Hematopathology / BONE MARROW STAGING IN CUTANEOUS T-CELL LYMPHOMA
Bone Marrow Histopathologic and Molecular Staging
in Epidermotropic T-Cell Lymphomas
Vincent Sibaud, MD,1,2 Marie Beylot-Barry, MD, PhD,2,3 Rodolphe Thiébaut, MD,4
Marie Parrens, MD,2,5 Béatrice Vergier, MD, PhD,2,5 Michèle Delaunay, MD,1 Claire Beylot, MD,2,3
Geneviève Chêne, MD, PhD,4 Jacky Ferrer,5 Antoine de Mascarel, MD,2,5 Pierre Dubus, MD, PhD,2,5
and Jean Philippe Merlio, MD, PhD2,5
Key Words: Cutaneous lymphoma; Mycosis fungoides; Clonality; Polymerase chain reaction; PCR; Bone marrow
DOI: 10.1309/QH6XLRF3MVUF2M8M
Abstract
This study was undertaken to determine the
prognostic value of bone marrow histopathologic and
molecular analyses in 53 patients with mycosis
fungoides and 7 with Sézary syndrome.
Bone marrow was involved in only 1 patient with
Sézary syndrome, clinical stage IVA, before bone
marrow biopsy. An ambiguous T-cell infiltrate was
observed in 8 patients but was not associated with
disease progression. The bone marrow specimen was
normal in 51 patients. Monoclonality was detected in
the skin specimen in 44 cases; an identical T-cell clone
in the blood specimen was found in 21 of them and, in
16 of the 21 patients, in bone marrow specimens
without histologic correlation. Multivariate analysis
confirmed that clinical stage and detection by
polymerase chain reaction of an identical T-cell clone
in skin and blood specimens had an independent
prognostic value. No further prognostic value was
observed for the presence of a T-cell clone in bone
marrow specimens. Our data do not support the need
for bone marrow examination in patients with mycosis
fungoides/Sézary syndrome.
Primary cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of lymphomas primarily involving the skin, of
which epidermotropic CTCLs represent the most frequent
type.1 Mycosis fungoides (MF), the most common epidermotropic lymphoma, represents an indolent disease with
prolonged evolution of several years or decades (5-year
survival, 87%).1 Sézary syndrome (SS) is a leukemic form of
epidermotropic CTCL characterized by infiltrated erythroderma
with circulating cerebriform T lymphocytes (Sézary cells) and
generalized lymphadenopathy. SS represents an aggressive
disease with a poor prognosis (5-year survival, 11%).1 Despite
these differences, MF and SS are considered to represent a
neoplastic proliferation of memory T cells that presumably
originates from and homes to the skin.1 They also have been
grouped in the same TNM staging classification for prognostic
studies.2 Peripheral blood involvement is a diagnostic criterion
for SS,1,3 and several criteria have been recommended recently
for the diagnosis of leukemic blood involvement by atypical or
Sézary cells (absolute Sézary cell count, CD4/CD8 ratio and/or
aberrant T-cell phenotype, increased lymphocyte count with
evidence of monoclonality, chromosomally abnormal clone).4
Indeed, blood monoclonal T cells have been detected by the
Southern blot technique and by polymerase chain reaction
(PCR) in cases of SS and in a variable proportion of cases of
MF, especially erythrodermic MF.5-8
Bone marrow biopsy seems more appropriate than bone
marrow aspirate for the diagnosis of visceral (M1) involvement of CTCL. 9-11 The detection rate of bone marrow
involvement at initial staging varied from 6%12,13 to 21.7 %
of CTCLs.14 At an early clinical stage, bone marrow involvement is uncommon,9,10 which has led some groups to perform
bone marrow biopsies in selected patients with advanced
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MF/SS,15 but no consensus has been established. For example,
in a recent report of 556 cases of primary cutaneous
lymphomas, bone marrow biopsy was included in the staging
procedure without specific guidelines for epidermotropic
CTCL.16 In epidermotropic CTCL, bone marrow involvement
is more frequent in the presence of an advanced cutaneous T
stage, but its independent prognostic value for survival has not
been demonstrated by multivariate analysis.12,15 Moreover, the
assessment of bone marrow infiltration often is a difficult challenge in patients with CTCL. Even after immunohistochemical analysis for the detection of T cells, a substantial proportion of bone marrow specimens may be considered abnormal
or atypical but cannot be judged sufficiently involved to permit
a diagnosis of T-cell lymphoma infiltration.14
PCR has permitted the detection of T-cell receptor
(TCR) gamma chain gene monoclonality in a variable
proportion of cutaneous specimens of MF/SS ranging from
53% to 90%.6,17-20 Since cutaneous T-cell monoclonality is
correlated with an advanced T stage of cutaneous lesions of
MF/SS (73%-100% for tumor or erythrodermic
lesions),6,19,21,22 such a molecular marker has been used to
search for the extracutaneous spread of malignant T cells,
especially in blood or lymph node specimens. Peripheral
blood T-cell monoclonality has been observed at various
stages of MF 5,6 and 2 studies have suggested its prognostic
value in epidermotropic CTCL, independent of the TNM
clinical stage.7,20 However, the Southern blot technique and
PCR have been used rarely for the study of bone marrow
specimens in patients with CTCL.23,24
Because no consensus has been reached to select patients
with epidermotropic CTCL for bone marrow examination, we
analyzed the diagnostic and prognostic value of bone marrow
examination by histopathologic and molecular techniques by
prospectively studying unselected patients with epidermotropic CTCL. Histopathologic data were compared with
TNM staging data before bone marrow examination. We used
a multiplex PCR followed by denaturing gel gradient electrophoresis (DGGE) to determine the similarity of TCR
gamma gene rearrangement in skin, blood, and bone marrow
specimens. Univariate and multivariate analyses were
performed to determine the prognostic value of clinical stage
and the detection of monoclonal T cells in skin, blood, and
bone marrow specimens for disease progression.
