Detection of minimal residual T cell acute lymphoblastic leukemia by flow cytometry

Journal of Immunological Methods, 132 (1990) 275-286

275

Elsevier JIM05692

Detection of minimal residual T cell acute lymphoblastic leukemia by flow cytometry * S t e v e n D. G o r e , M i c h a e l B. K a s t a n , S t e v e n N . G o o d m a n a n d C u r t I. C i v i n The Johns Hopkins Oncology Center, Baltimore, MD, U.S.A.

(Received 30 March 1990, revised received 4 June 1990, accepted 8 June 1990)

We have developed a flow cytometric assay for the determination of cellular expression of terminal deoxynucleotidyl transferase (TdT) and applied this to the detection of minimal residual T cell acute lymphoblastic leukemia (T-ALL). The flow cytometric assay for T d T demonstrated requisite specificity: T d T was localized to the nucleus, and was detected in M O L T 3 T lymphoblasts, clinical T - A L L samples, and normal bone marrow B lymphoid precursors, but in neither the K G l a myeloid leukemia cell line nor normal myeloid cells. Co-expression of T d T and the p a n T cell marker CD5 was used to quantify T lymphoblasts. 0.25 + 0.13% of normal adult bone marrow CD5 + cells were TdT+; these m a y represent early T lymphoid precursors. When admixed with normal bone marrow, CD5 + T d T + leukemic cells could be detected above background levels at an added concentration of 0.035% (95% confidence interval 0.028-0.043%). Long term follow-up of a large number of patients will be required to determine the clinical significance of a minimal burden of leukemic cells. Key words: Acute lymphoblastic leukemia; Flow cytometry; Minimal residual disease; Terminal deoxynucleotidyltransferase

Introduction Correspondence to: S.D. Gore, The Johns Hopkins Oncology Center, 600 N. Wolfe St., Baltimore, MD 21205, U.S.A. (Tel.: 301-955-8816; Fax: 301-955-8897). * Supported in part by Grants CA-32318, CA-06973, and CA-09071 from the National Cancer Institute, National Institutes of Health, and a grant from Becton-Dickinson Immtmoeytometry Systems. M.B.K. was a recipient of a fellowship grant from the Ladies Auxiliary of the Veterans of Foreign Wars. C.I.C. was a Scholar of the Leukemia Society of America. Presented in part at the 1989 meeting of the American Society of Clinical Ontology. Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myelogenons leukemia; BMT, bone marrow transplant; CR, complete remission; FACS, fluorescence activated cell sorter; FITC, fluoreseein isothioeyanate; PE, phycoerythrin; PCR, polymerase chain reaction; TdT, terminal deoxyaucleotidyl transferase.

Despite advances in molecular and cell biological techniques, "remission' of leukemia is still operationally defined b y a morphologic marrow blast count of < 5%. As m a n y as 10 s tumor cells m a y be harbored in the patient with 5% leukemic blasts (Van Bekkum, 1984). Sensitive techniques for reliable identification of leukemic cells during remission would potentially allow selection of patients who might benefit from further therapy: reinduction, intensive consolidation, or myeloablative therapy with bone marrow rescue. The enzyme terminal deoxynucleotidyl transferase is a marker of ALL. This template-independent intranuclear D N A replication enzyme appears to randomly attach deoxynudeotides to the

