Abstract. Hematopathology / FLOW CYTOMETRIC DNA PLOIDY ANALYSIS IN SÉZARY SYNDROME

Transcription

1 Hematopathology / FLOW CYTOMETRIC DNA PLOIDY ANALYSIS IN SÉZARY SYNDROME Flow Cytometric DNA Ploidy Analysis of Peripheral Blood From Patients With Sézary Syndrome Detection of Aneuploid Neoplastic T Cells in the Blood Is Associated With Large Cell Transformation in Tissue Sa Wang, MD, 1,2 Ning Li, MD, 2 Peter Heald, MD, 3 John M. Fisk, MD, 1 Oluwole Fadare, MD, 1,2 John G. Howe, PhD, 1 Jennifer M. McNiff, MD, 2,3 and Brian R. Smith, MD 1,4 Key Words: Cutaneous T-cell lymphoma; Sézary syndrome; Flow cytometry; DNA ploidy; Immunophenotype; Minimal residual disease; Large cell transformation DOI: /8B849FC6PHAP8FDD Abstract We reviewed and screened 219 cutaneous T-cell lymphoma (CTCL) cases for Sézary syndrome (SS); 63 met the criteria for SS. Of these, 17 (27%) demonstrated circulating aneuploid cells and 46 (73%) showed only euploid cells in blood samples. Of 17 aneuploid cases, DNA ploidy study was essential for initial blood-based diagnosis of SS in 4 (24%) and important in monitoring minimal residual disease after treatment in 9 (53%) in which neoplastic T cells showed otherwise unremarkable or nonspecific flow cytometric immunophenotypic findings. Tissue biopsy slides (predominantly skin and lymph node) at the time of DNA ploidy studies were available for 47 of 63 cases. Of 14 cases with circulating aneuploid cells, 11 (79%) showed large cell transformation (LCT; 6 [43%]) or markedly increased large cells (ILC; 5 [36%]) in tissue, whereas only 10 (30%) of 33 euploid cases showed LCT (4 [12%]) or ILC (6 [18%]) (P <.01). There was no significant difference in blood tumor burden, immunophenotype, or proliferation index between euploid and aneuploid groups or histologic high- and low-grade groups. DNA ploidy study by flow cytometry is important for blood-based diagnosis of SS and detection of minimal residual disease in aneuploid SS after treatment. Detection of aneuploid neoplastic T cells in peripheral blood samples of patients with CTCL is associated with LCT in skin, lymph node, or other tissues. Sézary syndrome (SS), the leukemic variant in the spectrum of cutaneous T-cell lymphomas (CTCL), is characterized by erythroderma, peripheral adenopathy, and circulating atypical mononuclear cells with cerebriform nuclei (Sézary cells). 1 The overall survival in patients with SS is markedly decreased compared with patients with other forms of CTCL, with a median of 2.5 to 5 years. 2,3 However, it also is well recognized that skin specimens from SS cases frequently show only equivocal histologic features of CTCL and/or nonspecific cutaneous histopathologic findings. 4,5 This highlights the need for an adjunctive diagnostic modality, eg, peripheral blood immunophenotyping and/or molecular genetic analysis. Immunophenotyping by flow cytometry has had an important role in the evaluation of CTCL and SS. Flow cytometric immunophenotyping studies have demonstrated an increased CD4/CD8 ratio and aberrant expression or loss of pan T-cell markers in many affected patients. 6-8 However, the use of flow cytometry for DNA ploidy analysis has been studied only minimally in this setting. Alteration in total cellular DNA content is rarely present in normal lymphocytes; thus, the presence of DNA aneuploidy is a good indicator of neoplasia and also might have prognostic significance in various tumors For example, in non-hodgkin lymphoma (NHL), DNA ploidy studies have suggested that aggressive NHL has a higher incidence of DNA aneuploidy than low-grade NHL, although the role of DNA aneuploidy as a prognostic factor for most large and small cell NHL remains controversial Because hyperdiploidy is a known characteristic of the malignant cells in some patients with CTCL, it is reasonable to hypothesize that DNA ploidy studies might have prognostic import in SS. Moreover, DNA ploidy studies provide an additional means of establishing the presence 774 Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD

2 Hematopathology / ORIGINAL ARTICLE of an abnormal T-cell clone in the absence of abnormal immunophenotypic studies. Two conferences were sponsored by the International Society for Cutaneous Lymphomas (ISCL) to gain consensus on clinically useful definitions and terminology in erythrodermic CTCL (E-CTCL). The hematologic criteria recommended for use in the diagnosis of SS are intended to identify patients with a worse prognosis compared with the other E-CTCL subsets. The ISCL recommended that a chromosomally abnormal T-cell clone identified in the blood of patients with E-CTCL be adopted as an independent hematologic criterion for SS, 15 based on studies showing that the presence of clonal abnormalities correlated with advanced-stage disease and a significantly reduced survival duration from the time of cytogenetic studies Such findings further suggest that identification of DNA aneuploidy by flow cytometry might have similar prognostic importance. Transformation of mycosis fungoides (MF)/SS to large cell lymphoma is a well-described phenomenon that has a very poor prognosis. 