HEMATOLOGICALONCOLOGY, VOL. 10,213-220 (1992)

EXPRESSION OF p170 PROTEIN IN MULTIPLE MYELOMA: A CLINICAL STUDY GIOVANNI UCCI, MARIO PETRINI*, ALBERTO RICCARDI, ROSANGELA INVERNIZZI, GIOVANNI CARULLI*,

RENATA LUONI, MONICA GIORDANO AND MARC0 DANOVA

Clinica Medica II, Dipartimenro di Medicinu Iniernu e Terupiu Medica, Universita di Pavia andlstituto di Ricovero e Cura (1 Carattere Scientific0 Policlinico S. Matteo, 27100 Pavia, and *Clinics Medica, Sezione di Ematologiu. Universita di Pisa. 56100 Pisa, Italy

SUMMARY The expression of the p 170 multidrug resistance protein by bone marrow plasma cells (BMPC) was assessed at clinical presentation in 53 patients with multiple myeloma (MM) using the C219 monoclonal antibody. Twenty-two of the 53 (41 per cent) patients had variable aliquots (1-60 per cent, median=6 per cent) of pi70 BMPC by immunocytochemistry. Five of 10 patients studied using bivariate flow cytometry had both diploid and hyperdiploid (DNA index ranged from 1.2 to 1.5) BMPC with hyperdiploid clones having significantly greater pl70 expression than diploid ones. Of the 37 patients evaluated for a response, 20 (54 per cent) had responded to induction chemotherapy. The presence of p170+ BMPC was a negative indicator for achieving response. The response rate was 75 per cent for p170 - and 25 per cent for p170+ cases ($ < 0.01), with no difference o n the basis of treatment schedule (melphalan and prednisone, 24 patients; peptichemio, vincristine and prednisone, 13 patients). No difference in response and survival duration was found between p170+ and p170- paticnts. In six of nine patients studied both at diagnosis and following induction chemotherapy the p170+ BMPC% increased irrespective of the type of treatment or outcome.

+

KEY WORDS

p170

Myeloma

Therapy

INTRODUCTION The p 170 glycoprotein (coded by the mdr- 1 gene on the long arm of chromosome 7) probably plays a role in the multidrug resistance (MDR) observed in tumour cells both in vitro and in vivo.’V2In fact, p170 is able to actively transport from a cell a number of widely used and structurally unrelated cytostatics, including the vinca alkaloids and the anthracyclines, thus protecting cells from damage. A small number of retrospective studies also suggest that there is a correlation between the expression of the pl70 glycoprotein and the resistance to cytostatics in both hematologic and non-hematologic human but the overall clinical relevance of this glycoprotein is unclear at present. We have assessed the clinical significance of pl70 expression in a prospective series of 53 patients with multiple myeloma (MM) at presentation, who were treated for induction using drug combinations one of which also included vincristine (an active substrate of p170). In 10 patients, the Research supported by C.N.R. (Consiglio Nazionale delle Ricerche, Roma, Progetto Finalizzato Oncologia, grant no. 88.00841.44), by M.U.R.S.T. (Minister0 dell’Universitae della Ricerca Scientificae Tecnologica, 40 and 60 per cent funding), by A.I.R.C. (AssociazioneItaliana per la Ricerca sul Cancro, Milano) and by I.R.C.C.S.Policlinico S. Matteo (Istituto di Ricovero e Cura a Carattere Scientific0Policlinico San Matteo, Pavia). Addressee for correspondence: Dr Giovanni Ucci, Clinica Medica 11, Policlinico S. Matteo, 27 100 Pavia, Italy.

0278-0232/92/040213-08 $09.00 0 1992 by John Wiley & Sons, Ltd.

Received January 1992 Revised February 1992

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study was integrated by analysing pl70 expression versus DNA content using bivariate flow cytometry (FCM). MATERIALS AND METHODS Fifty-three patients (28 male and 25 female, median age = 61 years) with untreated MM diagnosed between June 1986 and September 1990 were entered in the study (Table 1). p170 glycoprotein expression by BMPC was determined using the C219 monoclonal antibody and an immunocytochemical technique. In 10 patients p170 expression and DNA content of BMPC were simultaneously determined by bivariate FCM. A second immunocytochemical evaluation was in addition performed during the course of the disease in two untreated as well as in nine treated patients following induction chemotherapy.

