95 gene in pediatric
T-cell leukemia.
To look for larger intragenic alterations, the complete CD95
gene, successfully amplified in 65 patient leukemias by overlapping long-distance PCR, was subjected to restriction endonuclease analysis. Double-digestion with BamHI and HindIII
did not show alterations on ethidium bromide-stained agarose gels
(data not shown).
Abnormal SSCP patterns are found in some pediatric T-cell leukemias.
To estimate the sensitivity of the SSCP assay, the positive controls
made for each primer pair by using a single-base mismatched primer were
subjected to SSCP. The SSCP assay detected 15 of 18 (83%) of the
mutant PCR products (data not shown). To search for point mutations and
small deletions or insertions, PCR-SSCP analysis was performed on the
complete coding region and the 3 promoter of the CD95 gene in
the patient samples and leukemic cell lines. Abnormal SSCP patterns
were found in exon 3, (Fig 1A). The pattern seen in patient 7 was unique for this patient, because it was not
detected in 100 chromosomes of normal probands. A second pattern (patient 64) was found in 6 of 81 patients and in several healthy controls, suggesting a polymorphism. SSCP analysis of the 3
promoter showed abnormal bands in 1 patient (patient 14) not seen in
the 50 normal controls (Fig 1B). All other exons were normal in all patient samples and cell lines studied.
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| Fig 1.
Abnormal SSCP patterns are found in some pediatric T-cell
leukemias. PCR-SSCP analysis of exon 3 (A) and the proximal promoter (B) of CD95. C, normal human genomic DNA amplified using a
mutant primer with a single base mismatch (positive control);
7,14,64, patient leukemias; N, peripheral blood from
healthy controls. Abnormal migrating bands are seen in the positive
controls, the leukemias and in one healthy control (*).
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A heterozygous germline mutation in exon 3 of CD95 causing a 68Pro
68Leu change is associated with decreased CD95-mediated
apoptosis.
In patient 7, a C T transition was found at position 251 of
the cDNA causing a Pro68 Leu68 substitution (the locations of cDNA and protein are given in respect to the ATG start codon and the
start of the mature protein, respectively, according to Itoh et
al46) (Fig 2A). To
determine zygosity and germline involvement of the mutation, the
patient's mature T lymphocytes were immunomagnetically purified from
the T-ALL blasts based on CD3 expression. With this separation method
we routinely obtain greater than 95% purity of both cell populations
of interest. Sequencing of exon 3 amplificates of both cell populations
showed the mutation to be heterozygous and to involve the patient's
germline (Fig 2A). To assess whether CD95 expression was altered in
this patient in addition to the presence of the missense mutation, CD95
mRNA of the blasts was determined by RT-PCR. The blasts strongly
expressed CD95 compared with a different cryopreserved patient leukemia
and Jurkat cells not harboring CD95 mutations (Fig 2B). To determine
whether this mutation affected CD95-mediated apoptosis, specific
apoptosis was measured by flow cytometry after incubation of blasts
with anti-APO-1 and found to be markedly decreased
(Table 3).
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| Fig 2.
Mutation analysis of patient 7. (A) Heterozygous germline
mutation in exon 3 of CD95 causing a 68Pro 68Leu change.
Genomic DNA of PBMC was subjected to PCR using primers flanking
exon 3 of CD95, amplificates were subjected to SSCP analysis, abnormal migrating bands were cut-out from the gel, reamplified, and sequenced. T-ALL blasts and mature T lymphocytes were immunomagnetically purified,
exon 3 of CD95 amplified by PCR and sequenced. Nucleotide sequence is
compared to wild type. Protein is numbered in respect to the start of
the mature protein (according to Itoh et al46). (B) CD95
mRNA is strongly expressed compared with cryopreserved blasts from a
different patient and thawed Jurkat cells. mRNA was determined by
RT-PCR and compared with GAPDH.
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Table 3.
Decreased Sensitivity of T-All Blasts of Patient 7 Towards Anti-CD95 Induced Apoptosis Compared With Jurkat Cells
Without CD95 Mutations
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A homozygous germline mutation in the promoter of CD95 causes
disruption of an AP-2 binding consensus sequence, but does not decrease
constitutive CD95 expression.
A deletion/insertion was found in patient 14, involving nt 350
to nt 347 of the CD95 promoter and destroying an AP-2-binding consensus sequence (location of promoter nucleotides relative to the
ATG start codon according to Behrmann et al41)
(Fig 3A). To determine zygosity and
germline involvement of the mutation, the patient's mature T
lymphocytes were immunomagnetically purified from the leukemic blasts.
Sequencing of appropriate promoter amplificates of both cell
populations showed the mutation to be homozygous and to involve the
patient's germline (Fig 3A). To define the pattern of inheritance of
the mutation, the appropriate promoter region was sequenced in the
parents. As shown in Fig 3B, the father was homozygous for the mutation
and the mother homozygous for the wild type. To rule out paternal
disomy, two polymorphic markers flanking the CD95 gene, AFM205xe3 and
AFMb362yg5, were analyzed. The patient showed both paternal and
maternal alleles, thus ruling out paternal disomy (data not shown). To
assesss whether this promoter mutation affected expression, CD95 was
assayed on immunomagnetically separated leukemic blasts and mature T
cells of the patient from the time of initial presentation. CD95 was
expressed (Fig 3C) and was not decreased compared with patients'
leukemias not harboring this or other CD95 mutations
(Table 4).