Materials and Methods
Patient Selection and Staging System
Between May 1994 and November 2000, 79 patients
with CTCL referred to the dermatology departments of the
University Hospital of Bordeaux, Bordeaux, France, were
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included prospectively in this study according to the
following inclusion criteria: The clinical and histopathologic diagnosis of CTCL was confirmed by examination of
skin lesions and histopathologic review during inclusion of
the patients’ records in the files of the French Study Group
of Cutaneous Lymphoma. Diagnosis was made according
to the European Organization for Research and Treatment
of Cancer classification.1 Staging evaluation included at
least skin and general physical examination with special
attention to lymph nodes areas, CBC count, standard blood
and urinary laboratory tests, and chest radiograph. For most
patients (except those with MF stage IA), a total body
computed tomography scan was performed. The search for
Sézary cells in blood smears was performed for selected
patients with erythroderma, widespread plaque, skin tumor,
or clinically significant adenopathy. The initial clinical
stage of the patients with MF/SS was determined by using
the TNM classification adapted for MF, which classifies
according to skin, nodal, and visceral involvement. 2
Patients with patches or plaques (T1 and T2) without
specific nodal involvement are classified as IA, IB, or IIA,
while patients with more extensive disease such as cutaneous tumors (T3) or erythroderma (T4) without or with
nodal and visceral involvement are classified as IIB (T3
N0-N1 M0), III (T4 N0-N1 M0), IVA (T1-T4 N2-N3 M0),
and IVB (T1-T4 N0-N3 M1).
For 60 patients, simultaneous analyses of skin, blood,
and bone marrow samples were done during the initial
staging procedure, before administration of systemic therapy.
Histopathologic examination of the bone marrow was not
performed in 14 patients with stage IA to IIA MF/SS and 5
patients with stage IIB to IVA, so these patients were
excluded from the study.
Bone Marrow Specimen Processing
Bone marrow biopsy was performed at the iliac crest
using a Jamshidi needle, and the specimen was divided
immediately into 2 parts. About two thirds of the biopsy
specimen (10-13 mm) was immersed in Bouin fluid (as the
fixative) and processed for conventional histopathologic
analysis. One third of the biopsy specimen (5-6 mm) was
snap-frozen on dry ice in a 1.5-mL sterile tube and stored at
–80°C until DNA isolation was performed by a standard
phenol-chloroform extraction. Histopathologic analysis was
performed independently by 2 trained hematopathologists
(A.M., M.P.) and included examination of 4-µm sections
stained with H&E, Giemsa, and reticulin fiber silver staining.
Immunohistochemical analysis was performed on adjacent
sections by means of an LSAB procedure (DAKO, Les Ullis,
France) using antibodies for the detection of B-cell (CD20,
DAKO) and T-cell (CD3, DAKO) antigens. The analysis
focused on the detection of T-cell nodules and aggregates.
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
Under high magnification (×100), a careful search for
lymphoid interstitial infiltrates and dysplastic cells was
performed by comparison with anti-CD3 immunostained
sections. The lymphoid infiltrates were classified as atypical
based on the presence of an abnormal density of normal
small lymphocytes and/or on the presence of atypical small
or large lymphoid cells with cerebriform nuclei. Finally,
bone marrow specimens were classified into 1 of 3 groups
according to Salhany et al14: positive or involved, atypical
with lymphoid cells, and negative or normal. Discordant
cases were reviewed by the 2 pathologists, without knowledge of the molecular results or clinical outcome, at a multiheaded microscope for final classification.
TCR gamma Gene Rearrangement Analysis
The study of rearrangement of the TCR gamma gene
was performed by PCR-DGGE.18 The use of GC-clamp
primers and DGGE shows 1 or 2 specific dominant bands
for each monoclonal TCR gamma allele resulting in a
genetic imprint useful for comparison of the size and the
sequence of PCR products.18 In our laboratory, the sensitivity threshold for the detection of the 2 rearranged alleles
was 2.5% for the VIV-JI allele and 5% for the VIJI allele of
the Jurkatt cell line DNA diluted into polyclonal peripheral
blood lymphocyte DNA (provided by P. Cornillet, MD,
PhD, Reims, France). A positive diluted control was
included in each PCR assay, permitting a constant detection
threshold throughout the study above 5%. Thereafter,
samples were interpreted as polyclonal (presence of a
smear) or monoclonal (presence of 1 to 4 dominant bands
depending on the formation of homoduplexes or heteroduplexes).18 For each patient, DNA was extracted from a
frozen part of the initial cutaneous biopsy specimen that
corresponded clinically to the most infiltrated lesion.20
When a monoclonal pattern was observed, the cutaneous
clone was considered the reference clone because it originated from skin material that permitted the diagnosis of
CTCL. DNA also was extracted from peripheral blood
mononuclear cells purified by density gradient centrifugation and from the frozen part of the bone marrow biopsy
specimen. Their analysis by PCR-DGGE was run along
with the cutaneous sample.
