(CANCER RESEARCH 46, 2172-21 78, April 19861 Induction of Features Characteristic of Hairy Cell Leukemia in Chronic Lymphocytic Leukemia and Prolymphocytic Leukemia Cells' H.W.LömsZiegler-Heitbrock,2ReinholdMunker,BerndDörken,GerhardGaedicke,andEckhardThiel Institutefor Immunology, University ofMunich, Goethestrasse 31, 8 Munich 2 [H. W. L. Z-H.J; lnstitutefor Hematology, GSF, Landwehrstrasse 61, 8 Munich 2 (R. M., E. T.J; Medizinische Polikiinik, University ofHeidelberg, Hospitalstrasse 3, 69 Heidelberg[B. D.J; andZentrumfurKinderheilkunde, University ofUlm, Prittwitzstrasse 43. 79 Ulm (G. G.J, Federal Republic of Germany

ABSTRACT differentiation to convert cells of, e.g., CLL type of leukemic B-cell lymphoma into HCL-type cells. Previous studies dem In vitro maturationwas inducedwith 12-O-tetradecanoylphorbol-13- onstrated that in CLL clg and immunoglobulin secretion can acetate in leukemiccell samples from patients with chronic lymphocytic be induced, which suggests maturation towards plasmacytoma leukemia(n 10) and prolymphocyticleukemia(n = 4). The cells were studied for morphology, for immunologicalmarkers using the fluores type cells (7). Although these findings are not disputed, we and cence activated cell sorter, and for acid phosphatase isoenzymesusing others (8, 9) could previously demonstrate that, by detailed both cytochemistryand isoelectrofocusing. analysis, patterns emerged which are consistent with differen Morphologically the induced changes included appearance of cells tiation of CLL cells to HCL-type cells rather than to plasma with an excentric nucleus and basophilic cytoplasm and eventually of cytoma cells. In the present report we have extended this cells with many fine cytoplasmic projections (“hairs―).Analysis of im analysis by using flow cytometry and isoelectrofocusing on both munological markers by flow cytometry revealed that the monoclonal CLLandPLLleukemiclymphomacells.Ourfindingslend antibody defined cell surface moleculeHD6 (CD22), which is strongly further support to the notion that CLL and PLL cells can be expressed on hairy cell leukemia (HCL) but absent from plasmacytoma induced to develop into cells which are closely related to HCL and plasma cells, can be inducedor enhanced in the leukemicsamples. cells. In the study of acid phosphatase isoenzymes using cytochemistry we observedthe inductionof the tartrate resistant isoenzyme.Further, using isoelectrofocusing we could demonstrate the induction of the same band MATERIALS AND METhODS of tartrate resistant acid phosphatase with an isoelectricpoint of 9.0—9.7 as detected also in HCL. This particular isoenzyme is considered char Patients and Diagnosis.Peripheral blood samples from patients were acteristic of HCL but is absent in plasmacytoma.Our data demonstrate obtained through the courtesy ofDrs. H. Theml, Krankenhaus Schwab that chronic lymphocytic leukemia and prolymphocyticleukemia cells ing; B. Emmerich, Krankenhaus rechts der Isar, and W. Siegert, Kli can be induced to realize a common genetic program which bears char nikum Grosshadern, Munich, Federal Republic of Germany. Patients acteristics of HCL, indicating that these three entities are much more had not received specific cytostatic therapy for at least 4 weeks prior to closely related than previously thought. blood sampling. Patients with B-CLL are characterized by blood and bone marrow lymphocytosis. The B-CLL cells showed dense chromatin, INTRODUCTION little cytoplasm, and faint slg staining. In B-PLL, splenomegaly in the absence of enlarged lymph nodes was the prominent feature. B-PLL Leukemia cells can be addressed as clonal transformants of cells are large (>1 2 jzm) with a single prominent nucleolus and with the lymphohematopoietic system. Heterogeneity