Diagnosis, treatment and response Plan of study and treatment of MM is detailed elsewhere." Briefly, the diagnosis of MM required the presence of at least two of the following three features: (a) a serum and/or urine monoclonal component (MC); (b) a BMPC% greater than 20 on the trephine biopsy; (c) the presence of osteolytic lesions on plain skeletal X-ray. Induction was decided on the basis of stage" of the disease at diagnosis. Stage I patients were randomized to receive either no treatment (1 3 patients) or courses of melphalan (MPH) and prednisone (P) (MPH-P) (eight patients). All 11 stage 11 patients received MPH-P while stage I11 patients were randomized to receive either MPH-P (7 patients) or the association of peptichemio (PTC), vincristine (VCR) and P (PTC-VCR-P) (14 patients). A total of 40 of the 53 patients received cytostatics. Response to treatment was assessed after six courses of chemotherapy and defined as complete or partial ( > 50 per cent versus 25-50 per cent reduction of the initial MC level, respectively, in addition to stable or improved clinical and laboratory features)." Patients with an unchanged MC level and stable clinical features were classified as unresponsive and those with >25 per cent increase of the initial MC level and/or worsening of the clinical, laboratory or radiologic picture were considered to have progressive disease. Median follow-up of patients is 31 months. Immunocytochemicalstaining Cytospins of BM cells obtained by sternal aspiration, were prepared for each case after separation on a Ficoll-Hypaque density gradient. These were then air dried for 1 h at room temperature and stored at -20°C. Slides were thawed every 3-6 months and fixed in cold acetone for 10 min, air dried for 15-30 min and then processed with 1:lO diluted C219 monoclonal antibody (MoAb; p-glycoCHEKTM, Centrocor Inc., Malvern PA, U.S.A.) which binds an epitope of the p170 glycoprotein at the inner aspect of the cell membrane. Further processing was according to the APAAP method13using rabbit anti-mouse and APAAP mouse complex (Dakopatts, Denmark) employed at a final dilution of 1 :30 for 30 min. The slides were finally counterstained with Mayer's hematoxylin for 30 min. Bivariate flow cytometry Ten patients had BM samples available for bivariate FCM, using a method described in detail e1~ewhere.l~ Briefly, the unicellular BM cell suspension obtained by density separation was fixed in 70 per cent ethanol and stored at + 4°C. At time of analysis, cells were washed, resuspended in PBS and incubated with C219 MoAb (final dilution of 1:lOO) for 30 min at room temperature. This

MDR IN MULTIPLE MYELOMA

215

MoAb was chosen for FCM analysis since it seems the most suitable both for the detection of pl70 positive cells and for the simultaneous evaluation of pl70 expression and DNA ~ 0 n t e n t . l ~ After further washings, cells were exposed to 1 5 0 diluted goat anti-mouse FITC conjugated MoAb for 30 min at room temperature and finally suspended in propidium iodide (PI, 5 pg/ml) isotonic solution. In univariate FCM, PI red fluorescence (nuclear DNA) was plotted versus the number of cells measured. In bivariate FCM, green (FITC-C219) and red (PI) fluorescence were simultaneously detected by means of a FACS Star flow cytofluorometer (Becton Dickinson). DNA content was expressed as a DNA index calculated by the ratio between the median channel of the main peak of the BM sample under evaluation and that of normal peripheral blood lymphocytes, used as an internal reference standard. Controls and sample evaluation For both immunocytochemistry and bivariate FCM, the specificity of C219 staining was assessed in two steps. First, human colon carcinoma cell lines were immunostained. These were either resistant to doxorubicin (Lovo D x ) ' ~and C219 positive or sensitive (Lovo) to doxorubicin and C219 negative. Second, in each MM case negative controls were prepared by incubating the BM sample with an isotype matched aspecific normal mouse immunoglobulin (IgG2a) in place of the primary antibody. To evaluate BMPC positivity on imprints, slides were scored by two distinct observers who counted at least 200 BMPC. C219 positivity intensity was scored from 0 to 3 using the Lovo DX positivity as a reference. Only BMPC scoring 2 or 3 either on the membrane or within the Golgi body (or both) were counted as pl70 positive. To evaluate the FCM positivity intensity, the mean pl70 green fluorescence was measured in each case and expressed as arbitrary units (AU) following subtraction of the mean fluorescence of the MM negative control.