After these analyses, patients were classified into 4
groups: 1, polyclonal profile in the skin specimen whatever
the profile in blood and bone marrow specimens (polyclonal
or unrelated T-cell clone); 2, T-cell clone in skin specimen
without an identical clone in a peripheral blood lymphocyte
or bone marrow specimen (where PCR detected a polyclonal
profile or an unrelated T-cell clone); 3, identical T-cell clone
in skin and blood specimens but not in the bone marrow
specimen; and 4, identical T-cell clone in skin, blood, and
bone marrow specimens.
Statistical Analyses
Criteria for evaluating disease outcome have been
reported elsewhere.20 The prognostic value of clonality was
assessed using survival analysis methods. The entry dates for
our study sample were the dates of histologic diagnosis of
MF, and the patients were followed up every 6 months.
Patients were considered lost to follow-up if their last
follow-up occurred more than 6 months before April 2001.
Disease progression was evaluated retrospectively by the
physicians (V.S., M.B.B., M.D.) who followed up all
patients. The interval to disease progression was calculated
from the entry date to the date of diagnosis of events
(absence of response to treatment, progression of cutaneous
lesions or occurrence of extracutaneous involvement [both
leading to a change in the stage of the disease]) or to the date
of death. Patients with complete or partial remission
(absence or regression of more than 50% of initial skin
and/or extracutaneous involvement) until last clinical followup were “right censored,” ie, considered alive without any
sign of disease progression. We did not consider the different
treatments received by the patients. Indeed, treatments were
administrated according to the initial clinical stage and,
therefore, could not be evaluated as an independent variable
since no prospective therapeutic trial was conducted. Therefore, the multivariate analysis of clonality effect was
adjusted for age, sex, and clinical stage.
Univariate and multivariate analyses were performed
using a Cox proportional hazards model with SAS software
(proportional hazard regression procedure), version 6.12
(SAS Institute, Cary, NC).25 Results are reported using the
hazard ratio (HR), which is interpreted like the relative risk,
ie, above 1, the factor is a risk factor, and below 1, the factor
is a protective factor. P values were calculated using Wald
chi-squared statistic
Results
The study included 60 patients (43 men, 17 women).
The mean age was 59 years (range, 21-90 years). According
to the European Organization for Research and Treatment of
Cancer classification,1 the CTCLs were classified as 53 cases
of MF and 7 cases of SS.
Before bone marrow examination, according to the
TNM classification for MF/SS,2 the stages for the 53 patients
with MF were as follows: IA, 13; IB, 21; IIA, 3; IIB, 6; IIIA,
5; IIIB, 3; IVA, 1; and IVB, 1. Seven patients had SS,
defined by the association of erythroderma and a count of
Sézary cells in the blood smear of more than 1,000/µL. They
were at stage IVA (n = 6) or IIIB (n = 1). All patients with
stage III or IV MF/SS were erythrodermic, except 1 patient
with stage IVB MF because of meningeal involvement, as
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reported elsewhere.26 Patients with stage IA to IIA MF were
considered as having early-stage MF/SS, and patients with
stage IIB to IVB MF and patients with SS were considered
as having advanced-stage MF/SS.
Histopathologic Examination of Bone Marrow
The routine examination of bone marrow sections was
considered negative or normal in 51 patients. No lymphoid
infiltration was detected on immunostaining, and the reticulin fiber network was normal.
In 1 patient, bone marrow involvement by CTCL was
assessed by both reviewers on the presence of clusters of
atypical pleomorphic lymphoid cells with irregular nuclei
❚Image 1A❚ and ❚Image 1B❚. This infiltration also was underlined by a modification of reticulin fiber network. Moreover,
immunohistochemical analysis confirmed the CD3+ phenotype (data not shown).
In 8 patients, a borderline or atypical pattern was seen
by 1 of the 2 reviewers on the basis of abnormal numbers of
interstitial lymphocytes (n = 5), on the presence of dysplastic
lymphoid cells with cerebriform nuclei (n = 2), or both (n =
1). Immunostaining showed that these lymphocytes were
predominantly of the CD3+CD20– phenotype. There were
no abnormal nodules (more than 300 µm in diameter). Rare
small aggregates (<3 per bone marrow section) were seen in
2 specimens. A multiheaded review classified these specimens as normal (n = 3) or atypical (n = 5) ❚Table 1❚. In 4
patients, the borderline or atypical pattern was due to the
presence of interstitial dysplastic lymphoid cells ❚Image 1C❚
and ❚Image 1E❚ with a CD3+ phenotype ❚Image 1D❚ and
❚Image 1F❚. In the fifth atypical case, an abnormal number of
interstitial CD3+ lymphoid cells without atypia was seen
❚Image 1G❚ and ❚Image 1H❚.
Clinical features of the patients with atypical or involved
bone marrow are summarized in Table 1. Patients were at
various clinical stages of MF/SS, and 3 patients had SS at
initial examination. The patient with bone marrow involvement was at stage IVA before bone marrow biopsy and died
rapidly of visceral involvement. There was no difference in
disease progression between patients with atypical or
involved bone marrow and those with normal bone marrow.
T-cell monoclonality in skin, blood, and bone marrow
specimens according to the clinical stage of the disease in
MF/SS is shown in ❚Table 2❚.