of leukemias strong slg expression (10). reflects the different lineages, e.g., T, B, myeloid, monocytic, Cell Isolation. Peripheral blood mononuclear cells from these pa tients and from healthy controls were isolated from heparinized blood and the different stages of differentiation within these lineages. specimens by density gradient separation and stored in liquid nitrogen Classification of human B-cell tumors has made considerable after freezing in the presence of 10% dimethyl sulfoxide using a pro progress in recent years, due to the detailed analysis of immu grammable PTC 200 machine (Planer, Ltd., Sunberry-on-Thames, nological characteristics of the tumor cells (1—4).Newstimulus United Kingdom). Directly before use cells were rapidly thawed in a to this analysis came from (a) the generation of a variety of 3TC water bath, washed twice, and resuspended in complete medium monoclonal antibodies against cell surface molecules of human (RPMI 1640 with penicillin, streptomycin, L-glutamine,and 10% heat B-cells and (b) the use ofstudies involving in vitro differentiation inactivated fetal calf serum). induction of normal and malignant cells. Within the B-cell In Vitro Culture. Ten-mi volumes with cells adjusted to 2 X I0@/ml lineage HCL3 appears to be an unique type, which does not fit were cultured at 37C in a humidified 7% CO2 incubator in upright unconstrained into current differentiation schemes (5) and the No. 3013 tissue culture flasks (Falcon, Oxnard, CA) for up to 5 days. TPA (Sigma, Munich, Federal Republic of Germany) was dissolved normal counterpart of which remains to be clearly identified, in ethyl acetate (Merck AG, Darmstadt, Federal Republic of Germany) although recent evidence suggests that normal sIgG' cells from at 4 mg/mI and stored at —20'C. Directly before use a 25-al aliquot peripheral blood resemble HCL cells (6). If the HCL cell is was taken, the solvent was allowed to evaporate, and the TPA was directly related to other clonal transformants of the B-cell redissolved in 100 @zIofacetone to be diluted with 10 ml of complete lineage then it should be possible by in vitro induction of medium. This stock was used for I week. After addition to cultures the final concentration was 160 nM TPA and 0.01 % acetone. Controls were Received 8/6/85; revised I2/30/85: accepted I2/31/85. set up with the same amount of acetone. Cells were recovered on days The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in 3 and 5 and the cell number and the percentage of enlarged cells were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. determined in a hematocytometer, enlarged cells being defined as >10 I Supported by grants from the VW-Stiftung (Hannover, Federal Republic of @mforCLL samples and >15 @imlforPLL samples. The cells were Germany) and the Wilhelm-Sander-Stiftung (Munich, Federal Republic of Ger washed once and were used together with uncultured cells for all many. 2To whom requests for reprints should be addressed. subsequent analyses. Data from days 3 and 5 were comparable and 3 The abbreviations used are: HCL, hairycell leukemia; ACP, acid phosphatase; were combined for presentation. CLL, chronic lymphocytic leukemia; FACS, fluorescence activated cell sorter; l3HlThymidine Incorporation. Cultured cells were adjusted to 5 x IEF, isoelectrofocusing: IP, isoelectric point: PLL. prolymphocytic leukemia: 105/ml and 100 z1 each were seeded in triplicate wells of round TPA, I2-O-tetradecanoylphorbol-13-acetate: slg, surface immunoglobulin; clg, cytoplasmic immunoglobulin; MAB, monoclonal antibody; FITC, fluorescein bottomed microtiter plates (No. 163320; Nunc, Roskilde, Denmark). isothiocyanate. Cells were pulsed for 6 h with [3Hjthymidine (1 @tCi/well;approximately 2172