Statistical methods The different incidence of p170 expression among groups of patients was evaluated by means of Fisher's exact test. Differences in the percentage of p170+ BMPC among groups of patients was evaluated by a non-parametric test (Wilcoxon's rank test) which was also employed to compare green fluorescence (in arbitrary units) of the diploid peak with that of the hyperdiploid peak in aneuploid cases. Difference in response and survival duration on the basis of pl70 expression were determined by the log-rank test. RESULTS The obtained results are summarized in Table 1. Expression of p170 at diagnosis Using the C219 immunocytochemical technique p170 expression by BMPC was noted in 22 of the 53 (41 per cent) patients. The aliquot ofpl70 + BMPC ranged from 1 to 60 per cent (median = 6 per cent). Positivity was uniformly distributed on the surface of the cell membrane in most cases, while intense staining of the Golgi bodies was observed less frequently. The presence and the aliquot of p 170+ BMPC was not related to age, sex, MC type or BMPC%. pl70 was not detected in normal hemopoietic cells. Thirty-seven of the 40 patients who received chemotherapy were evaluated for response (Table 1). Five of the 20 (25 per cent) responsive and 13 of the 17 (76 per cent) unresponsive patients had

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G. UCCI ETAL.

Table 1. Expression of p170 glycoprotein in 53 patients with multiple myeloma at diagnosis in relation to their clinical features

Total [no. of pts. (%)I Stage I

I1 I11 IgG

IgA Micro n.s. Not treated

Treated PTC-VCR-P MPH-P Responsive Unresponsive

p170f

~170-

22 (41)

31 (59)

9 (43) 7 (64) 6 (29) 19 (52) 3 (25)

12 (57) 4 (46) 15 (71) I7 (48) 9 (75) 4 (100) l(lO0) 10 (77) 21 (53) 11 (79) 10 (39) 15 (75) 4(24)

-

3 (23) 19 (47) 3 (21) 16(61) 5 (25) 13 (76)

P

n.s.

n.s.

t0.01

PTC:peptichemio; VCR: vincristine; P: prednisone; MPH: melphalan. Three patients not evaluable for response.

p170+ BMPC (p > 0.01). p170 positivity was similar between the 26 patients treated with MPH-P and the 14 patients treated with PTC-VCR-P. Among p170+ patients, p170+ BMPC% was not different between responders (median 8 per cent) and non-responders (median 8.5 per cent). Response duration as well as overall survival were not influenced by the presence or the absence of p 170 BMPC. Median response duration was 15 months for p 170 patients and 13months for p 170- ones while median survival was 47 months and 56 + months, respectively.

+

+

Bivariate flow cytornetry Five out of 10 patients evaluated at presentation by bivariate FCM had both diploid and hyperdiploid clones with DI ranging from 1.2 to 1.5 (median= 1.3). Patients with diploid clones had a median C219 green fluorescence of 60.4 (57-70) AU, while patients with both diploid and hyperdiploid clones, the median C219 green fluorescence was 49.0 (38-55) AU for the diploid and 64 (52-73) AU for the aneuploid peaks (p < 0.05) (Figure 1). Changes in the p170 expression during follow-up In two stage I patients who received no chemotherapy, BMPC remained p170- at 14 and 18 months following diagnosis. In six of the nine treated patients who were studied both at diagnosis and at the time of response evaluation there was an increase (from 0-10 to 6 1 0 0 ) in the p170+ BMPC%. Of these, two were responsive to PTC-VCR-P while one was responsive and three unresponsive to MPH-P. In one MPH-P-resistant patient, the p170+ BMPC% further increased (from 9 to 42) when a response was achieved with PTC-VCR-P. The p170+ BMPC% was unchanged in one patient who responded to PTC-VCR-P and decreased (from 2-20 to 0-1 3) in two patients responsive to MPH-P.