0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

276 3' terminus of a DNA molecule (Bollum, 1979) and probably plays an important role in the rearrangement of immunoglobulin and T cell receptor genes during early lymphoid development (Alt and Baltimore, 1982; Landau et al., 1987). TdT is expressed in > 90% of lymphoblastic leukemias (Hoffbrand et al., 1977; Coleman and Hutton, 1981); however, the utility of TdT as a marker of residual leukemia has been limited by the presence of normal TdT ÷ B lymphoid precursors in bone marrow, the need to microscopically screen thousands of ceils, the relative instability of the enzyme, and the uncertain specificity of polyclonal antisera used to label TdT. Recent developments in preparation of cells for flow cytometry in our laboratory and others enable relatively rapid, sensitive detection of cytoplasmic and intranuclear proteins, even in rare subsets of heterogenous cell populations (SlaperCortenbach et al., 1988; Kastan et al., 1989a,b). Slaper-Cortenbach et al. (1988) demonstrated that TdT could be detected by flow cytometry in sampies from patients with acute lymphoblastic leukemia following fixation in buffered formalin acetone; however, leukemic blast populations did not appear to stain uniformly for TdT (range of positive blasts 28-78%), and normal lymphoid precursors were not examined. We have measured intranuclear TdT expression following fixation and permeabilization of cells with paraformaldehyde and Triton X-100, using monoclonal anti-TdT. We have demonstrated that positive signals in this assay are specific for lymphoid precursor cells in normal bone marrow and a lymphoid leukemia cell line. Once appropriate specificity was demonstrated, we established a two-color flow cytometric assay for the detection of minimal residual T cell acute lymphoblastic leukemia (T-ALL). This assay, based on earlier immunofluorescence microscopic work of Van Dongen et al. (1984) and Janossy et al. (1988) takes advantage of the extremely small numbers of TdT ÷ T cells in normal bone marrow (Janossy et al., 1980; Van Dongen et al., 1985). Cells were simultaneously analyzed for expression of CD5, a pan-T cell marker, and TdT. Mixing experiments showed the sensitivity of the assay to exceed standard Southern blotting and to be within one log of a recently described application of the

polymerase chain reaction (PCR) for detection of residual T-ALL (Hansen-Hagge et al., 1989). A small number of patients with T-ALL in remission has been monitored using this assay. In all patients with sufficient data, the number of marrow T lymphoblasts has remained normal, and all remain in clinical remission.

Materials and methods Cells

Bone marrow cells from patients with T-ALL in remission or acute myelogenous leukemia (AML) recovering from intensive chemotherapy were obtained at the time of clinically indicated marrow aspirations, and bone marrow was obtained from consenting healthy volunteers, as approved by the Institutional Review Board and Department of Health and Human Services. Heparinized marrow was kept overnight at room temperature before isolation of low density mononuclear cells (1.077 g/ml, Ficoll-Hypaque, Pharmacia, Piscataway, N J). MOLT3, a T lymphoblast cell line, and KGla, a myeloid leukemia cell line, were grown in RPMI 1640 (Whittaker Bioproducts, Walkersville, MD) supplemented with 10% fetal bovine serum (Whittaker), 2 mM glutamine (Whittaker) and gentamicin (Elkins-Sinn, Cherry Hill, NJ) 20 pg/ml, under 5% CO 2 at 37°C. Antibodies

Unlabeled and fluorescein (FITC)-conjugated monoclonal anti-human TdT were the generous gifts of Dr. F. Bollum (Supertechs, Bethesda, MD). These preparations are cocktails of two IgG2a antibodies and one IgG1. Phycoerythrin (PE)-conjugated and unlabeled monoclonal antibodies to CD5, CD7, CD10, CD15, and FITC-conjugated control mouse IgG1 and IgG2, were kindly provided by Dr. M. Loken (Becton-Dickinson, Mountain View, CA), and ICH3, a CD34 IgG2a, by Dr. F. Katz (Great Ormond Street, London). FITCconjugated monoclonal rat anti-mouse IgG1 and anti-mouse IgG2 were the kind gifts of Dr. D. Buck (Becton-Dickinson). When unlabeled antibodies were used to stain ceil surface antigens, PE-conjugated, affinity purified isotype-specific

277 goat anti-mouse antibodies (Southern Bioteclmology Associates, Birmingham, AL) were used as secondary labeling reagents.