19,20 The presence of large cells exceeding 25% of the total lymphoid infiltrate in the skin or lymph node biopsy specimen has been used to define large cell transformation (LCT). 21 The incidence of such transformation has been reported to range between 8% and 55%. 19,22 Molecular mechanisms responsible for LCT have not been elucidated, although immunologic and molecular evidence based on T-cell receptor analysis indicates that transformed MF/SS represents evolution of the original malignant clone rather than a new clone of neoplastic T cells. 23,24 Early studies on DNA ploidy in patients with CTCL suggested that DNA aneuploidy might correlate with decreased survival; however, correlation with LCT was not studied. 25,26 So et al 27 reported a transformed SS case that showed a direct relationship between cell size and aneuploidy by flow cytometric analysis in peripheral blood lymphocytes, suggestive of a correlation between near tetraploidy and LCT. In the present study, we retrieved all CTCL cases from a 12-year period from our flow cytometry database. In the majority of these cases, DNA ploidy study was performed as part of the routine blood evaluation for CTCL in our institution. Cases that met the hematologic criteria for SS, coupled with DNA ploidy study and/or tissue biopsy (skin, lymph node and/or other tissues) findings, were analyzed. Our objective was to study the value of flow cytometric DNA ploidy analysis in the diagnosis and prognosis of SS. We hypothesized that aneuploidy in CTCL/SS would identify patients with a higher likelihood of LCT and, therefore, a worse prognosis. Materials and Methods Patients All patients undergoing evaluation for blood involvement by previously diagnosed CTCL during a 12-year period (January 1991-March 2003) were retrieved from our flow cytometry database. We identified 219 cases of clinically and pathologically documented CTCL. In the majority of these cases, DNA ploidy study was performed as part of the initial diagnostic workup for patients with CTCL when they first came to our institution. Flow cytometric immunophenotyping was used to determine the CD4+ T-cell count, CD4/CD8 ratio, and percentage of phenotypically abnormal lymphocytes in the blood. All cases were categorized according to the 2002 hematologic criteria recommended for blood-based diagnosis of SS by the ISCL 15 and complemented by the European Organization for Research and Treatment of Cancer group criteria. 28 Specifically, patients were considered to have blood involvement by CTCL on the basis of one or more of the following: (1) atypical lymphocytes more than 10% of peripheral blood mononuclear cells 29 ; (2) CD4/CD8 ratio of 10 or higher caused by an increase in circulating T cells; (3) increased circulating T cells with aberrant loss of expression of pan T-cell markers by flow cytometry, including aberrant expression of CD2, CD3, CD4, or CD5 or coexpression of CD4 and CD8; (4) CD4+CD7 cells of more than 40% of the cells in the lymphocyte gate if CD7 negativity was the only phenotypic abnormality identified; (5) increased lymphocyte count with evidence of a T-cell monoclone in the blood by Southern blot or polymerase chain reaction (PCR) technique; or (6) a chromosomally abnormal T-cell clone identified by DNA ploidy, cytogenetics, or other studies. Of the 219 cases, 63 (28.8%) met the diagnostic criteria for blood involvement by CTCL, and these 63 cases form the basis for the study. All studies were carried out in accordance with guidelines from the Human Investigation Committee, Yale University School of Medicine, Yale New Haven Hospital, New Haven, CT. Peripheral Blood Lymphocyte Flow Cytometric Immunophenotyping Peripheral blood lymphocytes were analyzed using a FACScan cytometer (Becton Dickinson, San Jose, CA). Three-color immunofluorescence analyses were performed using fluorescein isothiocyanate, phycoerythrin-, and peridinin chlorophyll protein conjugated antibodies. At least 10,000 lymphocytes were collected for each antibody combination. For each blood sample, isotype-matched negative control samples conjugated to each fluorochrome were used. A wide panel of monoclonal antibodies directed against T-cell antigens was tested routinely Table 1. All 3-color analyses included CD4 as one of the antigens studied. DNA Ploidy Analysis Cells isolated on Ficoll-Hypaque were fixed in ice-cold 70% methanol and stained with a saturating concentration (40 µg/ml) of propidium iodide and 100 µg/ml of RNase. Two Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD 775

