Bone Marrow Histopathologic and Molecular Staging in Epidermotropic T-Cell Lymphomas

Transcription

1 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, PhD 2,5 Key Words: Cutaneous lymphoma; Mycosis fungoides; Clonality; Polymerase chain reaction; PCR; Bone marrow DOI: /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 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 Am J Clin Pathol 2003;119: DOI: /QH6XLRF3MVUF2M8M 1

2 Sibaud et al / BONE MARROW STAGING IN CUTANEOUS T-CELL LYMPHOMA 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 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. 415 Am J Clin Pathol 2003;119: DOI: /QH6XLRF3MVUF2M8M

3 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 al 14 : 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 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, 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 Am J Clin Pathol 2003;119: DOI: /QH6XLRF3MVUF2M8M 3

4 Sibaud et al / BONE MARROW STAGING IN CUTANEOUS T-CELL LYMPHOMA 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 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 T- cell 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, 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 417 Am J Clin Pathol 2003;119: DOI: /QH6XLRF3MVUF2M8M

5 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 involvement (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 Am J Clin Pathol 2003;119: DOI: /QH6XLRF3MVUF2M8M 5

6 Sibaud et al / BONE MARROW STAGING IN CUTANEOUS T-CELL LYMPHOMA 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 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 T- cell clone in skin and blood specimens (groups 3 and 4) was significant in comparison with polyclonality in the skin 419 Am J Clin Pathol 2003;119: DOI: /QH6XLRF3MVUF2M8M

7 Hematopathology / ORIGINAL ARTICLE Table 1 Characteristics of Patients With Bone Marrow Involvement or Atypical Bone Marrow Histologic Features Bone Marrow Histologic Sex/Age (y) Initial Clinical Stage PCR Results * Features at Final Review Outcome (Follow-up After Diagnosis, mo) M/35 Mycosis fungoides IA 1 Atypical Alive, complete remission (37) F/59 Mycosis fungoides IA 4 Atypical Alive, complete remission ( 35) M/50 Mycosis fungoides IB 1 Atypical Alive, partial remission (39) M/64 Mycosis fungoides IB 1 Normal Alive, partial remission (36) M/63 Mycosis fungoides IB 1 Atypical Alive, partial remission (35) F/74 Mycosis fungoides IB 4 Normal Alive, progression (47) M/71 Sézary syndrome (IVA) 3 Normal Alive, progression (38) M/73 Sézary syndrome (IVA) 3 Atypical Progression, death due to lymphoma (6) M/60 Sézary syndrome (IVA) 4 Involved 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 15 (41) 15 (41) 2 (5) 5 (14) 37 IIB to IV 1 (4) 8 (35) 3 (13) 11 (48) 23 Total 16 (27) 23 (38) 5 (8) 16 (27) 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, ; P =.04) and with a T-cell clone only in the skin specimen (group 2) (HR = 2.8; 95% confidence interval, ; 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 However, the initially described adverse prognostic value of bone marrow or visceral involvement 9,10 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 T- cell 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 Am J Clin Pathol 2003;119: DOI: /QH6XLRF3MVUF2M8M 7

8 Sibaud et al / BONE MARROW STAGING IN CUTANEOUS T-CELL LYMPHOMA 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 blood 20,33,34 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 Univariate Analysis Multivariate Analysis Variable No. (%) of Events HR 95% CI P HR 95% CI P Clinical stage Early (n = 37) 9 (24) Advanced (n = 23) 19 (83) < Clonality None (n = 16) 2 (13) In skin only (n = 23) 10 (43) In skin and blood, with or 16 (76) without clonality in bone marrow (n = 21) 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, ; 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: DOI: /QH6XLRF3MVUF2M8M

9 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 1 Oncodermatology Unit, Dermatology Department, Saint-André University Hospital of Bordeaux, Bordeaux, France; 2 Histology and Molecular Pathology Laboratory, Victor Segalen University, Bordeaux; 3 Dermatology Department and 5 Pathology Department, Haut-Lévêque University Hospital of Bordeaux, Pessac, France; and 4 Clinical Research and Epidemiology Unit, INSERM U330, University Hospital of Bordeaux. 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