Immunofluorescence assay and flow cytometry Cell surface antigens were labeled, directly or indirectly, with PE-conjugated antibodies as previously described (Loken et al., 1987). Cell preparation for TdT staining was as described for c-myb, c-myb and c-fos (Kastan et al., 1989b). All steps were performed at 4°C. Following cell surface labeling, 106 cells were fixed in 2 mi 1% paraformaldehyde (Polysciences, Warrington, PA) in phosphate-buffered saline (PBS) for 15 rain, centrifuged (200 × g, 10 rain) and resuspended in 2 ml 0.1% Triton X-100 (TX-100, Sigma, St. Louis, MO) in 10 mM Hepes-saline (pH 7.4) containing 4% newborn bovine serum (Whittaker), (IFA buffer), for 3 rain. The permeabilized cells were centrifuged (450 × g, 10 min), then incubated with 0.1 ml unlabeled or FITC-conjugated anti-TdT (diluted 1/10 in IFA buffer; optimal dilution for this assay; data not shown), for 1 h. Control samples were incubated with isotype-matched irrelevant mouse monoclonal antibodies, or FITC-conjugated isotype-matched control mouse antibodies (see below). 2% human AB + serum was included in all antibody incubation steps to block nonspecific binding. 2% rat serum was also included in indirectly labeled experiments. The cells were washed with 2 ml 0.1% TX-100/IFA, and the wash repeated (directly labeled samples) or the cells resuspended in 0.1 ml FITC-rat anti-mouse IgG1, (diluted 1/100 in IFA buffer; indirectly labeled samples). The latter cells were then washed twice in 2 ml 0.1% TX-100/IFA. All samples were resuspended in 1 rod IFA buffer. Although the anti-TdT monoclonal antibody cocktail contains both IgG2a and IgG1, preliminary experiments using this preparation showed that no staining of MOLT3 cells (a TdT ÷ cell line) was seen when anti-mouse IgG2a was used as a secondary antibody, suggesting that the epitopes on the TdT molecule recognized by the IgG2a antibodies in the cocktail were not stable to the fixation technique (data not shown). Therefore, in subsequent experiments using indirect labeling, monoclonal FITC-anti-mouse IgG1 was

used as a secondary antibody, and MOPC 21, an irrelevant IgG1 was used as a control antibody. Because the IgG2a antibodies could contribute to non-specific binding when directly FITC-conjugated, FITC-conjugated control mouse IgG2 plus IgG1 (2 : 1, the ratio of isotypes in the antiTdT cocktail) was used as a control in direct labeling experiments. Two-color immunofluorescence was analyzed on a FACScan flow cytometer after appropriate compensations of the FITC and PE channels using Calibrite beads according to manufacturer's instructions (Becton-Dickinson). Cells prepared using paraformaldehyde-TX-100 lose orthogonal light scattering properties, while retaining relative forward light scatter. Signals with significant residual orthogonal light scatter represent cell fragments and yield nonspecific autofluorescence (unpublished data). Such signals were eliminated during data analysis using an electronic gate. For detection of minimal leukemia, an electronic gate was set to selectively analyze CD5 ÷ cells (T cells) for TdT expression. 17,000-20,000 CD5 + events were then analyzed. The percentage of total T cells (CD5 +) which were also positive for TdT was determined. Samples and controls for nonspecific fluorescence were run in duplicate, and the average control value was subtracted from the average sample value. The percent of CD5 ÷ cells co-expressing TdT was then multiplied by the fraction of total marrow mononuclear cells expressing CD5 to quantify the T lymphoblasts as a percentage of mononuclear cells.

Mixing experiments In order to estimate the sensitivity of this technique for detecting occult T lymphoblasts in remission marrows, mixing experiments were performed. MOLT3 T lymphoblasts or fresh cells from a patient with T-ALL were mixed at various concentrations with heparinized normal bone marrow. Mixing of cells was performed before density centrifugation in order to closely approximate the clinical situation. Low density mononuclear cells were then prepared and processed as described above.

Statistical analysis Sample means were compared using Student's t

278 test. To identify the concentration of MOLT3 cells which could be detected when mixed with normal bone marrow, the results of four mixing experiments were combined as follows: the log concentration of admixed MOLT3 cells was plotted versus the log percent of C D 5 + T d T ÷ cells detected. We then fit a bilinear model by least squares linear regression to the log-transforms of the MOLT3 and C D 5 + T d T + cell concentrations (excluding the zero points) on each experiment. The slope of the initial segment was constrained to zero, and the intercept of that line, the join point of the two lines, and the slope of the second line were estimated. The overall join point was estimated as the reciprocal variance weighted average of the individual join points. This represents the minimum MOLT3 concentration that the assay could detect above background.