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80 Ci/mmol; NET 027Z; New England Nuclear, Dreieich, Federal detect a very faint staining of CLL cell cytoplasm, which was, however, Republic of Germany). They were harvested on glass fiber filters using scored negative in comparison to the bright staining seen, for instance, an automated Titertek cell harvester and filters were counted in a beta in plasma cells. The immunoglobulin isotype of sig was determined by counter after immersion in scintillation fluid. direct immunofluorescence using FITC coupled F(Ab)2 fragments spe Cytochemistry. Acid phosphatase staining was performed on cytospin cific for human IgG, IgM, Kand X, respectively (Dakopatts, Hamburg, preparations fixed with formalin according to the method of Barka and Federal Republic of Germany). For statistical analysis Student's t test Anderson (1 1). Tartrate resistance of the enzyme was tested in the was used. presence of tartrate (2 mg/ml; No. 804; Merck) after readjusting the pH to 5. RESULTS Isoelectrofocusing. Samples to be analyzed in IEF were washed twice in phosphate buffered saline and the cell pellets were stored at —70'C. Characteristics of the Leukemic Cells. Listed in Table 1 are Thawed material was resuspended and adjusted to 108 cells/ml and patients grouped according to diagnosis. All patients were over processed as described in detail elsewhere (12). Briefly, cells were the age of 50 years and in more advanced stages of disease. homogenized by sonic disruption (30 s, 20 W). The 20,000 x g Leukocyte counts were beyond 50,000/mm3 in all CLL patients supernatant was made I % with Triton X-100 (30 mm at room temper and in 3 of 4 of the PLL patients. Concomitant with the high ature). This crude extract was stored at —70'C without loss of ACP leukemic cell counts, the nonleukemic cells represented by the activity. Isoenzyme analysis was performed by isoelectrofocusing on horizontal thin layer slabs as described. For the visualization of the OKT3-positive T-cells usually were below 10% with an average isoenzymes the gels were immersed for 3 h at room temperature into a percentage of 5. 1%. In all samples that could be evaluated the substrate solution corresponding to that used for the cytochemical leukemic B-cells were monoclonal with respect to light chain reaction. For enzyme inhibition 75 mM tartrate was added to this expression, while for 5 cases of CLL the slg density was too staining solution. The isoelectric point of tartrate resistant isoenzyme low to allow for such analysis (which was performed with was determined from the pH gradient of the IEF gels. conventional fluorescence microscopy in this study). Staining Immunofluorescence.All immunofluorescence procedures were per of the clg showed no or only faint fluorescence of cytoplasm. formed in immunofluorescence buffer consisting of phosphate buffered In cytomorphology diagnosis was mostly unequivocal. In 9 of saline, 2.5% fetal calf serum, and 0.02 NaN3. Stained cells were ana 10 CLLs the cells were small with dense chromatin and little lyzed after fixation with 1% paraformaldehyde (13) by flow cytometry cytoplasm, and in PLL the large cells exhibited a prominent using a FACS-Il (Becton-Dickinson, Mountain View,CA). The FACS single nucleolus. Only one case of CLL exhibited 10% cells was operated at 488 nm excitation wavelength with the photomultiplier tube at 650—800V. Fluorescence intensity was expressed on a scale of with lymphoplasmacytoid features, i.e., an excentric nucleus 256 channels with the median representing the channel that covers50% and basophilic cytoplasm (patient Sc). of the cells. These calculations were performed with a nuclear data ND slg on more than 60% of the cells in a sample was detected 100 module. in 7 of 10 CLLs and in all PLLs. In CLL slg density, when Indirect immunofluorescence was used for the mouse monoclonal expressed in arbitrary median channels according to FACS, antibodies directed against lymphocytecell surface determinants (Table gave an average of 14 median channels; only one case (Ha) 2). These MABs were OKT3 (14), directed against T-cells, and 63D3 exhibited a median of 72 channels. By contrast, PLL cells (15), which recognizes monocytes and is used as an isotype control exhibited high slg densities with an average of 160 median (IgGI) for HD6. HD6 reacts most strongly with HCL cells and less channels. For HD6, conventional fluorescence microscopy de intensely with certain B-cell lymphomas and with a B-cell subpopula tected 12 and 21 % positive cells in 2 samples of CLL (patients tion (16). The HD6 MAB was recently assigned to the CD22 cluster of Sc and Hu) but no staining in the remaining 8 samples. By leukocyte differentiation antigens (17). The reagents were used at contrast, FACS analysis revealed low percentages of faintly optimal concentrations for 30 mm on ice, followed by two washes and positive cells in 6 additional cases of CLL (Table 2, Column incubation on ice with goat anti-mouse immunoglobulin FITC (Tago, Burlingame, CA) diluted 1:50. 2). For the PLL cells fluorescence microscopy and FACS gave Direct immunofluorescence was used for determination of slg and similar results and more than 60% of the cells were positive for clg. Suspension cells or acetone-fixed cytospin preparations were in HD6 in 3 of 3 PLL samples tested. Intensity of staining was cubated for 30 mm at 4'C with goat anti-human immunoglobulin very weak in CLL (median, 0.6) and stronger in PLL (median, F(Ab)2 FITC (Kallestad, Austin, TX) diluted 1:32, followed by three 5.3)(Table 2, Column 6). The average median for HD6 staining washes before microscopic analysis. Using this procedure we could in 4 cases ofHCL was 11.0 ±6.5 (SD).