3 Wang et al / FLOW CYTOMETRIC DNA PLOIDY ANALYSIS IN SÉZARY SYNDROME aliquots of each sample were stained; the second was mixed in a 1:1 ratio with normal human mononuclear cells that were processed identically and served as an internal standard. After staining, the samples were protected from light and analyzed within 2 hours. Flow cytometric ploidy analysis was performed with a FACScan flow cytometer using CellFIT software (Becton Dickinson). For each sample, 10,000 to 20,000 cells were analyzed. Single cells were discriminated from cell clumps by a doublet discrimination model (pulse area vs pulse width). The DNA histograms were evaluated using the SFIT mode. The DNA index was obtained by dividing the modal fluorescence channel of the G 0 /G 1 peak of the abnormal cells by the modal fluorescence channel of the residual G 0 /G 1 normal cells present in the sample. 30 Standardization of the instrument was carried out using the DNA Quality Control kit (product No , Becton Dickinson) according to manufacturer instructions, based on the use of chicken erythrocyte nuclei and calf thymocyte nuclei; linearity was always between 1.95 and 2.05, and the coefficient of variation (CV) for chicken erythrocyte nuclei was less than 3.0% and for calf thymocyte nuclei was less than 5.0%. As noted, normal human mononuclear cells were run with each sample, and the CV of the G 0 /G 1 peak was uniformly less than 3.0%. Similarly, the CV of the aneuploid peaks noted in the study was less than 4.5%. An aneuploid peak was considered present only if a separate, distinct G 0 /G 1 peak could be identified; widening of the CV was not considered a criterion for the presence of a small aneuploid population. Data were collected in list mode to allow for later reanalysis and comparison with follow-up samples from the same patient. Polymerase Chain Reaction DNA was extracted from peripheral blood mononuclear cells by column isolation (Qiagen, Hilden, Germany). The T- cell receptor (TCR) γ chain gene was amplified using 2 reactions with primers V γ I plus V γ III/IV plus J γ 1/2 (product sizes, approximately base pairs [bp]) and V γ I plus V γ III/IV plus JP γ 1/2 (product sizes, bp). Amplification of the TCRβ chain gene was carried out in 5 separate reactions with the following primer combinations: V plus J1, V plus J2, D1 plus J1, D1 plus J2, D2 plus J29 (product sizes, approximately bp). 31 Of the 63 patients identified as having blood involvement by CTCL, 31 had TCR gene rearrangement studies (PCR or Southern blot), and 29 of these met criteria for monoclonality (2 had negative PCR results and insufficient samples for Southern blot analysis). Of the 156 patients without definitive evidence of blood involvement by CTCL, 51 had TCR PCR studies and/or Southern blot studies performed, and all results were negative. Table 1 Monoclonal Antibodies Used to Determine Immunophenotypes of Patients With Cutaneous T-Cell Lymphoma * Monoclonal Antibody Isotype CD2 Leu-5b (S5.2) IgG2a CD3 Leu-4 (SK7) IgG1 CD4 Leu-3a (SK3) IgG1 CD5 Leu-1 (L17F12) IgG2a CD7 Leu-9 (4H9) IgG2a CD8 Leu-2 (SK1) IgG1 CD14 Leu-M3 (MφPa) IgG2b CD16 Leu-11c IgG1 CD19 Leu-12 (4G7) IgG1 CD20 Leu-16 (L27) IgG2a CD25 IL-2r (2A3) IgG1 CD30 Ber-H8 IgG1 CD45RA HI 100 IG2b CD45RO UCHL1 IgG2a CD56 B159 IgG1 CD57 Leu-7 (HNK-1) IgM HLA-DR L243 (G46-6) IgG2a * All from Becton Dickinson, Mountain View, CA. Southern Blot Analysis High-molecular-weight DNA extracts from peripheral blood were digested with restriction enzymes EcoRI, HindIII, and BamHI. Blots were hybridized sequentially to phosphorus 32 labeled probe C β for TCRβ and ph60 for TCRγ. Samples that showed the presence of 2 bands distinctly stronger than the other rearranged fragments with 2 or more restriction digests were scored as positive, and those that showed only a germline or typical polyclonal ladder were recorded as negative. 32 For 11 of the 63 patients identified as having blood involvement by CTCL, Southern blots were performed, and all met the criteria for monoclonality. Histopathologic Examination In all cases, the diagnosis was based on routinely processed, H&E-stained, 6-µm sections from formalin- or B-5 fixed, paraffin-embedded tissue specimens. All available biopsy slides for the patients from around the time of the DNA ploidy study, including skin, lymph node, bone marrow, liver, or lung, were reviewed. The morphologic features of LCT were based on accepted criteria 20,21 and defined as the presence of large cells exceeding 25% of the infiltrate throughout or forming microscopic nodules. A marked increase in the number of large cells (ILC) was defined as large cells representing more than 5% but fewer than 25% of cells and a lack of large sheet formation in all biopsy tissues examined microscopically. Classic CTCL/MF cases with fewer than 5% large cells with or without nonspecific inflammatory findings were classified as no increase of large cells (NILC). Statistical Analyses Statistical analyses were performed using BMDP Statistical Software (SPSS, Chicago, IL). One-way analysis of variance 776 Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD

4 Hematopathology / ORIGINAL ARTICLE was performed for 2 separate groups of patients (aneuploid SS and euploid SS) and 3 variables (absolute values of CD4+ T cells, absolute value of abnormal lymphocytes identified by flow cytometric immunophenotyping/dna ploidy study, and CD4/CD8 ratio) for tumor burden. We used χ 2 test 2 3 contingency tables to evaluate the relationship between DNA ploidy with flow cytometric immunophenotyping (normal; aberrant expression of CD2, CD3, CD4, CD5, CD8; or CD7 ) and tissue histologic grades (LCT, ILC, and NILC), respectively. Results DNA Ploidy Analysis in Initial Diagnosis of Sézary Syndrome in Peripheral Blood From a total of 219 patients with clinically and pathologically (skin biopsy) established CTCL, 63 patients (28.8%) met the hematologic criteria for blood-based diagnosis of SS as defined earlier. Clonal TCR gene rearrangement was confirmed in 29 of these cases by PCR or Southern blot analysis. DNA aneuploidy was present in 17 (27%) of 63 Table 2, including 15 with hyperdiploidy (DNA index ranging from 1.05 to 1.9), 1 with hypodiploidy (DNA index, 0.9), and 1 with mixed biclonal aneuploidy (hyperdiploidy, DNA index, 1.4/hypodiploidy, DNA index, 0.9). In most of these cases, more than one subsequent DNA ploidy study was performed, and in all such cases, repeated DNA ploidy indices were identical to the original. Figure 1 shows 2 examples of aneuploid DNA histograms in these patients, with DNA indices of 1.77 and 1.87 and abnormal cell fractions of total peripheral blood leukocytes of 24.4% and 61.1%, respectively. The diagnosis of blood SS could be established without DNA ploidy studies in 13 of 17 cases at initial evaluation by applying the aforementioned hematologic criteria. These 13 cases showed a marked increase of T cells with an increased CD4/CD8 ratio and/or aberrant expression of pan T-cell markers (CD2, CD3, CD4, CD5) or a significantly increased CD4+CD7 proportion (>40% of lymphocyte gate). However, in case 1 (Table 2), the blood showed normal WBC, lymphocyte, and CD4+ T-cell counts, the CD4/CD8 ratio was not increased (0.8), and the T cells were phenotypically unremarkable. The SS diagnosis was made only with the DNA ploidy study. In cases 3, 8, and 9, the peripheral blood lymphocytes showed aberrant pan T-cell marker expression or positive PCR results; however, the total CD4+ T cells and the CD4/CD8 ratio were not sufficiently increased to warrant an SS diagnosis according to published criteria. In these cases, DNA ploidy studies were considered highly contributory to the final SS diagnosis. Use of DNA Ploidy Analysis in Determination of Residual Disease Following Therapy Normal or nonspecific (CD7 as the sole aberrant marker) immunophenotyping of CTCL cells was present in 9 Table 2 Laboratory Data for Patients With Cutaneous T-Cell Lymphoma/Sézary Syndrome With Aneuploid Circulating Neoplastic T Cells Detected by Flow Cytometry Case No./Sex/ DNA WBC Count CD4+ Abnormal CD4/CD8 Immunophenotyping TCR Gene Histologic Age (y) Index ( 10 9 /L) * T Cells (%) Lymphocytes (%) Ratio by Flow Cytometry Rearrangement Findings 1/F/ Nonspecific NA NA 2/F/ CD7 CD2 NA MF 3/M/ CD7 + Nondiagnostic 4/M/ CD7 + NA 5/M/ CD7 NA LCT 6/F/ Nonspecific + ILC 7/F/ CD7dim+ + LCT 8/M/33 0.9/ CD2 CD7 + ILC 9/M/ CD7 NA ILC 10/M/ CD7 NA LCT 11/M/ CD7 + MF 12/F/ CD7 NA NA 13/M/ CD3dim+CD7 NA LCT 14/F/ Nonspecific + ILC 15/M/ CD3 CD2 CD7 NA LCT 16/F/ CD2 CD7 + LCT 17/M/ CD3dim+CD7dim+ NA ILC ILC, increased numbers of large cells (see the Materials and Methods section for further definition); LCT, large cell transformation; MF, mycosis fungoides; NA, not available; TCR, T-cell receptor; +, positive;, negative. * Data are given as Système International units; to convert to conventional units (/µl), divide by TCR gene clonality was confirmed by polymerase chain reaction, Southern blot analysis, or both. Abnormal lymphocyte percentage could not be assessed due to lack of an abnormal immunophenotype; these cases were excluded from tumor burden statistics. Histologic slides were not available; however, a large T-cell population was identified by side and forward scatter in flow cytometric studies. This case was excluded from the analysis of the incidence of large cell transformation. Case 8 had mixed biclonal aneuploidy. Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD 777

5 Wang et al / FLOW CYTOMETRIC DNA PLOIDY ANALYSIS IN SÉZARY SYNDROME (53%) of 17 cases. In these cases, DNA ploidy studies were essential in identifying and quantifying residual disease following treatment as total CD4+ T cells and/or CD7 CD4+ cells dropped below diagnostic levels. The percentage of aneuploid cells fluctuated with multimodality treatment and disease progress. At some points in some patients, it reached a minimum of 1% of the total WBC count but was never below the detectable level throughout the follow-up period. The only exception was case 15, in which LCT occurred; the patient subsequently did well with marrow ablation salvaged by autologous hematopoietic stem cell transplantation. This patient s aneuploid cells were no longer detectable after that procedure and during a 2-year follow-up period. The lack of identifiable clonal T cells by DNA ploidy analysis also was confirmed by TCR gene rearrangement study by PCR on 3 occasions after the autologous transplant. These observations A B No. of Cells 1,600 1, No. of Cells DNA Content suggest that it is rare to totally eliminate the abnormal cells from blood in CTCL/SS, except with the most aggressive therapeutic approaches. Comparison of Tumor Burden and Flow Cytometric Immunophenotyping Between Aneuploid and Euploid SS Patients The aneuploid group included 7 women and 10 men (Table 2). The median age at the time of the study was 56 years (range, years). The euploid group included 20 women and 26 men with a median age of 64 years (range, years). Although several patients in the aneuploid group were relatively young (cases 8, 10, 14, and 16), there was no statistically significant difference in age or sex between the aneuploid and euploid groups. The absolute CD4+ T-cell count in aneuploid SS patients ranged from 218/µL to 25,160/µL ( ,600 1, DNA Content CD4 CD7 CD3 CD7 CD2 Figure 1 A, Two examples of aneuploid DNA histograms with DNA indices of 1.77 and 1.87 and abnormal cell fractions of total peripheral blood leukocytes of 24.4% and 61.1%, respectively. B, Examples of abnormal immunophenotyping results for circulating cutaneous T-cell lymphoma cells. 778 Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD

6 Hematopathology / ORIGINAL ARTICLE 10 9 /L; mean ± SD, 4,786/µL ± 6,678/µL [4.79 ± /L]) compared with 402/µL to 68,495/µL ( /L; mean ± SD, 7,971/µL ± 13,886/µL [ /L]) in euploid patients (P =.25) Table 3. The absolute abnormal lymphocyte count was calculated by multiplying the total number of lymphocytes by the percentage of abnormal lymphocytes identified by flow cytometric immunophenotyping after excluding cases with a normal immunophenotype. The mean ± SD for aneuploid SS patients was 4,136/µL ± 5,879/µL and for euploid patients, 6,874/µL ± 9,856/µL, again, not statistically different. Similarly, there was no statistically significant difference in total CD4+ T-cell count or CD4/CD8 ratio between the 2 groups. Of the 17 patients in the aneuploid group, 6 (35%) showed aberrant expression of CD2, CD3, CD4, CD5, or other markers (CD8, CD56), in contrast with 25 (54%) of 46 in the euploid group. CD7 negativity as the sole aberrant marker was found in 8 aneuploid cases (47%) and 12 euploid cases (26%). Neither of these differences was statistically significant by the χ 2 test. Aneuploidy Is Associated With a High Incidence of LCT in CTCL/SS Skin and/or other tissue histologic slides were available for review in 14 of 17 aneuploid and 33 of 46 euploid SS cases at the time of DNA ploidy studies. Of these 47 total cases with tissue samples for review, LCT was found in 10 (21%), marked ILC was seen in 11 (23%), and classic MF or nondiagnostic of CTCL histologic features (NILC) were found in 26 (55%). Of 14 aneuploid cases, 6 (43%) showed LCT and 5 (36%), ILC histologic features, whereas of 33 euploid cases, 4 (12%) showed LCT and 6 (18%), ILC histologic features. The incidence of LCT histologic features alone and the incidence of showing LCT or ILC histologic features were statistically significant between the aneuploid and euploid groups (P <.05 in both cases). As shown in Table 4, aneuploidy was present in 6 (60%) of 10 LCT cases and 5 (46%) of 11 ILC cases but only 3 (12%) of 26 nontransformed SS cases (P <.05). Image 1 shows cases of classic MF (Image 1A), marked ILC histologic features (Image 1B), and the histologic features of LCT (Image 1C) in 3 aneuploid patients. Comparison of tumor burden and flow cytometric immunophenotyping results was made among the LCT, ILC, and NILC groups (Table 4). Although the patients with LCT showed a slightly higher mean absolute CD4+ and abnormal lymphocyte counts, no statistically significant difference was found. Flow cytometric immunophenotypes subdivided into normal ; aberrant expression of CD2, CD3, CD4, CD5, CD8 ; and CD7 as the sole aberrant marker also were compared among the 3 histologic groups; no statistically significant difference was found. Table 3 Comparison of Tumor Burden and Flow Cytometric Immunophenotyping in Patients With Sézary Syndrome With Circulating Aneuploid and Euploid Neoplastic T Cells * Aberrant Expression CD7 as Only Absolute CD4+ T-Cell Absolute Abnormal CD4/CD8 Normal Pan T-Cell of CD2, CD3, CD4, Abnormal T-Cell Count (/µl) Lymphocyte Count (/µl) Ratio Marker Expression CD5, or Others Marker Aneuploid (n = 17) 4,786 ± 6,678 4,136 ± 5, ± (18) 6 (35) 8 (47) Euploid (n = 46) 7,971 ± 13,886 6,874 ± 9, ± (20) 25 (54) 12 (26) * Tumor burden was estimated by using the absolute CD4+ T-cell and abnormal lymphocyte counts and the CD4/CD8 ratio. Student t tests were performed for each parameter between the aneuploid and euploid groups; flow cytometric immunophenotyping results were compared between the 2 groups for normal and aberrant marker expression using a χ 2 test 2 3 contingency table. Cases with normal flow cytometric immunophenotyping results or negative CD4 expression were excluded from tumor burden statistical analysis. Data for the absolute CD4+ T-cell and abnormal lymphocyte counts and the CD4/CD8 ratio are given as mean ± SD, and for marker expression, as number with expression (percentage). CD4+ T-cell counts are given in conventional units; to convert to Système International units ( 10 9 /L), multiply by Other abnormal markers included CD56, CD57, and CD8 coexpression on CD4+ T cells. Table 4 Comparison of DNA Ploidy, Tumor Burden, and Flow Cytometric Immunophenotyping Results Among Different Histologic Grades in Tissue Biopsy Specimens * Absolute Abnormal Aberrant Expression CD7 as Only DNA Aneuploidy/ Absolute CD4+ T-Cell Lymphocyte Normal Pan T-Cell of CD2, CD3, CD4, Abnormal T-Cell DNA Index Count (/µl) Count (/µl) Marker Expression CD5, or