Results

Flow cytometric detection of TdT In order to determine whether the flow cytometric methodology utilized for measurement of intranuclear oncoproteins also allowed specific detection of nuclear TdT, binding of an anti-TdT monoclonal antibody cocktail to the T lymphoblast cell line MOLT3 was compared to the myeloid leukemia line K G l a . In Fig. 1A, histograms representing FITC fluorescence are shown comparing TdT staining to control (MOPC 21), using the indirect immunofluorescence technique. MOLT3 lymphoblasts were strongly positive for TdT, while staining of the myeloid line with antiTdT was at control level. When MOLT3 cells prepared by this method and stained with anti-TdT were examined by fluorescence microscopy, staining was intranuclear (Fig. 1C), displaying the convoluted nuclear structure of MOLT3 cells. Binding of directly conjugated FITC-anti-TdT to K G l a cells was slightly higher than control FITC-IgG, but was clearly less than binding to MOLT3 and was well below the fluorescence levels considered positive in the assays described below (Fig. 1B). Further evidence for the specific staining of T d T was obtained by investigation of normal human bone marrow. Fig. 2A shows the mononuclear fraction of normal adult bone marrow

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Fig. 1. Flow cytometric determination of TdT expression in cell lines. A: histogram analysis comparing FITC fluorescence of MOLT3 T lymphoblasts using anti-TdT (solid line) or irrelevant mouse antibody (dotted line) and KGla myeloid leukemia cells stained with anti-TdT (dashed line) using indirect immunofluorescence. B: histograms of MOLT3 cells stained with FITC-anti-TdT (solid line), FITC-control IgG (dotted line) and KGla cells stained with FITC-anti-TdT (dashed line). C: immunofluorescencemicrograph of MOLT3 cells stained with anti-TdT under conditions of flow cytometric assay. Original magnification620 x.

stained for T d T or control (indirect technique). An intermediate peak of nonspecific FITC staining is seen between unstained and brightly stained cells. This 'nonspecific' peak appears with control antibody (Fig. 2A) or anti-TdT (Fig. 2B), whereas the brightly stained peak occurs only with antiTdT. Two color immunofluorescence with CD15PE and FITC-anti-TdT (Fig. 2D) revealed that the 'nonspecific' binding of FITC-Ig was to granulocytes and their precursors. This double staining allows definitive identification of the 'true' TdT positive cells (non-granulocytes) on the cytogram. Microscopically, the staining of granulocytes was cytoplasmic. Because the majority of normal bone marrow TdT ÷ cells are CD34+CD10 + B lymphocyte precursors (Loken et al., 1987), further two color experiments were

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done using these surface markers. As seen in Fig. 3, the majority of T d T + cells stained for CD34 and CD10 (88% and 86%, respectively in a representative experiment), as expected for early B lymphoid progenitors. G a t e d acquisition of CD34 ÷ and CD10 + cells revealed that 40 + 2% of CD34 ÷ and 29 + 7% of CD10 ÷ cells (n = 3) ex-

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pressed TdT, consistent with previous data obtained b y use of an immunoperoxidase assay for T d T on FACS-separated early B precursors (Loken et al., 1987). When directly conjugated FITC-anti-TdT was used to stain bone marrow mononuclear cells, the non-specific granulocyte binding of F I T C occa-

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280 sionally o v e r l a p p e d the ' t r u e ' T d T + signals. T w o c o l o r labeling with P E - c o n j u g a t e d l y m p h o i d specific antibodies a n d F I T C - a n t i - T d T a ll o w e d the resolution of T d T ÷ l y m p h o i d cells f r o m the g r a n u l o c y t e precursors w h i c h do n o t express l y m p h o i d antigens (see Fig. 5 f o r example).