studiedType Table 1 Basicdata on the leukemic lymphomas

of x 10-'@ density'@ (OKT3 leukemiaPatientSexAge(%)CLLHa (yr)StageaLeukocytes mm3slg isotypeslg (median channels)Non-leukemicpositive) T-cells

Be M 78 0 171 —,— 3 2 Hu M 60 II 84 y,K 3 5 Ku M 78 IV 260 u,K 16 2 Sc M 70 II 83 —,— 4 10 HU M 72 II 72 —,— 2 4 Lo M 69 II 69 —,— 11 3 Ne M 63 II 99 —,— I 4 We M 79 II 71 u,y,K 19 6 9PLLWen HeM M63 630 IV130 70u,K —,K72 103―

Th M 77 II 222 u,y,K >250 5 Huh F 75 II 14 y,K 89 11 BerM F59 65IV III288 57u,y,K u>250 503 4 aStagesaccordingto the system of Rai et al. (33). b Average leukocyte count for all patients was 120.7 ±82.8 (SD) X I0-'/mm3. CAverage medians were 14 ±21 for all CLL patients and 160 ± 105 for all PLL patients. d Average percentage of T-cells for all patients was 5.1 ±2.9. 2173 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1986 American Association for Cancer Research. HCL PHENOTYPE INDUCTION IN CLL AND PLL CELLS

Table 2 TPAlymphomas% induced changes in HD6 binding ofleukemic B-cell (median)ControllO%UnculstainedIntensity of staming channelsDonorUnculturedculture ControlTPA cul 3 cultureinducedturedculturetureinducedCLLHa8@12 TPA

50004Be229 42115Hu3011 75216Ku268 39113Sc 27 25/10Lo01HU35 011 2 12‘1 02 03 34003Ne58 27013We156 8010He157 31114Mean l.6aPLLWenND40±SD15.6 ±11.87.5 ±3.5 34.5 ±18•1b0.6 ±0.70.8 ±0.63.3 ±

49ND66Th 76 Huh7741Ber659 6451 20 310/3 ‘8 54 311 69325Mean 2.7HCL(n=4)Mean±SD48.5±±SD68.7 ±5.930.0 ±16.5 56.3 ±17.65.3 ±2.13.8 ±1.56.5 ±

19.411.0±6.5 aDirectinspectionusing the fluorescencemicroscoperevealedpositivity in only 2 cases(patientsHu and Sc). b Significant compared to fresh cells with P < 0.01. C Significant compared to fresh cells with P < 0.001.