Others Marker LCT (n = 10) 6 (60) / ,405 (318-68,495) 9,670 (286-64,726) 1 (10) 6 (60) 3 (30) ILC (n = 11) 5 (45)/ ,045 (521-47,242) 6,660 (387-44,532) 3 (27) 4 (36) 4 (36) NILC (n = 26) 3 (12)/ ,665 (548-42,167) 5,423 (480-39,880) 5 (19) 14 (54) 7 (27) ILC, increased number of large cells; LCT, large cell transformation; NILC, no increased number of large cells (see the Materials and Methods section for further definition of ILC and NILC). * Cases with normal flow cytometric immunophenotyping results or negative CD4 expression were excluded from tumor burden statistical analysis. Data for the absolute CD4+ T- cell and abnormal lymphocyte counts are given as mean (range), and for DNA aneuploidy and marker expression, as number with expression (percentage). CD4+ T-cell counts are given in conventional units; to convert to Système International units ( 10 9 /L), multiply by The case with large cells on flow cytometric side and forward scatter but without tissue biopsy slides available for review was not included. Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD 779

7 Wang et al / FLOW CYTOMETRIC DNA PLOIDY ANALYSIS IN SÉZARY SYNDROME Because CD25 expression measured immunohistochemically on skin biopsy specimens might be useful in diagnosing transformation, CD25 expression detected by flow cytometry on peripheral blood cells was compared among different histologic grades. Positive CD25 expression on circulating CD4+ cells, including dim expression, was found in 7 (70%) of 10 LCT cases, 6 (55%) of 11 ILC cases, and 14 (54%) of 26 NILC cases; differences were not statistically significant. There also was no difference in age or sex distribution between patients with and without LCT. Cell proliferation indices (S phase and G 2 phase) also were compared for the LCT, ILC, and NILC groups; no statistically significant correlation was found. In summary, in patients with a diagnosis of SS, LCT was not related to tumor burden as measured by the aforementioned parameters but was associated with abnormal flow cytometric DNA ploidy study results (P <.05). No significant A C changes in immunophenotype or proliferation indices of the neoplastic T cells were found to be associated with LCT. Discussion Compared with other hematologic malignant disorders, there is relatively limited information on the chromosomal characteristics of malignant cells in MF/SS because of the rarity of the disease, difficulty in obtaining malignant cells from skin biopsy specimens, the relative paucity of malignant cells in the circulation, the low frequency of metaphase cells in routine culture, and poor morphologic features of the chromosomes in many cases. Although some studies report that clonal chromosomal abnormalities correlate with disease activity and stage, 18,33-35 the complexity of the karyotype, lack of recurring abnormalities, and difficulty in interpretation of data B Image 1 Skin biopsy specimens from aneuploid patients. A, Classic mycosis fungoides (H&E, 100; inset, H&E, 400). B, Markedly increased numbers of large cells (see the Materials and Methods section for further definition) (H&E, 100; inset, H&E, 400). C, Large cell transformation (H&E, 100; inset, H&E, 400). 780 Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD

8 Hematopathology / ORIGINAL ARTICLE from various clinical stages of disease limit its use in routine pathologic diagnosis. Flow cytometry DNA ploidy analysis can be performed on nondividing cells and can analyze relatively small numbers of cells and, therefore, provides a means for obtaining general chromosomal/ploidy information in many diseases. 36 Flow cytometric methods have been applied to measure nuclear DNA content and cell cycle kinetics as prognostic indicators or for minimal residual disease detection in childhood acute leukemia, 9 multiple myeloma, 37 and various solid tumors. 10,11 In this article, we report our experience with DNA ploidy studies by flow cytometry in identifying SS at our institution during a 12-year period in which DNA indices were measured routinely by flow cytometry for all initial MF/CTCL blood evaluations. The primary goal of these DNA ploidy studies was to detect and quantify abnormal circulating lymphocytes because many cases of CTCL will not show an abnormal immunophenotype, and gene rearrangement studies, while very sensitive for detection of monoclonal cells, are difficult to make quantitative. We found DNA aneuploidy in 17 (27%) of 63 cases with a blood-based diagnosis of SS; normal or nonspecific (CD7 as the sole aberrant marker) immunophenotyping was found in 9 of these 17 cases. In all aneuploid cases, DNA ploidy studies were valuable in identifying minimal residual disease following the course of treatment. MF transformation is the presence of large cells exceeding 25% of the CTCL infiltrate throughout a tissue specimen or the presence of microscopic large cell nodules. 20,23,38 It is associated with an aggressive clinical course and shortened survival. 