According to PCR results, polyclonality (group 1) was
observed in the skin specimens of 16 (27%) of 60 patients
with MF/SS ❚Image 2❚. A T-cell clone was detected only in
the skin specimens of 23 (38%) of 60 patients (group 2). A
similar T-cell monoclonal rearrangement was detected in the
skin and blood specimens but not in the bone marrow specimen in 5 (8%) of 60 patients (group 3) and in all samples of
16 (27%) of 60 patients (group 4) (Image 2). The 7 patients
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with SS belonged to group 3 (n = 2) or 4 (n = 5). All patients
with SS had an increase in the count of Sézary cells in blood
smears and a blood T-cell clone. Conversely, the patients
with MF in which a blood T-cell clone was detected (groups
3 and 4) did not have a high count of Sézary cells in blood
smears or an increase of the absolute lymphocyte count.
When patients with early-stage disease were distinguished from those with advanced-stage disease, monoclonality in skin, blood, and bone marrow specimens showed a
significant association (Pearson chi square = 8.4; P < .004).
Indeed, 22 (59%) of 37 patients with early-stage disease had
a skin T-cell clone (groups 2, 3, and 4) vs 22 (96%) of 23
patients with advanced-stage disease. In only 1 patient with
advanced MF/SS (stage IIIB), was polyclonality found in the
skin specimen. Identical skin and blood T-cell clones (groups
3and 4) were detected in 7 (19%) of 37 patients with earlystage MF/SS vs 14 (61%) of 23 patients with advanced-stage
MF/SS. Sixteen (76%) of 21 patients with blood clonality
had an identical clone in the bone marrow specimen and
belonged to group 4, whereas no patient had an identical Tcell clone in the bone marrow specimen without blood
involvement.
For 12 patients with either a polyclonal (n = 4) or a
monoclonal T-cell skin (n = 8) profile, a different or unrelated T-cell monoclonal rearrangement was detected in blood
and also in bone marrow for 8 of these 12 patients (Table 2,
Image 2). Moreover, 7 patients had an unrelated T-cell clone
in bone marrow specimens that was not detected in skin and
blood specimens with a polyclonal skin profile (n = 5) or a
different reference skin T-cell clone (n = 2). Interestingly,
patients with unrelated blood and/or bone marrow T-cell
clones mostly belonged to the early-stage MF/SS group
(15/19). In 6 of 12 patients with an unrelated blood T-cell
clone, successive blood molecular analyses showed the same
monoclonal profile in 5 of them (data not shown).
When comparing the final histopathologic and molecular bone marrow analyses, no correlation between bone
marrow monoclonality and atypical or involved histologic
features was found. An identical clone was observed in 14
(26%) of 54 bone marrow specimens with normal histologic
features and in 2 (33%) of 6 bone marrow specimens with
atypical or involved histologic features (Table 1).
Outcome Study According to PCR Results
The median follow-up time in the study was 38 months
(interquantile range, 18-48 months). Only 1 patient with MF
stage IB was lost to follow-up after 6 months. Six patients
with MF and 2 with SS died of their disease.
Since death was a rare event, survival did not permit
evaluation of the prognostic value of T-cell clonality within
the median follow-up time. Prognosis was evaluated in terms
of disease progression as opposed to complete or partial
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
A
B
C
D
❚Image 1❚ Histopathologic features of bone marrow sections in mycosis fungoides (MF)/Sézary syndrome (SS). A, Bone
marrow involvement in a 60-year-old patient with SS, stage IVA, before bone marrow examination. Hypercellularity and
lymphoid aggregates or small nodules are shown (arrows) (H&E, ×400). B, Same patient as in Image 1A. Dysplastic small
lymphoid cells form aggregates with cerebriform or hyperchromatic nuclei (arrows) (H&E, ×1,000). C, Atypical bone marrow in
a patient with MF. Interstitial lymphoid infiltration is present in a normocellular bone marrow sample. An abnormal number of
lymphoid cells exhibits normal features (arrows) or cerebriform nuclei (arrowheads) (H&E, ×1,000). D, Same specimen as in
Image 1C. Immunostaining underlines the T-cell interstitial infiltrate and shows small aggregates in several areas
(immunoperoxidase procedure for CD3, ×1,000).
response, as previously reported.20 At the end of the study,
28 patients had experienced disease progression: 13 had no
response to treatment and 15 had cutaneous and/or extracutaneous spread of the disease. Interestingly, disease progression occurred in 26 of 28 patients within the first year of
follow-up. Among these 28 patients, 7 died of lymphoma,
whereas the eighth death was unrelated to the disease. Only
2 patients who died had bone marrow histologic and molecular involvement (n = 1) or bone marrow molecular involve-
ment (n = 1). Considering the PCR results, 2 (13%) of 16
patients in group 1 had disease progression, compared with
10 (43%) of 23 in group 2, 3 (60%) of 5 in group 3, and 13
(81%) of 16 in group 4.
Univariate analysis was performed to identify factors
associated with a higher risk for disease progression ❚Table
3❚. Neither age nor sex was associated with disease progression. Conversely, advanced-stage disease was associated
significantly with a higher risk of progression than early-stage
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E
F
G
H
❚Image 1❚ (cont) E, Atypical bone marrow in a patient with SS. Rare lymphoid cells with dysplastic features are present (arrows).