TPAEffectontheLeukemicCells.Basicfindings,obtained significantly higher T-cell contamination of the leukemia cell after culture of the leukemic cells in the presence of TPA, are population (cf Table 3, Footnote d). summarized in Table 3. Cell size increased in all samples but Morphologically the CLL cells changed to cells with an one (Wen) as evidenced by microscopic evaluation and con excentric nucleus and basophilic cytoplasm with interspersed firmed by forward angle light scatter using the FACS (not plasma cell-type cells and eventually to cells with several short shown). The average percentages of enlarged cells for all sam (spike-like or hairy) projections. In PLL changes included de ples were 44.1 ±23.8 in TPA cultures and 3.7 ±6.2 in control velopment of vacuoles, increase in basophilicity, and develop cultures. This increase in cell size, however, was not linked to ment of many cellular projections. In one case each of CLL and proliferation since (a) no increase in cell number was observed PLL, we were able to detect cells with such “hairs―witha (percentage ofrecovery ofinput; 57.1 ±18.9%, control cultures; frequency higher than 25% (patients Hu and Th; cf@Fig. 1), 58.1 ±24.5%, TPA cultures) and (b) [3H]thymidine incorpo while in other samples less than 3% were identified as hairy ration did not reveal any increase in 8 of 12 samples tested. cell-type cells according to this criterion in Pappenheim stains. The samples showing some response (experimental value di clg could be induced in most samples of CLL (Table 3) while vided by control value = stimulation index >2) were those with slg decreased in both control and TPA cultures to the same extent, as evidenced by FACS (not shown). Table 3TPA inducedcells@3HJThychanges in leukemic lymphoma The changes in HD6 staining induced by TPA in the CLL ofType Appearance and PLL cells are summarized in Table 2. As already discussed, midine uncultured CLL cells showed low levels or no staining for HD6, incorpo rationLargeAcidleukemiatient(cpm)cellsofPa the intensity of which usually did not exceed 1 median channel phosphataseCLLHa (%)clg according to FACS. Control culture without TPA resulted in a decrease in the percentage of cells stained. After culture in the @ Be 75d + Hu 342 60 + + Ku 32 29 + + Sc 2,119 69 — + HU ND 53 + + Lo 92 80 + + Ne 104 72 ND + We 657 17 — + +PLLWen HeNDC 4,61520 61@a —

— Th 8.014 63 ND + Huh 14,589 55 — + Ber136 492 35— — + @ a more than 10% of cells with bright cytoplasmic immunofluorescence induced. @ a morethan 10%of cellswith intensestaininginduced;thepercentageof acid phosphatase positive cells after TPA treatment was greater than 50% in 12 samples. C ND, not determined. di.. d Given are the cpm minus the control values, which were on the average 826 ± I210 cpm for all samples. TPA induced [3Hjthymidine incorporation in mononuclear cells from 2 healthy donors was 9250 ±8380 cpm. The responsive cases (Sc, He, Th, Huh) were those with significantly higher T-cell contamination L. (8.8% OKT3 positive cells) compared to the unresponsive cases (3.6% OKT3 positive cells, P < 0.05) (cf Table I). Fig. 1. Morphologyof a TPA treated CLL cell. 2174

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presence of TPA there was a striking increase of HD6 staining Table 4 TPA induction ofa tartrate resistant phosphatase as detected by cytochemistry as evidenced by FACS analysis (Fig. 2). TPA induced or en positivefor tartrate resist hanced HD6 staining of the CLL cells both in percentage of (%)CLL ant ACP positive cells (average, 34.5%) and in intensity (average median, 3.3) (Table 2). An increase by more than 10% of cells stained Type2)Uncultured ofcell cultureCells (n = 5) PLL (n = 0 for HD6 was seen in 8 of 10 CLLs and by more than 20% in 5 Controlcultured 1.4+ l.5a 1.5 +0.7 of 10 CLLs. Intensity increased by 3 channels or more in 5 of 20.5a TPA cultured0 70.8 + 9.6 67.5 +