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| Fig 3.
Mutation analysis of patient 14. (A) Homozygous germline
mutation in the promoter of CD95 causing disruption of a consensus sequence for AP-2 binding. Genomic DNA of PBMC was amplified by PCR using primers 1-1F/1-1R (covering nt 408 to nt 202 of
the CD95 promoter; nucleotides numbered relative to the ATG start codon
according to Behrmann et al41). The amplificate was
subjected to SSCP analysis, abnormal migrating bands were cut-out from
the gel, reamplified, and sequenced. T-ALL blasts and mature T
lymphocytes were immunomagnetically purified, PCR-amplified as above,
and sequenced. Nucleotide sequence is compared to wild type. Bases involved in the mutation are highlighted by asterisks. (B) The mutation
is inherited from the father. Genomic DNA of PBMC from the mother (M),
the father (F), and the patient (P) were amplified as under (A).
The mutation is inherited from the homozygous father, whereas the
mother has wild-type alleles only. (C) The mutation in the CD95
promoter does not decrease constitutive CD95 expression. CD95
expression on cryopreserved PBMC was determined by three-color immunofluorescence analysis as described in Materials and Methods. For
electronic gating T-ALL blasts were identified by forward/side scatter
characteristics of viable lymphocytes and surface expression of CD7 but
not CD3. Mature T cells were CD3+ and
CD7+.
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No homozygous loss of CD95 in T-ALL cell lines.
No homozygous loss of CD95 was present in the cell lines because the
gene could be amplified in all cell lines examined (data not shown).
No CD95 mutations in relapsed T-ALL.
To determine if CD95 mutations are acquired during disease evolution,
relapsed T-ALL were investigated. No mutations were found by SSCP in
the 10 relapses studied (data not shown).
A known polymorphism is detected with the expected frequency.
Sequence analysis of the abnormal SSCP bands detected as the same
pattern in exon 3 in 6 patients and 1 healthy control showed the
presence of a polymorphism already described47: 222A G
(data not shown; numbering of cDNA in respect to the ATG start codon,
according to Itoh et al46).
| |
DISCUSSION |
We hypothesized that CD95 (APO-1/Fas) mutations may occur in childhood
T-lineage leukemia based on several lines of evidence. First, CD95 is
of major importance for controlling T-cell homeostasis. Second, the
existence of benign lymphoproliferative syndromes shows that
dysfunction of CD95 causes accumulation of T lymphocytes. Third, most
primary T-lineage leukemias are resistant to CD95-mediated apoptosis.
Fourth, evidence is emerging that lymphoid malignancies may be
associated with CD95 mutations.48 Finally, the CD95 system mediates cytotoxicity of important drugs used in the therapy of ALL and
drug resistance is common in T-ALL.27-32
We found mutations in 2 of 81 patients. It is unlikely that many
mutations have been missed because our PCR-SSCP assay has a sensitivity
of at least 83% for detecting point mutations, as estimated by the
detection of positive controls created by single-base mismatched
primers. This sensitivity is comparable with most published SSCP
assays. Nevertheless, detection of primer mutations are not necessarily
predictive of the detectability of mutations elsewhere within the
amplicon. Therefore, as in all SSCP assays, some mutations might have
been missed. The long PCR/restriction enzyme assay will detect most
gross intragenic alterations of CD95 such as large deletions (unless
they involve a primer binding site) or insertions (as long as their
size does not prevent amplification), although rearrangements such as
translocations would not be amplified. Homozygous loss of CD95 could
have been masked by contamination of the leukemic samples with normal
cells. This, however, is unlikely, because no homozygous loss was
detected in the homogenous cell lines. Because we have examined only
the 3 promoter and did not examine the majority of cases for
CD95 expression, we cannot rule out a higher incidence of CD95
alterations outside the coding and 3 promoter region. However,
most T-ALL have been shown to express CD95.5
In T-ALL with CD95 mutations, these mutations might contribute to
leukemogenesis and chemoresistance. In patient 7, the heterozygous alteration of CD95 was not detected in 100 chromosomes of normal probands, making a polymorphism unlikely. This mutation without loss of
expression was associated with apoptosis resistance. Interestingly, this mutation is located adjacent to a stretch of DNA that encodes for
amino acid residues important for ligand binding to CD95.49 It remains to be proven whether this mutation decreases ligand binding,
interferes with receptor signaling or is silent. Whether the
development of two unrelated malignancies at young age, one of which
very rare at this age, is related to the germline mutation of this
dysmorphic patient with a family history of Hodgkin's disease, has to
be investigated.