Note their characteristic cerebriform nuclei (H&E, ×1,000). F, Same specimen as in Image 1E. Immunostaining shows that
these cells were scattered among hematopoietic cells since this area was the most infiltrated throughout the section
(immunoperoxidase procedure for CD3, ×1,000). G, Atypical bone marrow in a patient with SS. Abnormal numbers of small and
normal-appearing lymphoid cells are present (arrows). No atypical cells were seen (H&E, ×1,000). H, Same specimen as in
Image 1G. Immunostaining shows an abnormal number of T cells (immunoperoxidase procedure for CD3, ×1,000).
disease (HR = 5.9; P < .0001). The results of molecular
analyses also were found to be a prognostic factor (P =
.0004). When considering all patients with cutaneous monoclonality (groups 2, 3, and 4), a higher risk of progression
was found by comparison with skin polyclonality (HR = 3.6;
P = .04), while monoclonality in skin only (group 2) did not
reach prognostic significance compared with skin polyclonality (group 1) (HR = 4.1; P = .07). The presence of an identical T-cell clone in the skin and blood specimens, whatever
the bone marrow status (groups 3 and 4), was associated
with a higher risk of progression compared with group 1
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patients (HR = 11.3; P = .001) and with group 2 patients
(HR = 4.0; P = .04) Alternatively, patients with skin, blood,
and bone marrow T-cell clones (group 4) were not statistically different from patients with skin and blood T-cell
clones (group 3) (HR = 0.98; P = .07).
In multivariate analyses, only clinical stage and the
presence of a T-cell clone in skin and blood specimens
(groups 3 and 4) were found as significant independent variables (P = .01 and .05, respectively). The presence of a Tcell clone in skin and blood specimens (groups 3 and 4) was
significant in comparison with polyclonality in the skin
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
❚Table 1❚
Characteristics of Patients With Bone Marrow Involvement or Atypical Bone Marrow Histologic Features
Sex/Age (y)
M/35
F/59
M/50
M/64
M/63
F/74
M/71
M/73
M/60
Initial Clinical Stage
PCR Results*
Mycosis fungoides IA
Mycosis fungoides IA
Mycosis fungoides IB
Mycosis fungoides IB
Mycosis fungoides IB
Mycosis fungoides IB
Sézary syndrome (IVA)
Sézary syndrome (IVA)
Sézary syndrome (IVA)
1
4
1
1†
1‡
4
3
3
4
Bone Marrow Histologic
Features at Final Review
Atypical
Atypical
Atypical
Normal§
Atypical
Normal§
Normal§
Atypical
Involved
Outcome (Follow-up After Diagnosis, mo)
Alive, complete remission (37)
Alive, complete remission ( 35)
Alive, partial remission (39)
Alive, partial remission (36)
Alive, partial remission (35)
Alive, progression (47)
Alive, progression (38)
Progression, death due to lymphoma (6)
Progression, death due to lymphoma (10)
PCR, polymerase chain reaction.
* Number indicate group assignments: 1, polyclonal profile in the skin whatever the profile in blood and bone marrow; 3, identical T-cell clone only in skin and blood; 4,
identical T-cell clone in skin, blood, and bone marrow.
† Unrelated clone in bone marrow only.
‡ Unrelated similar clone in blood and bone marrow.
§ Initially evaluated as atypical by at least 1 of 2 reviewers (A.M., M.P.).
❚Table 2❚
T-Cell Monoclonality in Mycosis Fungoides/Sézary Syndrome According to the Initial Clinical Stage in 60 Cases*
Clinical Stage
Group 1
Group 2
Group 3
Group 4
Total
I and IIA
IIB to IV
Total
15 (41)
1 (4)
16 (27)
15 (41)
8 (35)
23 (38)
2 (5)
3 (13)
5 (8)
5 (14)
11 (48)
16 (27)
37
23
60
*
Data are given as number (percentage). Groups are as follows: 1, skin polyclonality; 2, T-cell clone in skin only; 3, identical clone in skin and blood only; 4, identical clone in
skin, blood, and bone marrow.
specimen only (group 1) (HR = 5.5; 95% confidence
interval, 1.14-26.3; P = .04) and with a T-cell clone only in
the skin specimen (group 2) (HR = 2.8; 95% confidence
interval, 1.1-7.7; P = .04).
Discussion
There is little recent information in the literature about
bone marrow examination in CTCL. The TNM classification
adapted for MF/SS relies on cutaneous T staging, nodal N
staging, and bone marrow or visceral M involvement.
Univariate analyses have identified adverse prognostic
factors such as age older than 60 years,9,12 the type of skin
involvement (T stage),9,10,12,15 the occurrence of large cell
transformation,27,28 extracutaneous spread (bone marrow or
visceral involvement),9,10 more than 5% Sézary or atypical
cells in the peripheral blood,11 and the lactate dehydrogenase
level.10,12 In fact, clinical stage is determined mainly on the
type and extent of skin lesions (T stage), which repeatedly
has been found as an independent prognostic factor in multivariate analyses.9,10,15
Bone marrow has been reported as the most common
extranodal site of CTCL involvement in several series.11-13
However, the initially described adverse prognostic value of
bone marrow or visceral involvement9,10 was not confirmed
by 2 multivariate analyses.12,15 Therefore, we tried to evaluate the rationale for bone marrow examination at diagnosis
in unselected patients with CTCL by using both histologic
and molecular analyses.