10 cases. In PLL, where HD6 expression was stronger to begin Mean ±SD. with, staining decreased in most control culture samples (only acetone added). TPA induced an increase by 3 channels in one case and in another by 2 channels. Finally when analyzing the effect of TPA on PLL cells in comparison to control cultures there was an increase in HD6 staining induced by TPA in 3 of 4 samples. Thus TPA is able to induce or enhance HD6 staining in ,@ terms of both fluorescence intensity and percentage of positive -@ cells in most samples of CLL. PLL cells, with higher staining :.4. to begin with, show a culture induced decrease which is over come by the addition of TPA in some cases. Isotype control for IgGl demonstrated no induction of binding of 63D3 to TPA treated leukemia cells in that control cultured samples gave @ 0.5% positives with a median of —0.6andTPA cultures gave -—@ 0.2% positives with a median of 0.2 (n = 3). Acid Phosphatase Analysis after Culture with TPA. Since the morphology after TPA treatment and the induction of HD6 are suggestive of a phenotype of HCL, we studied an isoenzyme 3 characteristic of HCL, namely the tartrate resistant acid phos phatase. Acid phosphatase could be induced after TPA culture Fig. 3. Cytochemical staining for tartrate resistant ACP in a TPA cultured (Table 3) in all samples of CLL and in 3 of 4 PLL. In 12 of CLL. Strong perinuclear staining (arrow) is seen in a cell with “hairy―morphol ogy. these 13 cases the percentage of acid phosphatase positive cells after TPA exceeded 50%. Tartrate resistance was tested in cytochemistry in 5 samples of CLL and in 2 samples of PLL. In every instance the staining was found to be tartrate resistant (cf Table 4 and Fig. 3), while staining of normal mononuclear cells was inhibited by tartrate. The faint, scanty staining seen TR in uncultured samples of leukemic lymphoma was also sensitive to tartrate. Since there is more than one tartrate resistant isoenzyme of AcP ACP (18), we used IEF in order to investigate whether the enzyme induced by TPA in CLL and PLL and the ACP detected in HCL are identical with respect to isoelectric point. An

@ •1

G) @0 @@ E ‘ —@w―@- I z ?g@ .@\ @a b4 0 Fig. 4. Isoelectrofocusing of ACP from control and TPA cultured cells. The TPA cultured cells (Lane a) exhibit an additional ACP band in the basic (upper) portion of the gel, which is absent in control cultured cells (Lane b). TR ACP, FLuorescenceIntensity tartrate resistant ACP; I, ACP type I. Fig. 2. Effect ofTPA on cell surface staining with HD6 monoclonal antibody. Given is fluorescence intensity (linear scale) versus cell number. Superimposed curves on the left, staining of uncultured and control cultured cells; right curve, example of the IEF pattern induced by TPA in cells from a staining of the TPA treated cells (patient Be). case with PLL is given in Fig. 4. While ACP activity is detected 2175

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with a major band in the acidic lower portion of the gel in both DISCUSSION TPAandcontrolculturesamples,additionalstainingisinduced In the present report we have analyzed two types of leukemic by TPA in the basic portion (Fig. 4, Lane a). Further results B-cell lymphoma, i.e., CLL and PLL. The classification was are shown as schematic drawings (Fig. 5). In Fig. 5A two cases based on clinical findings, on cytomorphology, on distribution of CLL and one PLL are given. Again ACP activity with an IP and density of immunoglobulin, and on restriction to K or A of 4.8 to 6.8 is detected in control and TPA cultures, although chains. Isotype analysis ofslg demonstrated monoclonality with control cultured cells analyzed in cytochemistry never gave a respect to light chains in 9 cases. In S samples of CLL this significant ACP staining, in that only few cells (less than 5%) determination was not possible, which is reflective of the low contained a few faint granules. ACP activity with an IP of 9.0— slg density in CLL compared to PLL as quantitated by FACS 9.7 was found in TPA treated cells of the two CLLs and of one analysis. In order to avoid significant T-cell contamination in PLL.Intwoinstancesthecontrolculturedcellsalsoexhibited our samples, we preferentially used cases with high leukocyte a low degree ofthis activity (Fig. 5, Lanes 2, 5). The same bands counts, such that the average T-cell percentage was 5.1%. Still, at an IP of 9.0—9.7as in TPA induced CLL and PLL cells are analysis of [3Hjthymidine incorporation data revealed that the also found in uncultured samples from HCL (Fig. 5A, Lane 9). autologous T-cells might be somehow involved in proliferation No cytochemical staining for ACP and no band with a basic IP seen in some instances. Saiki et a!. (19) reported on the allo in IEF were detected with TPA culture cells from a centrocytic geneic T-cell dependence of CLL B-cell proliferation. Whether lymphoma and from purified T-cells from two donors (data not in our samples T-cells give help to the monoclonal B-cells to shown). We then tested the tartrate resistance of the ACP proliferate or proliferate themselves remains to be shown. Fur directly on the gel. Incubation of the gel in the presence of ther, it is not excluded that T-cells help in the TPA induced tartrate during the enzyme staining procedure prevented almost differentiation of leukemic B-cells. all staining of ACP in the range of IP 4.5—6.8(Fig. SB). In vitro differentiation in B-cell tumors can be induced by a By contrast, the bands at IP 9.0—9.7found in TPA treated variety of substances, e.g., lipopolysaccharide, poke-weed mi CLL and PLL cells and in uncultured HCL were virtually togen, and TPA (7, 20—23).In previous studies using TPA, it unaffected. These experiments directly demonstrate that the IP could be demonstrated that CLL cells can be driven to accu 9.0—9.7bands represent the tartrate resistant ACP isoenzyme mulate clg (heavy and light chain), to synthesize J-chain (24), characteristic of HCL. to increase mRNA for secretory 1gM over mRNA for membrane