In patient 14, a homozygous alteration was found in the promoter. This
alteration was not found in 50 normal individuals suggesting a bona
fide mutation rather than a polymorphism. The mutation's unusual
feature of involving the germline in a homozygous fashion prompted us
to investigate the parents. Surprisingly, the mutation was inherited
from the homozygous father. We ruled out uniparental (paternal) disomy
although a small partial paternal disomy might escape detection if the
distance of the flanking polymorphic markers is large enough.
Alternatively, the maternal allele might have been lost by
microdeletion during early zygote development. This mutation destroys a
consensus sequence for binding the transcription factor AP-2
without affecting constitutive CD95 expression. In T cells, AP-2 has
been shown to influence the expression of the tumor necrosis factor (TNF-) gene50,51 and might play a role in the
constitutive expression of the IL3 gene.52 Drug treatment
of leukemic cells increases CD95 expression and susceptibility to
CD95-mediated apoptosis.27,28 Leukemia cells
cross-resistant to APO-1 and cytotoxic drugs show diminished
upregulation of CD95 that contributes to drug resistance in these
cells.29,30 It has to be shown whether the chemoresistant
blasts of this patient show diminished CD95 upregulation and, if so,
whether the destruction of the AP-2 binding site contributes to this
regulation defect. The similarity between this patient with two T-ALL
in rapid succession, a CD95 germline mutation with possible attenuation
of CD95 upregulation, an ALL-1 gene deletion at initial
presentation, and the lymphoma-bearing lpr/lpr mouse
overexpressing L-MYC16 is intriguing and warrants
further study to show a permissive effect of this CD95 mutation on
leukemogenesis.
It is surprising that both mutations identifed in this study were
present in the germline, as germline mutations in other oncogenes and
tumor-suppressor genes are rare in childhood ALL. Interestingly, the
death domain, which is mutated in most cases of ALPS with CD95
mutations,21-25 is not altered in T-ALL.
The two ALL patients with CD95 mutations were both drug resistant. This
might indicate that CD95 mutations are more important for
chemoresistance than for leukemogenesis. As mentioned, recent evidence
suggests that the CD95 system mediates cytotoxic drug action; and CEM
cells cross-resistant to APO-1 and cytotoxic drugs show a defect in
activation of the CD95 system.27 However, CD95 in these
cells was not mutated in the present study, indicating that
chemoresistance due to alterations in the CD95 system may be determined
by mechanisms other than CD95 mutations. Our study population included
a high percentage of steroid-poor responders. Steroid sensitivity is
mediated by mechanisms other than the CD95 system. It is therefore
conceivable that, despite the predictive value of the steroid response
for therapy failure, alterations in cytotoxic mechanisms different from
the steroid pathway may contribute to therapy failure. Thus, examining
more chemoresistant patients might show a higher incidence of CD95
mutations.
The low incidence of CD95 mutations in untreated T-ALL in this cohort,
heavily biased towards high-risk patients, suggests that mutations in
CD95 do not contribute to leukemogenesis in most T-ALL. This is in line
with the absence of reported cytogenetic or molecular genetic
aberrations in T ALL involving 10q23 where CD95 is
located.53 Also, coinheritance of a lymphoid
malignancy with a germline CD95 mutation causing impaired apoptosis has
been reported in only one familiy with ALPS and Hodgkin's
disease.22 Furthermore, in mouse models, loss of CD95 does
not give rise to malignancy unless a second insult is
provided.15,16 We found an APO-1-resistant phenotype
without mutations in CD95 and with normal CD95 expression in one
leukemia (patient 36). This finding is supported by the fact that most
T-ALL are resistant to CD95-mediated apoptosis,4,5 whereas
only very few have mutated CD95. Thus, apoptosis defects in T-ALL may
be caused by molecules other than CD95. In a limited number of
relapses, half of them paired with their initial disease, no CD95
mutations were found. This argues against CD95 mutations being
accumulated in late stages of disease evolution.
In conclusion, CD95 is mutated in some de novo childhood T-ALL and
these alterations, which differ markedly from those described in ALPS,
might be of functional relevance. In most de novo T-ALL, CD95
alterations do not contribute to leukemogenesis and, in a limited
number of relapsed T-ALL, no involvement of CD95 mutations in the
evolution of the disease was seen. It remains to be determined whether
CD95 mutations are involved in chemoresistance of T-ALL.
| |
FOOTNOTES |
Submitted September 11, 1997;
accepted January 7, 1998.
Supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and
the Tumorzentrum Heidelberg/Mannheim, Germany.
Address correspondence to Klaus-Michael Debatin, MD,
Universitäts-Kinderklinik Ulm, Prittwitzstrasse 43, 89075 Ulm,
Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr B. Jannsen, Institut für Humangenetik und
Anthropologie, Universität Heidelberg, Germany for DNA of healthy controls and helpful discussions, and Dr Bender-Götze,
Kinderpoliklinik der Universität München, Germany and Dr I. Richter, Kinderklinik und Poliklinik der Universität Rostock,
Germany for providing patient specimens and information.
| |
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Abstract
Introduction
Abstract