Despite a careful histologic review of bone marrow
specimens, bone marrow involvement was not observed at
initial staging in 59 of 60 patients with MF/SS, which is in
contrast with the data of Salhany et al,14 who found 21.7%
bone marrow involvement in patients with unusual advanced
skin disease and a rapid fatal outcome for CTCL. An atypical lymphoid infiltrate was seen by at least 1 pathologist in 9
patients with MF/SS, but a concordant diagnosis between the
2 reviewers was found in only 5 of the 9 cases, showing the
poor reproducibility in recognition of this borderline group,
even with the aid of immunohistochemical analysis. Moreover, such a borderline group was not associated with clinical stage, clonality stage, or prognosis. The difficulties in
identifying subtle bone marrow infiltration by peripheral Tcell lymphoma have led several groups to develop molecular
testing of bone marrow samples to assess clonal infiltration
by comparison with the original neoplastic T-cell clone.29,30
Such bone marrow molecular staging has not been evaluated previously for prognosis in CTCL. In the present
study, bone marrow monoclonality was more frequent in
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1
2
3
4
5
6
7
8
9
10 11 12
13 14 15
❚Image 2❚ Examples of molecular staging in patients with
epidermotropic T-cell lymphoma as determined by a parallel
polymerase chain reaction–denaturing gradient gel
electrophoresis analysis of skin, blood, and bone marrow
samples. Lanes 1 to 3, group 1 patient with skin (lane 1) and
bone marrow polyclonality (lane 3) and the presence of an
unrelated T-cell clone in blood (lane 2); lanes 4 to 6, group 2
patient with a T-cell clone in skin (lane 4) but not in blood (lane
5) or bone marrow (lane 6); lanes 7 to 9, another group 2
patient with a T-cell clone in skin (lane 7), blood polyclonality
(lane 8), and an unrelated T-cell clone in the bone marrow
(lane 9); lanes 10 to 12, group 3 patient with the same
monoclonal biallelic T-cell rearrangement in skin (lane 10) and
blood (lane 11) but not in bone marrow (lane 12); lanes 13 to
15, group 4 patient with an identical monoclonal profile in skin
(lane 13), blood (lane 14), and bone marrow (lane 15). Groups
are as follows: 1, skin polyclonality; 2, T-cell clone in skin only;
3, identical clone in skin and blood only; 4, identical clone in
skin, blood, and bone marrow.
advanced-stage MF/SS (48%) than in early-stage disease
(13%). However, bone marrow T-cell monoclonality was not
correlated with bone marrow histologic data, suggesting a
lack of sensitivity of histopathologic analysis. Moreover, we
performed a parallel study of skin, blood, and bone marrow
specimens by DGGE since each rearranged clonal allele
generates a dominant band with specific size and
sequence.17,18,31 Similar to the heteroduplex-loaded temperature gradient gel electrophoresis used by others,6,32 such
comparison increases the specificity of distinguishing unrelated T-cell clones that may be observed in blood20,33,34 or in
bone marrow specimens (present study).
The presence of the relevant T-cell clone in bone
marrow specimens was observed in 76% of patients who had
the same T-cell clone in blood and skin specimens,
suggesting that monoclonal T cells are present in the blood
rather the in bone marrow. It seems unlikely that blood in the
bone marrow sample would account for the detection of
clonal T cells in the bone marrow specimen, since the
patients, except those with SS, had normal peripheral blood
lymphocyte counts with a normal percentage of lymphoid
cells. In such patients, monoclonal bands were detected only
within purified mononuclear blood cells but not in whole
blood DNA (unpublished results). Although the presence of
a relevant bone marrow T-cell clone always was associated
blood clonality, bone marrow involvement may represent a
specific infiltration by malignant blood T cells, which in turn
may originate from the skin, explaining the correlation
between clinical and molecular stages in our series.
However, a positive bone marrow molecular stage did not
reach statistical significance for prognosis in comparison
with the positive skin and blood molecular stages.
Indeed, we and others have found a prognostic value for
skin and blood monoclonality in patients with MF/SS,
although differences in evaluating the prognosis exist among
these series.7,19,20 This was especially underlined for tumor
❚Table 3❚
Factors Predictive of Disease Progression in 60 Cases of Mycosis Fungoides (28 Events)*
Description
Variable
No. (%) of Events
Clinical stage
Early (n = 37)
Advanced (n = 23)
Clonality
None (n = 16)
In skin only (n = 23)
In skin and blood, with or
without clonality in bone
marrow (n = 21)
Univariate Analysis
HR
95% CI
9 (24)
19 (83)
1.0
5.9
—
2.6-13.2
2 (13)
10 (43)
16 (76)
1.0
4.1
11.3
—
0.90-18.7
2.6-49.6
Multivariate Analysis
P
HR
95% CI
1.0
<10–4
4.10–4
—
.07
10–3
—
3.2
—
1.3-7.7
1.0
3.7
5.5
—
0.75-18.5
1.14-26.3
P
—
.01
.05
—
.11
.03
CI, confidence interval; HR, hazards ratio.
* In a proportional hazards model, univariate analysis, monoclonality in skin and blood vs monoclonality in skin, blood, and bone marrow: HR = 0.98; P = .97; multivariate
analysis, monoclonality in skin and blood with or without clonality in bone marrow vs monoclonality in skin only: HR = 2.8; CI, 1.1-7.7; P = .04. Events were defined as
absence of response to treatment, progression of cutaneous lesions or occurrence of extracutaneous involvement (both leading to a change in the stage of disease), and death.