b o n e m a r r o w . P r e v i o u s r e p o r t s h a v e suggested that n o cells w h i c h co-express the p a n - T cell m a r k e r C D 5 a n d T d T are p r e s e n t in n o r m a l b o n e m a r r o w (Van D o n g e n et al., 1985; S m i t h an d Kitchens, 1989). N o r m a l b o n e m a r r o w was stained with C D 5 - P E a n d F I T C - a n t i - T d T , a n d electronic gating was used to e x a m i n e large n u m b e r s of C D 5 + cells ( 1 7 , 0 0 0 - 2 0 , 0 0 0 / s a m p l e ) . To avoid any p o t e n t i a l p r o b l e m s o f cross-reactivity of s e c o n d a r y antibodies, directly c o n j u g a t e d a n t i b o d i e s were used for cell m e m b r a n e a n d i n t r a n u c l e a r staining. T h e level o f F I T C f l u o r e s c e n c e in M O L T 3 cells

Sensitivity of detection of minimal numbers of T lymphoblasts. Before using this assay for the d e t e c t i o n of a b n o r m a l T lymphoblasts, it was necessary to e s t i m a t e the n u m b e r of T d T + T ceils in n o r m a l

TABLE I ANALYSIS OF CD5 + TdT + CELLS IN BONE MARROW Sample

Date

Status

Treatment

% T cells

TdT + T cells (%) a

TdT + T cells (%) b

phase

(CD5 + )

T cells

Mononuelear cells

Normal n ffi13

26.3 + 9.3

0.25 + 0.13

0.060 + 0.036

AML recovery n= 8

22.6+21.2

0.10+0.14 ¢

0.013-t-0.13 d

Patient 1

10/20/88 11/10/88 12/01/88 05/01/89

CR1 CR1 CR1 REL

Post-induct. Post-eonsol. Pre-allo-BMT d90-post-BMT

3.2 13.8 5.7 75.0

0.45 0.18 0.16 95.0 *

0.014 0.025 0.010 71.2 *

Patient 2

10/31/88 12/20/88 05/10/89 01/10/90

CR2 CR2 CR2 CR2

Pre-anto-BMT d30-post-BMT dlS0-post-BMT d420-post-BMT

1.5 7.0 2.2 5.2

1.26 * < 0.01 0.64 * 2.03 *

0.018 < 0.010 0.014 0.106

Patient 3

11/10/88 12/07/88 03/31/89 08/21/89 10/31/89 03/19/90

CR1 CR1 CR1 CR1 CR1 CR1

Maintenance Maintenance Maintenance Maintenance Maintenance Maintenance

9.5 5.8 5.0 4.5 5.0 5.4

0.11 0.24 0.39 0.60 * 0.44 0.76 *

0.10 0.014 0.020 0.027 0.022 0.041

Patient 4

01/20/89 08/22/89 01/23/90 03/26/90

CR1 CR1 CR1 CR1

Maintenance Maintenance Maintenance Maintenance

4.7 6.1 5.8 6.7

0.42 0.48 2.14 * 4.17 *

0.020 0.030 0.125 0.280 *

Patient 5

07/10/89 09/08/89

CR1 CR1

d30-post-allo-BMT d90-post-BMT

2.0 3.0

0.62 * 0.64 *

0.012 0.019

Patient 6

08/22/89

CR2

Pre-auto-BMT

9.0

13.3 *

1.22 *

a Flow cytometric determination of % of gated CD5 + cells positive for TdT (see materials and methods section). b Value calculated as fraction of T cells expressing TdT (column 6) multiplied by percent of mononuclear cells expressing CD5 (column 5). c Significantly different from normal controls P < 0.03. d Significantly different from normal controls P < 0.001. * > 2 standard deviations above normal controls.