217

MDR IN MULTIPLE MYELOMA

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DISCUSSION In this prospective study, MM patients having variable aliquots of p170+ plasma cells in their bone marrow were distinctly more resistant to induction chemotherapy than patients whose BMPC did not express p170. This is in keeping with similar observations in hematologic4739317 as well as solid3,' tumours. The main interest in relating p 170 expression by tumour cells with response to chemotherapy is that there is no other biologic parameter that is both easily evaluable and predictive of response to induction treatment. For example, a low proliferative activity of tumour cells is linked with poor response in MM" and acute leukemia (AL),19*20 but traditional cell kinetics is usually difficult to determine. Hyperdiploid clones are easily identified with DNA FCM in MM at diagnosis and, more frequently, late in the course of MM,2' but their relevance to response has not been settled.

218

G. UCCI ETAL.

Both ours and other^'^ experience with dual parameter analysis of cells using FITC-C219 fluorescence have demonstrated that hyperdiploid clones have greater p 170 expression than diploid ones (Figure I). This aspect needs further consideration since a higher p170 expression has been linked with greater resistance to cytostatic drugs2*and, possibly, a worse prognosis' in soft tissue sarcomas. p170 expression lowered response rate irrespective of the inclusion or exclusion of VCR, which, of the cytostatics used in this study, is the only one proven to be actively transported from the cell by p170. Elucidating this point is intriguing since VCR cannot be assumed to play a major role in the induction of response in MM, particularly when used in association with more effective alkylating drugs. Therefore, it may be possible that p170 also plays a role in inducing resistance to MPH or to PTC. However, resistance to alkylating agents seems to be independent of p170 expression and possibly attributed to altered glutathione metabolism and increased levels of glutathione transferase in resistant ~ e l l s . * ~On * * ~the other hand, p 170 and glutathione-based detoxifying enzymes are expressed simultaneously in the same ~ e l l s .Furthermore, ~~,~~ C2 19 MoAb reacts with a highly conserved amino-acid sequence common to all MDR proteins, including the isoform 3, whose role in drug resistance is debated at the p r e ~ e n t . A ~ ~working - ~ ~ hypothesis might be that the expression of the p170 glycoprotein not only indicates resistance to specifically transported drugs but represents a marker of a more malignant and consequently drug-resistant phenotype. Duration of response and survival were similar in MM patients with and without p I70 + plasma cells, while it has been reported to be distinctly shorter in p170+ than in p170- AL patient^.'^^^' This is easily explained by the different clinical significance that response has in MM versus AL. In fact, in MM response is an operational condition mainly defined by the reduction in MC, while the disease is usually still present and its course might be further influenced by effective rescue strategies. On the contrary, obtaining response is the major factor favouring survival in AL. In fact, in AL the true relevance of both maintenance in responsive patients and of salvage regimens in refractory and relapsing patients is strongly questioned.32 A further biological finding with possible clinical relevance is that the aliquot of p170+ BMPC increased in the majority of patients following induction chemotherapy, independent of the type of treatment or response. The selection of p170+ BMPC by chemotherapy raises two issues. First, it casts doubts on the effectiveness of cytostatic maintenance therapy in MM, which could not only be clinically irrelevant,33but also biologically dangerous. In fact, continuous maintenance in responsive cases might contribute to the selection of p170+ BMPC clones which are potentially refractory to salvage chemotherapy. Second, attempts to overcome pl70-linked resistafice during the early stages of therapy might be justified in order to destroy all possibly resistant cells. This can be accomplished either by pl70 or by increased dosages of cytostatics coupled with rescue strategies such as hemopoietic growth factors and RM t r a n ~ p l a n t a t i o n . ~ ~ REFERENCES 1. Carulli, G., Petrini, M. Multidrug resistance: focus in hematology.Huemutologicu 1990,75,363-374. 2. Nooter, K., Herweijer, H. Multidrug resistance (mdr) genes in human cancer. Br. J . Cancer 1991, 63, 663-669. 3. Ozols, R. F., Cunnion, R. E., Klecker, R. W., et ul. Verapamil and adriamycin in the treatment of drug-resistant ovarian cancer patients. J. Clin. Oncol. 1987,5,641447. 4. Epstein, J., Xiao, H., Oba, B. K . P-glycoproteinexpression in plasma cell myeloma is associated with resistance to VAD. Blood 1989,74,913-917. 5. Dalton, W. S., Grogan, T. M., Meltzer, P. S. et uI. Drug-resistance in multiple myeloma and non-

Hodgkin's lymphoma: detection of P-glycoproteinand potentialcircumventionby addition o f verapamil to chemotherapy. J . Clin Oncol. 1989,7,415424.