A pH

In AcP .9

-8

.7 — — @ I — .--- — ..... —

I = @6 z = .5

.4 E:J D@ 2 EEJ E3@ EiJ EiJ @J EEJ E@ @-0―a@― Fig. 5. A, schematic drawing of isoelectro @ focusing analysis of ACP from 3 leukemic PIL I Qj : : H@. @ t———————IPAPU. ACPLL 0Qj AC T@@Lt@ ?C@L % samples. After culture with TPA an ACP with an IP of around pH 9 is induced; eventually a faint staining is also seen in control cultures. AC, acetone;for control the solvent for TPA was added to cultures. TR, tartrate resistant. B, direct demonstration of tartrate resistance of ACP on the gel. From a duplicate run of IEF one gel was developed without (left) and pH one with tartrate (right) in the reaction mix B ture. Only the band in the range of pH 9 is TR@ 9 AcP — — — preserved in the presence of tartrate. 8

7 I =

I = S

5 T = 4 @@@ D:J m rn cij: rn rn Origin Hc@ @L @LL 0 T@ T@ 2176

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1gM (25), and to increase the amount of mitochondria and tartrate resistant. endoplasmic reticulum. Further, morphologically plasmacytoid The detection also in control cultured CLL cells of some features and rare plasma cells were observed (7), suggesting tartrate resistant ACP activity by IEF but not by routine cyto maturation towards plasma cells. Our results, by contrast, dem chemistry underlines the power of the IEF approach for detec onstrate that CLL and PLL cells can be induced to acquire tion of this enzyme. The appearance of tartrate resistant ACP, hairy projections, suggesting maturation to HCL-type cells. however, is not a general phenomenon observed after culture The fact that, in spite of extensive studies with TPA in CLL, (±TPA)ofleukemicornonleukemiclymphocytes,sincenormal the hairy cell phenotype was not observed might be explained T-cells and a case of centrocytic-centroblastic lymphoma did by the low frequency of leukemic samples that show such a not exhibit this isoenzyme after such treatment. We also oh readily detectable .morphological change in stained smears. served isoenzymes with IP of 4.5—6.8in both TPA and control Support to our concept comes from the appearance of the HD6 cultures and eventually uncultured cells. This activity in our defined antigen after TPA induction of differentiation. The hands was not detected in cytochemical stains of control cul HD6 MAB CD22 was produced against HCL cells, defines a tures and uncultured cells (data not shown), which is probably cell surface molecule with an apparent molecular weight of also due to different sensitivities of the methods as discussed 130,000 and 140,000 (17), and most intensively stains HCL (12). but also, although less intensely, PLL cells, centrocyte derived Ofthe 14 cases in the present report only one was unrespon B-cell tumors, and a subpopulation of normal B-cells. No cell sive to TPA with respect to every parameter tested. Although surface staining of plasmacytomas is observed (16). In CLL this leukemia sample was a PLL, our data, which demonstrate only a few cases stain with this reagent in a low percentage of responsiveness in the other 3 PLL, do not support the conclu cells. In the present report we could detect by FACS some sion by Okamura et a!. (21) that PLL-type cells are poor additional cases with faint staining not detectable by conven responders to TPA. tional fluorescence microscopy. After culture in the presence of Our data demonstrate that the 2 types ofleukemic lymphoma TPA the cell surface staining for HD6 was either induced or studied are capable of acquiring some features of HCL. Thus enhanced in CLL and PLL in terms of