281 stained with anti-TdT the day of the experiment was used as a threshold for quantifying T d T + signals; in several specimens of A L L blasts tested, all samples had T d T expression at least as great as M O L T 3 (data not shown). As seen in Table I, 0.25 _+ 0.13% of T cells in normal marrows expressed T d T (n = 13). The percent of total marrow mononuclear cells which co-expressed CD5 and T d T was 0.06 _+ 0.04. Because it has been suggested that CD7 is the earhest marker of T lineage commitment (Furley et al., 1986; Pittaluga et al., 1986), several marrows were stained with CD7-PE versus FITC-anti-TdT or a 'cocktail' of CD5-PE plus CD7-PE versus FITC-anti-TdT. The numbers of C D 7 + T d T ÷ cells were not significantly different from the number identified as C D 5 + T d T + (data not shown). T d T + T cells were also quantified in 'regenerating' bone marrow of patients with A M L who had received intensive, aplasia-inducing induction or consohdation chemotherapy between 1 and 3 months prior to the specified marrow aspiration (Table I). 0.10 + 0.14% of T cells in these patients were T d T ÷ (0.013 + 0.013% of total mononuclear cells, n = 9). Thus, patients with regenerating marrows appeared to have fewer T d T ÷ T cells than normal volunteers ( P < 0.03). Variable numbers of T lymphoblasts were mixed with normal bone marrow to determine the sensitivity of this technique to detect contamination of normal marrow by leukemic cells. In Fig. 4, the results of four experiments in which M O L T 3 cells were admixed with normal bone marrow cells are shown. When the results of the four mixing experiments with M O L T 3 cells were analyzed as described in the materials and methods section, using a bilinear model to estimate the point at which C D 5 + T d T ÷ cells were detectable over background, the sensitivity of the assay was estimated to be 0.035% (3.5 cells/10,000, 95% confidence interval 0.028-0.043%). Mixing experiments using samples of fresh cells from a patient with T - A L L showed a similar level of sensitivity (data not shown).

Longitudinal monitoring of T-ALL patients in remission In a pilot study to test the feasibility of this assay in clinical use, we have prospectively moni-

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tored several pediatric and adult patients with T - A L L in clinical remission using C D 5 / T d T double immunofluorescence and flow cytometry. Samples were considered abnormal if the percentage of mononuclear cells co-expressing CD5 and T d T exceeded 0.13%, two standard deviations above the mean for normal volunteers. Although regenerating bone marrow appeared to have fewer T d T + T cells, the higher value based on normal subjects was used as a more conservative comparison. Preinduction phenotype was not available on all patients. The results are seen in Table I. Four patients remain in clinical remission. Patient 1, an adult in first remission (CR1), relapsed 90 days after allogeneic bone marrow transplant (BMT). Unfortunately, bone marrow samples post-BMT were not available to study until the marrow which showed morphologic relapse. Patient 6, an adult in CR2, had clear flow cytometric evidence of residual tumor prior to autologous B M T (13% T d T + T cells; 1.5% of mononuclear cells T d T + T cells, Fig. 5), but succumbed to bacterial sepsis during her transplant. Of the remaining four patients, patient 4 has recently developed an increased n u m b e r of

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CD5+TdT + cells (0.28% of mononuclear cells) and thus far remains in clinical remission. None of the others has developed increased T lymphoblasts and all remain in clinical remission, with follow-up between 8 and 18 months. Patients 2, 3, and 5 have developed elevated levels of TdT + T cells as a percent of total T cells, although the total number of marrow TdT ÷ T cells remains within the normal range. The significance of this elevated fraction of T cells expressing TdT, not seen in regenerating marrows or concurrently run normal volunteers, is unclear.

Discussion

TdT continues to play an important role in the lineage diagnosis of acute leukemias. We have applied a recently developed technique for fixation and permeabilization of cells enabling flow cytometric detection of intranuclear antigens (Kastan et al., 1989a,b) to the determination of TdT expression. This TdT assay is rapid and meets several important criteria for specificity: the assay detects TdT expression in the lymphoblast cell line MOLT3 and in bone marrow B cell precursors, but not in the K G l a myeloid leukemia line, and TdT staining is intranuclear. Because orthogonal light scattering properties of cells are not retained using this technique, two-color im-