20,21 Clinicopathologic or biologic criteria predictive of transformation are unknown, except for the expression of CD25 antigen (interleukin 2 receptor) on the abnormal cells in tissue specimens, which may identify a subset of at-risk patients with MF. 39 The molecular mechanisms by which some MF cases undergo transformation are undefined. Li et al 40 found overexpression of p53 protein in transformed MF, although this expression was not correlated with p53 gene mutation. The t(2;5)(p23;q35) chromosomal translocation reported in CD30+ anaplastic large cell lymphoma is not involved in the molecular pathogenesis of MF transformation. 41 Cytogenetic studies related to LCT have been attempted in only rare cases 27,42-44 ; complex but not constant numeric and structural chromosomal abnormalities have been reported. In the present study, we demonstrated that DNA aneuploidy identified in circulating lymphocytes is highly associated with LCT or a marked ILC found in biopsy tissue samples (skin, lymph node, bone marrow, and/or other solid organs) in contrast with euploid cases. Of note, no specific immunophenotypic characteristics of circulating T cells, including CD25 expression, or proliferation indices of the circulating neoplastic T cells were associated with LCT. In aneuploid SS patients with LCT, the DNA index measured in circulating neoplastic T cells ranged from 1.1 to This disagrees with the hypothesis that only near-hypertetraploid neoplastic clones would be associated with LCT. 26 As is well known, a high incidence of clonal cytogenetic abnormalities have been found in CTCL/SS 18,34,35 ; however, the typical complexity of the karyotypes, the lack of recurring abnormalities, and the difficulty in interpretation of data have limited the clinical application of cytogenetics in this disease. Although some studies report particular recurrent cytogenetic abnormalities 18 and possible association with adverse disease activity, 45 little is known about the association of abnormal chromosome number with LCT. Our findings suggest that the unbalanced chromosomal aberration in CTCL/SS, reflected as abnormal DNA ploidy, is highly associated with the likelihood of LCT. The study of DNA ploidy by flow cytometry has significant value in establishing the diagnosis of blood involvement by CTCL and SS and detecting minimal residual disease in aneuploid SS following therapy. Most important, detection of aneuploid neoplastic T cells in the peripheral blood of patients with CTCL is significantly associated with LCT in skin, lymph node, or other tissues and, because of the prognostic implications of transformed MF, might suggest a diligent search for further evidence of such transformation. From the Departments of 1 Laboratory Medicine, 2 Pathology, 3 Dermatology, and 4 Internal Medicine, Yale University School of Medicine, Yale New Haven Hospital, New Haven, CT. Address reprint requests to Dr Smith: Dept. of Laboratory Medicine, Fitkin 617, Yale University School of Medicine, 333 Cedar St, PO Box , New Haven, CT References 1. Lutzner M, Edelson R, Schein P, et al. Cutaneous T-cell lymphomas: the Sézary syndrome, mycosis fungoides and related disorders. Ann Intern Med. 1975;83: Sausville EA, Eddy JL, Makuch RW, et al. Histopathologic staging at initial diagnosis of mycosis fungoides and the Sézary syndrome. Ann Intern Med. 1988;109: 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: Buechner SA, Winkelmann RK. Sézary syndrome: a clinicopathologic study of 39 cases. Arch Dermatol. 1983;119: Trotter MJ, Whittaker SJ, Orchard GE, et al. Cutaneous histopathology of Sézary syndrome: a study of 41 cases with a proven circulating T-cell clone. J Cutan Pathol. 1997;24: Willemze R, Van Vloten WA, Hermans J, et al. Diagnostic criteria in Sézary s syndrome: a multiparameter study of peripheral blood lymphocytes in 32 patients with erythroderma. J Invest Dermatol. 1983;81: Harmon CB, Witzig TE, Katzmann JA, et al. Detection of circulating T cells with CD4+CD7 immunophenotype in patients with benign and malignant lymphoproliferative dermatoses. J Am Acad Dermatol. 1996;35: Bogen SA, Pelley D, Charif M, et al. Immunophenotypic identification of Sézary cells in peripheral blood. Am J Clin Pathol. 1996;106: Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD 781

9 Wang et al / FLOW CYTOMETRIC DNA PLOIDY ANALYSIS IN SÉZARY SYNDROME 9. Olah E, Balogh E, Kajtar P, et al. Diagnostic and prognostic significance of chromosome abnormalities in childhood acute lymphoblastic leukemia. Ann N Y Acad Sci. 1997;824: Nikolaeva TG, Dobrynin IaV, Letiagin VP, et al. The experience in the use of DNA flow cytometry to predict the natural history of malignant neoplasms [in Russian]. Vestn Ross Akad Med Nauk. January 2002: Magennis DP. Nuclear DNA in histological and cytological specimens: measurement and prognostic significance. Br J Biomed Sci. 1997;54: Duque RE, Andreeff M, Braylan RC, et al. Consensus review of the clinical utility of DNA flow cytometry in neoplastic hematopathology. Cytometry. 1993;14: Braylan RC. Flow-cytometric DNA analysis in the diagnosis and prognosis of lymphoma. Am J Clin Pathol. 1993;99: Horii A, Yoshida J, Hattori K, et al. DNA ploidy, proliferative activities, and immunophenotype of malignant lymphoma: application of flow cytometry. Head Neck. 1998;20: 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: Johnson GA, Dewald GW, Strand WR, et al. Chromosome studies in 17 patients with the Sézary syndrome. Cancer. 