both percentage of CLL and PLL are very similar with respect to the genetic positive cells and intensity of staining. It could be argued that program that they can realize in response to TPA and this the increase in intensity of staining as detected by FACS merely genetic program includes features of HCL. The assignment of reflects the increase in cell surface area with a constant density HCL to the B-cell lineage has been clearly established by the of binding sites. Although determinations of cell surface area presence ofintrinsic slg (e.g., Ref. 28) and by the demonstration have not been performed, our fluorescence microscopy read of immunoglobulin-gene rearrangement (29, 30). The precise ings, which were always done in parallel to FACS analysis, also location of this type of cell in a B-cell differentiation scheme, revealed an overt increase in intensity of staining, thus sup however, remains to be determined. Our data, showing the porting the assumption that TPA induces not only a higher conversion of CLL and PLL cells into HCL-type cells, would number but also a higher density of HD6 defined cell surface suggest a close relationship between these 3 types of lymphoma, molecules in CLL and PLL. a conclusion also supported by the earlier finding of a tartrate Two other MABs with a specificity similar to that of HD6, resistant ACP in cases of PLL (31). Thus HCL appears to be namely RFA-4 (26) and FMC7 (27), have been reported. Mo more closely related to other malignant transformations of the lecular weights of the antigens recognized are not published, B-cell lineage than previously thought. Previous studies (32) such that a comparison with HD6 remains incomplete. Induc have shown that TPA can induce morphological changes in tion of FMC7 has been reported in the study of a single case of HCLcells,whilesurfaceglycoproteinsstudiedin gelelectro CLL (23). This finding is in concert with our results; it was, phoresis did not change. It remains to be shown whether however, interpreted as maturation towards immunocytoma- or analysis with monoclonal antibodies and the use of other in PLL-type cells. Still, the appearance of a cell surface marker ducers ofdifferentiation reveal evidence for maturation of HCL which is absent from plasmacytoma cells argues against differ cells to plasma cell-type cells or whether HCL-type cells rep entiation to the level of the plasma cell. Accumulation of clg is resent mature cells of a separate branch of the B-cell lineage not in conflict with the concept of maturation towards HCL that includes cells of the CLL type. type cells, since some cases of HCL have been demonstrated to contain clg (5). ACKNOWLEDGMENTS Further support to our conept of induction of a phenotype similar to HCL comes from the isoenzyme studies. The cyto We acknowledgethe excellent technical assistance ofC. Wagenpfeil, chemical staining revealed the appearance, in the majority of E. B. Weiss, G. Scheffel, and A. Zimmer and the provision of patient cells in almost every sample tested, of a tartrate resistant acid specimens by Drs. Theml, Emmerich, and Siegert. phosphatase, as reported earlier (8, 9). Using IEF we were able to demonstrate the induction by TPA of an isoenzyme similar REFERENCES to HCL in the leukemic cells from CLL and PLL patients. This 1. Anderson, K. C., Bates, M. P., Slaughenhoupt, B. L., Pinkus, G. 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H. W. Löms Ziegler-Heitbrock, Reinhold Munker, Bernd Dörken, et al.

Cancer Res 1986;46:2172-2178.

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