munofluorescence is required to confirm the lineage of TdT + signals. Although the use of monoclonal anti-TdT antibodies reduces nonspecific staining, granulocyte precursors bind FITC nonspecifically when fixed and permeabilized, and appropriate control antibodies must still be used to distinguish nonspecific FITC uptake from specific TdT labeling. Simultaneous staining with lineage-specific PE-conjugated monoclonal antibodies (CD15, CD5, CD19) easily resolves specific TdT staining from non-specific granulocyte binding of FITC-Ig. By labeling ceils with PE-conjugated lymphoid-specific antibodies and FITC-anti-TdT, double-positive cells are identified as immature lymphoid cells (normal or leukemic). Electronic gating on the PE-positive cells enables the examination of large numbers of lymphoid cells for TdT expression with ~essentially no contaminating myeloid cells. Recently, Slaper-Cortenbach et al. (1988) re ported TdT detection by flow cytometry in buffered formalin acetone-fixed cells. While this technique appears to preserve some right angle light scattering properties of cells, it is somewhat surprising that patient samples with T and B lineage ALL displayed only 28-70% TdT + blast cells. It has been our experience that blast cells from ALL are > 90% TdT ÷ after paraformaldehyde-Triton X-100 treatment (data not shown). When concentrations of Triton X-100 below 0.1%

283 were used to permeabilize cells, intranuclear staining for c-myb was absent or variable (Kastan et al., 1989a), a finding we have repeated with TdT staining of MOLT3 cells (data not shown). In our hands, fixation of cells in methanol results in suboptimal TdT staining (data not shown). It is possible that buffered formalin acetone fixation gives variable permeabilization of nuclear membranes; however a direct comparison of the two techniques was not performed. Having validated specific detection of intranuclear TdT by flow cytometry, we combined cell surface labeling with the pan-T cell marker CD5 with TdT staining to detect TdT ÷ T lymphoblasts. Previous studies have shown that normal bone marrow has very few TdT ÷ T cells (Janossy et al., 1980; Van Dongen et al., 1985; Smith and Kitchens, 1989). In order to increase the ability to detect small numbers of TdT + T cells, electronic gating was used to examine large numbers of cells which express CD5. This gate excludes the non-specific binding of FITC by granulocyte precursors. A small, but reproducible number of CD5 ÷TdT + cells was detected in normal marrow (0.25 + 0.13% of T cells, 0.06 ___0.02% of mononuclear cells). Bone marrows from patients recovering from intensive chemotherapy for AML appeared to have fewer TdT ÷ T cells than did normal bone marrows. Mixing experiments in which MOLT3 T lymphoblasts or fresh cells from a patient with T-ALL and normal bone marrow were simultaneously stained for the pan-T marker CD5 and TdT demonstrated reliable detection of 0.035% admixture (3.5/10,000 cells). Previous flow cytometric assays of minimal leukemia have been limited by the use of a single membrane antigen to label cells. Van Dongen et al. (1984) could detect 2% admixture of CD1 ÷ cells, while Janossy and co-workers (1988), staining for CD7, could not detect less than a 10% admixture of CD7 + cells. In contrast, both groups have detected T lymphoblasts with sensitivity of 0.01% using immunofluorescence microscopy of cytocentrifuge preparations double stained for a T cell antigen and TdT. The sensitivity of our flow cytometric assay is comparable to the latter techniques, but has the technical advantage of not requiring difficult visual evaluation of thousands of cells.