1985;55: Vonderheid EC, Kantor GR, Pequignot EC, et al. Analysis of clinical, histologic and immunopathologic parameters in erythrodermic variants of cutaneous T-cell lymphoma with implication for staging. In: Lambert WC, Giannotti B, van Vloten WA, eds. Basic Mechanisms of Physiologic and Aberrant Lymphoproliferation in the Skin. New York, NY: Plenum Press; 1994: Thangavelu M, Finn WG, Yelavarthi KK, et al. Recurring structural chromosome abnormalities in peripheral blood lymphocytes of patients with mycosis fungoides/sézary syndrome. Blood. 1997;89: Dmitrovsky E, Matthews MJ, Bunn PA, et al. Cytologic transformation in cutaneous T cell lymphoma: a clinicopathologic entity associated with poor prognosis. J Clin Oncol. 1987;5: Diamandidou E, Colome-Grimmer M, Fayad L, et al. Transformation of mycosis fungoides/sézary syndrome: clinical characteristics and prognosis. Blood. 1998;92: Salhany KE, Cousar JB, Greer JP, et al. Transformation of cutaneous T-cell lymphoma to large-cell lymphoma: a clinicopathologic and immunologic study. Am J Pathol. 1988;132: Greer JP, Salhany KE, Cousar JB, et al. Clinical features associated with transformation of cerebriform T-cell lymphoma to a large-cell process. Hematol Oncol. 1990;8: Wolfe JT, Chooback L, Finn DT, et al. Large-cell transformation following detection of minimal residual disease in cutaneous T-cell lymphoma: molecular and in situ analysis of a single neoplastic T-cell clone expressing the identical T-cell receptor. J Clin Oncol. 1995;13: Wood GS, Hoppe RT, Warnke RA, et al. Evidence that mycosis fungoides (MF) and transformed MF arise from the same T-cell clone [abstract]. J Cutan Pathol. 1991;18: Ralfkiaer E, Larsen JK, Christensen IJ, et al. DNA analysis by flow cytometry in cutaneous T cell lymphomas. Br J Dermatol. 1989;120: Bunn PA Jr, Whang-Peng J, Carney DN, et al. DNA content analysis by flow cytometry and cytogenetic analysis in mycosis fungoides and Sézary syndrome. J Clin Invest. 1980;65: So CC, Wong KF, Siu LL, et al. Large cell transformation of Sézary syndrome: a conventional and molecular cytogenetic study. Am J Clin Pathol. 2000;113: 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: Fraser-Andrews E, Seed P, Whittaker S, et al. Extracorporeal photopheresis in Sézary syndrome: no significant effect in the survival of 44 patients with a peripheral blood T-cell clone. Arch Dermatol. 1998;134: Orfao A, Ciudad J, Gonzalez M, et al. Flow cytometry in the diagnosis of cancer. Scand J Clin Lab Invest. 1995;55: Diss TC, Watts M, Pan LX, et al. The polymerase chain reaction in the demonstration of monoclonality in T cell lymphomas. J Clin Pathol. 1995;48: Lefranc MP, Forster A, Rabbitts TH. Rearrangement of two distinct T-cell gamma-chain variable-region genes in human DNA. Nature. 1986;319: Lima M, Almeida J, dos Anjos Teixeira M, et al. Utility of flow cytometry immunophenotyping and DNA ploidy studies for diagnosis and characterization of blood involvement in CD4+ Sézary s syndrome. Haematologica. 2003;88: Whang-Peng J, Bunn PA, Knutsen T, et al. Clinical implications of cytogenetic studies in cutaneous T-cell lymphoma (CTCL). Cancer. 1982;50: Nowell PC, Finan JB, Vonderheid EC. Clonal characteristics of cutaneous T cell lymphomas: cytogenetic evidence from blood, lymph nodes and skin. J Invest Dermatol. 1982;78: Clausen OP, Olsen G, Tjonnfjord GE. Flow cytometry: use in cell biology and benefits in clinical medicine [in Norwegian]. Tidsskr Nor Laegeforen. 2000;120: Pope B, Brown R, Gibson J, et al. The bone marrow plasma cell labeling index by flow cytometry. Cytometry. 1999;38: 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: Stefanato CM, Tallini G, Crotty PL. Histologic and immunophenotypic features prior to transformation in patients with transformed cutaneous T-cell lymphoma: is CD25 expression in skin biopsy samples predictive of large cell transformation in cutaneous T-cell lymphoma? Am J Dermatopathol. 1998;20: Li G, Chooback L, Wolfe JT, et al. Overexpression of p53 protein in cutaneous T cell lymphoma: relationship to large cell transformation and disease progression. J Invest Dermatol. 1998;110: Li G, Salhany KE, Rook AH, et al. The pathogenesis of large cell transformation in cutaneous T-cell lymphoma is not associated with t(2;5)(p23;q35) chromosomal translocation. J Cutan Pathol. 1997;24: Vonderheid EC, Fang SM, Helfrich MK, et al. Biophysical characterization of normal T-lymphocytes and Sézary cells. J Invest Dermatol. 1981;76: D Alessandro E, De Pasquale A, Ligas C, et al. Cytogenetic findings in terminal large cell transformation in a case of Sézary syndrome. Cancer Genet Cytogenet. 1992;58: Matutes E, Schulz T, Dyer M, et al. Immunoblastic transformation of a Sézary syndrome in a black Caribbean patient without evidence of HTLV-I. Leuk Lymphoma. 1995;18: Karenko L, Sarna S, Kahkonen M, et al. Chromosomal abnormalities in relation to clinical disease in patients with cutaneous T-cell lymphoma: a 5-year follow-up study. Br J Dermatol. 2003;148: Am J Clin Pathol 2004;122: DOI: /8B849FC6PHAP8FDD