421 Am J Clin Pathol 2003;119:414-423
8
DOI: 10.1309/QH6XLRF3MVUF2M8M
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
burden in the blood of patients with erythrodermic CTCL.8
The presence of a T-cell clone in the blood has been included
for the blood rating of patients with erythrodermic CTCL in
parallel with other hematologic criteria to further identify
patients with a worse prognosis.4 Our prospective study of
unselected patients with CTCL suggests that the blood molecular burden may also be of prognostic significance in patients
with nonerythrodermic CTCL when a direct comparison with
the reference cutaneous T-cell clone is performed.20 This
blood molecular burden could be measured more precisely by
real-time quantitative PCR using allele-specific primers,
although the specificity of this approach needs to be evaluated
along with the DGGE technique for comparison between skin
and blood T-cell clones.35 The standardization of PCR techniques in prospective therapeutic trials also will permit inclusion of treatment variables in the statistical analysis.
Finally, our results do not argue for systematic
histopathologic or molecular analysis of bone marrow specimens in patients with MF/SS. Patients with an adverse prognosis already were identified by clinical staging and by skin
and blood molecular analyses. The presence of a T-cell clone
in the blood sample seems sufficient to identify patients with
an adverse prognosis, whatever clone may be present in bone
marrow. This clone also may represent a biologic parameter
to monitor therapeutic efficacy by PCR techniques in
sequential skin or blood samples.
From the 1Oncodermatology Unit, Dermatology Department,
Saint-André University Hospital of Bordeaux, Bordeaux, France;
2Histology and Molecular Pathology Laboratory, Victor Segalen
University, Bordeaux; 3Dermatology Department and 5Pathology
Department, Haut-Lévêque University Hospital of Bordeaux,
Pessac, France; and 4Clinical Research and Epidemiology Unit,
INSERM U330, University Hospital of Bordeaux.
Supported by a grant from the Clinical Research Program,
University Hospital of Bordeaux, Bordeaux, France, and from the
Ligue Contre le Cancer, Comité de Dordogne, Périgueux, France.
Address reprint requests to Dr Merlio: Equipe Histologie et
Pathologie Moléculaire, EA 2406, Université Victor Segalen
Bordeaux 2, Case 8, Bat 3B, 146 rue Léo Saignat, Bordeaux, France.
References
1. Willemze R, Kerl H, Sterry W, et al. EORTC classification for
primary cutaneous lymphomas: a proposal from the Cutaneous
Lymphoma Study Group of the European Organization for
Research and Treatment of Cancer. Blood. 1997;90:354-371.
2. Bunn PA Jr, Lamberg SI. Report of the Committee on Staging
and Classification of Cutaneous T-Cell Lymphomas. Cancer
Treat Rep. 1979;63:725-728.
3. Bakels V, Van Oostveen J, Gordijn R, et al. Diagnostic value
of T-cell receptor beta gene rearrangement analysis on
peripheral blood lymphocytes of patients with erythroderma.
J Invest Dermatol. 1991;97:782-786.
4. Vonderheid EC, Bernengo MG, Burg G, et al. Update on
erythrodermic cutaneous T-cell lymphoma: report of the
International Society for Cutaneous Lymphomas. J Am Acad
Dermatol. 2002;46:95-106.
5. Bakels V, Oostveen J, Gordijn R, et al. Frequency and
prognostic significance of clonal T-cell receptor beta-gene
rearrangements in peripheral blood of patients with mycosis
fungoides. Arch Dermatol. 1992;128:1602-1607.
6. Muche JM, Lukowsky A, Asadullah K, et al. Demonstration
of frequent occurrence of clonal T cells in the peripheral
blood of patients with primary cutaneous T-cell lymphoma.
Blood. 1997;90:1636-1642.
7. Fraser-Andrews E, Woolford A, Russel-Jones R, et al.
Detection of a peripheral blood T-cell clone is an independent
prognostic marker in mycosis fungoides. J Invest Dermatol.
2000;114:117-121.
8. Scarisbrick J, Whittaker S, Evans A, et al. Prognostic
significance of tumor burden in the blood of patients with
erythrodermic primary cutaneous T-cell lymphoma. Blood.
2001;97:624-630.
9. Sausville EA, Eddy JL, Makuch RW, et al. Histopathologic
staging at initial diagnosis of mycosis fungoides and the Sézary
syndrome: definition of three distinctive prognostic groups.
Ann Intern Med. 1988;109:372-382.
10. Marti R, Estrach T, Reverter J, et al. Prognostic clinicopathologic factors in cutaneous T-cell lymphoma. Arch
Dermatol. 1991;127:1511-1516.
11. Kim Y, Bishop K, Varghes A, et al. Prognostic factors in
erythrodermic mycosis fungoides and the Sézary syndrome.
Arch Dermatol. 1995;131:1003-1008.
12. Diamandidou E, Colome M, Fayad L, et al. Prognostic factor
analysis in mycosis fungoides/Sézary syndrome. J Am Acad
Dermatol. 1999;40:914-924.
13. Marti RM, Estrach T, Reverter JC, et al. Utility of bone
marrow and liver biopsies for staging cutaneous T-cell
lymphoma. Int J Dermatol. 1996;35:450-454.
14. Salhany KE, Greer JP, Cousar JB, et al. Marrow involvement
in cutaneous T-cell lymphoma: a clinicopathologic study of 60
cases. Am J Clin Pathol. 1989;92:747-754.
15. Zackheim HS, Amin S, Kashani-Sabet M, et al. Prognosis in
cutaneous T-cell lymphoma by skin stage: long-term survival
in 489 patients. J Am Acad Dermatol. 1999;40:418-425.