Similar screening techniques could be devised for monitoring of B lineage TdT + ALL and select cases of AML. Although normal bone marrow has substantial and variable numbers of TdT + B cell precursors, 'asynchronous' combinations of early and late B lineage antigens are expressed simultaneously in as many as 50% of cases of B lineage ALL (Hurwitz et al., 1988). In such cases, threecolor immunofluorescence using anti-TdT could provide a powerful tool for detection of residual disease. Similarly, simultaneous analysis of cells for early myeloid antigen expression and TdT could be used to monitor cases of AML which express this enzyme. Measurements of minimal numbers of leukemic cells by various methodologies share problems of sensitivity and specificity. Immunologic monitoring of peripheral blood from patients with ALL for increased numbers of TdT ÷ cells (single parameter study) has been shown to have poor positive and negative predictive values (29% and 91% respectively) (Hetherington et al., 1987). A number of investigators have used clonogenic assays to demonstrate early colonies with blast-like morphology from remission marrows (Allouche et al., 1986; Consolini et al., 1986; Estrov et al., 1986; Phillip et al., 1987). Although in some instances the colonies have been demonstrated by cytogenetics to be derived from the leukemic clone (Consolini et al., 1986; Estrov et al., 1986), positive identification of the colonies as leukemia-derived is often lacking, and the sensitivity of these assays has not been determined. Molecular biologic approaches which attempt to detect a marker clonal gene rearrangement during remission offer the advantage of diagnostic specificity for the leukemic clone. However, standard Southern blotting of remission marrow DNA lacks sensitivity (0.5%) (Zehnbauer et al., 1986), although sensitivity is somewhat improved when combined with immunologic techniques to enrich the lymphoid compartment (0.1%) (Bregni et al., 1989). Use of the polymerase chain reaction to amplify minute amounts of DNA sequences has the potential to detect 1/105-1/106 cells (Lee et al., 1987; Saiki et al., 1988) but has been hampered by the lack of a universal gene rearrangement in ALL such as the BCR-ABL rearrangement of chronic myelogenous leukemia (Kawasaki et al.,

284

1988) or the BCL2 rearrangement of follicular non-Hodgldns lymphomas (Loh et al., 1989). New approaches to overcome this limitation require the synthesis of oligonucleotide primers complementary to the individual gene rearrangement found in each patient at diagnosis (Hansen-Hagge et al., 1989; Lob et al., 1989; Yamada et al., 1989). This technique has recently been shown to have a sensitivity of 0.001% (Hansen-Hagge et al., 1989) for T-ALL. Although PCR is likely to become useful for an increasing number of patients as techniques improve, the need for DNA sequencing and oligonucleotide synthesis makes the routine use of PCR for monitoring of ALL by clinical laboratories unlikely. The less sensitive flow cytometric technique gives results within 1 day of marrow aspiration and can readily be performed by a clinical laboratory. The flow assay may be especially useful in detecting occult bone marrow lymphoblastic lymphoma (a related TdT ÷ T cell malignancy) at diagnosis, and in monitoring the response of patients with T-ALL in the early phases of chemotherapy, while the leukemic DNA is being sequenced and appropriate primers for PCR are being synthesized. In the small cohort of T-ALL patients we have followed, only one patient has relapsed to date, and marrow from that patient was not available for evaluation after BMT but prior to clinical relapse. Patient 6 had easily detectable residual T lymphoblasts pre-ABMT, but unfortunately did not survive transplantation. The remaining four patients remain in clinical remission, and all but one have maintained normal numbers of CD5+TdT + cells (the fourth patient having recently developed a single abnormal value). Determination of the positive and negative predictive values of this technique will require prospective monitoring of a large patient cohort. Important questions about the clinical significance of involvement of human bone marrow with small numbers of leukemic cells remain. Questions to be answered include the log involvement of bone marrow with leukemia which predicts for relapse, and the timing of such studies in a patient's treatment course which optimizes predictive value. Sawyers et al. (1990) have used PCR for BCR-ABL mRNA to follow patients with chronic myelogenous leukemia following BMT. 4/19 pa-

tients developed detectable levels of BCR-ABL mRNA, but all patients remain in clinical remission. Long term prospective follow-up of a large number of patients will be required to answer these questions. Such studies would ideally include comparison of immunologic, clonogenic, and molecular methods for monitoring residual leukemia. A second generation of studies would then be required to determine whether clinical intervention once minimal disease is demonstrated impacts on patient outcome. It is possible that a combination of biologic techniques (for example, use of PCR to document the clonality of a rare cell population purified by immunologic methods) may be needed to provide the requisite sensitivity and specificity to allow meaningful intervention in patient management; however, only with large prospective clinical studies will the biological and clinical significance of small numbers of residual leukemia cells be demonstrated.

Acknowledgements We thank the directors and staff of the adult leukemia, bone marrow transplantation, and the pediatric oncology services for providing bone marrow specimens, and Mr. Kelly Stone for excellent technical assistance.

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