16. Fink-Puches R, Zenahlik P, Bäck B, et al. Primary cutaneous
lymphomas: applicability of current classification schemes
(European Organization for Research and Treatment of Cancer,
World Health Organization) based on clinicopathologic features
observed in a large group of patients. Blood. 2002;99:800-805.
17. Wood GS, Tung RM, Haeffner AC, et al. Detection of clonal
T-cell receptor gamma gene rearrangements in early mycosis
fungoides/Sézary syndrome by polymerase chain reaction and
denaturing gradient gel electrophoresis (DGGE). J Invest
Dermatol. 1994;103:34-41.
18. Theodorou I, Delfau-Larue M, Bigorne C, et al. Cutaneous
T-cell infiltrates: analysis of T-cell receptor gamma gene
rearrangement by polymerase chain reaction and denaturing
gradient gel electrophoresis. Blood. 1995;86:305-310.
19. Delfau-Larue M, Dalac S, Lepage E, et al. Prognostic
significance of a polymerase chain reaction–detectable
dominant T-lymphocyte clone in cutaneous lesions of patients
with mycosis fungoides. Blood. 1998;92:3376-3380.
20. Beylot-Barry M, Sibaud V, Thiébaut R, et al. Evidence that an
identical skin and blood T-cell clone is an independent
prognostic factor in primary cutaneous T-cell lymphomas.
J Invest Dermatol. 2001;117:920-926.
© American Society for Clinical Pathology
9
Am J Clin Pathol 2003;119:414-423 422
9
DOI: 10.1309/QH6XLRF3MVUF2M8M
Sibaud et al / BONE MARROW STAGING IN CUTANEOUS T-CELL LYMPHOMA
21. Bachelez H, Bioul L, Flageul B, et al. Detection of clonal Tcell receptor gamma gene rearrangements with the use of the
polymerase chain reaction in cutaneous lesions of mycosis
fungoides and Sézary syndrome. Arch Dermatol.
1995;131:1027-1031.
22. Curco N, Servitje O, Llucia M, et al. Genotypic analysis of
cutaneous T-cell lymphoma: a comparative study of Southern
blot analysis with polymerase chain reaction amplification of
the T-cell receptor–gamma gene. Br J Dermatol. 1997;137:673679.
23. Veelken H, Wood G, Sklar J. Molecular staging of cutaneous
T-cell lymphoma: evidence for systemic involvement in early
disease. J Invest Dermatol. 1995;104:889-894.
24. Dommann S, Dommann-Scherrer C, Dours-Zimmermann M,
et al. Clonal disease in extracutaneous compartments in
cutaneous T-cell lymphomas: a comparative study between
cutaneous T-cell lymphomas and pseudo-lymphomas. Arch
Dermatol Res. 1996;288:163-167.
25. Allison P. Survival Analysis Using the SAS System: A Practical
Guide. Cary, NC: SAS Institute; 1995.
26. Beylot-Barry M, Dubus P, Vergier B, et al. Meningeal
involvement by a transformed mycosis fungoides following
Hodgkin’s disease. Br J Dermatol. 1999;141:909-913.
27. Diamandidou E, Colome-Grimmer M, Fayad L, et al.
Transformation of mycosis fungoides/Sézary syndrome: clinical
characteristics and prognosis. Blood. 1998;92:1150-1159.
28. Vergier B, de Muret A, Beylot-Barry M, et al, for the French
Study Group of Cutaneous Lymphomas. Transformation of
mycosis fungoides: clinicopathological and prognostic features
of 45 cases. Blood. 2000;95:2212-2218.
423 Am J Clin Pathol 2003;119:414-423
10 DOI: 10.1309/QH6XLRF3MVUF2M8M
29. Gebhard S, Benhattar J, Bricod C, et al. Polymerase chain
reaction in the diagnosis of T-cell lymphoma in paraffinembedded bone marrow biopsies: a comparative study.
Histopathology. 2001;38:37-44.
30. Weirich G, Funk A, Hoepner I, et al. PCR-based assays for
the detection of monoclonality in non-Hodgkin’s lymphoma:
application to formalin-fixed, paraffin-embedded tissue and
decalcified bone marrow samples. J Mol Med. 1995;73:235241.
31. Meyer JC, Hassam S, Dummer R, et al. A realistic approach
to the sensitivity of PCR-DGGE and its application as a
sensitive tool for the detection of clonality in cutaneous T-cell
proliferations. Exp Dermatol. 1997;6:122-127.
32. Bottaro M, Berti E, Biondi A, et al. Heteroduplex analysis of
T-cell receptor gamma gene rearrangements for diagnosis and
monitoring of cutaneous T-cell lymphomas. Blood.
1994;83:3271-3278.
33. Delfau-Larue MH, Laroche L, Wechsler J, et al. Diagnostic
value of dominant T-cell clones in peripheral blood in 363
patients presenting consecutively with a clinical suspicion of
cutaneous lymphoma. Blood. 2000;96:2987-2992.
34. Dippel E, Klemke D, Hummel M, et al. T-cell clonality of
undetermined significance. Blood. 2001;98:247-248.
35. van der Velden VH, Wijkhuijs JM, Jacobs DC, et al. T cell
receptor gamma gene rearrangements as targets for detection
of minimal residual disease in acute lymphoblastic leukemia
by real-time quantitative PCR analysis. Leukemia.
2002;16:1372-1380.
© American Society for Clinical Pathology