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6. Carulli, G., Petrini, M., Marini, A,, et al. P-glycoprotein expression in multiple myeloma. Haernatologica 1990,75,288-290. 7. Carulli, G., Petrini, M., Marini, A., Vaglini, F., Caracciolo, F., Grassi, B. P-glycoprotein and drug resistance in acute leukemias and in the blastic crisis of chronic myeloid leukemia. Haernatologica 1990, 75,516-521. X. Chan, H. S. L., Thorner, P. S., Haddad, G., Ling, V. lmmunohistochemical detection of p-glycoprotein: prognostic correlation in soft tissuc sarcoma of childhood. J . Clin. Oncol. 1990,8,689-704. 9. Miller, T. P., Grogan, T. M., Dalton, W. S., Spier, C. M., Scheper, R. J., Salmon, S. E. P-glycoprotein expression in malignant lymphoma and reversal of clinical drug resistance with chemotherapy plus highdose verapamil. J . Clin. Oncol. 1991,9, 17-24. 10. Riccardi, A,, Ucci, G., Luoni, R., Cassano, E., Castello, A., Ascari, E. Multiple myeloma: a treatment program which includes stratification of patients into three disease stage and randomizations for both induction and maintenance therapy. Basic data on patients recruited between January, 1987, and May, 1988. In: Caronia, F., Malleo, C., Marceno, R., eds. Ernatologia '88, Atti I1 Meeting Internazionale 'Ernatologia 'M',Ustica 26 Giugno-3 Luglio, 1988. Bologna: Editoriale Grasso, 1990: 8 15-832. 11. Durie, B. G. M., Salmon, S. E. Aclinical staging system for multiple myeloma. Cancer 1975,36,842-854. 12. Cohen, H. J., Silberman, H. R., Larsen, W. E., Johnson, L., Bartolucci, A. A., Durant, J. R. for the Southeastcrn Cancer Study Group. Combination chemotherapy with intermittent I -3(bis-2chloroethy1)-1 mitrosurea (BCNU), cyclophosphamide and prednisone for multiple myeloma. Blood 1979,54,824836. 13. Cordell, J. L., Falini, B., Erber, W. N., et a/. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP) complexes. J . Histochem. Cytochem. 1984,32,219-229. 14. Danova, M., Giordano, M., Erba, E., et al. Flow cytometric analysis of multidrug resistance associated antigen (p-glycoprotein) and DNA ploidy in human colon cancer. J. Cancer Res. Clin. Oncol. 1992, in press. 15. Krishnan, A., Sauerteig, A., Stein, J. H. Comparison of three commercially available antibodies for flow cytometric monitoring of P-glycoprotein expression in tumor cells. Cyfometry 1991, 12,731-742. 16. Grandi, M., Geroni, C., Giuliani, F. C. Isolation and characterization of a human colon adenocarcinoma cell line resistant to doxorubicin. Br. J . Cancer 1986,54,515-521. 17. Riccardi, A., Invernizzi, R., Ucci, G. et al. La p170 nel mieloma multiplo e nelle leucemie acute. Haematologica 1991,76(suppl3), 177-180. IS. Riccardi, A., Montecucco, C. M., Danova, M., Ucci, G., Merlini, G. P., Ascari, E. Rate of M-component changes and plasma cell labeling index in 25 patients with multiple myeloma treated with Peptichemio. Cancer Treat. Rep. 1985,69,971-975. 19. Riccardi, A., Danova, M., Montecucco, C . M., et al. Acute non lymphoblastic leukemia: reliability and prognostic significance of bone marrow S phase size determined with propidium iodide DNA flow cytofluorometry. Scand. J . Haernatol. 1986,36, 11-17. 20. Riccardi, A., Giordano, M., Danova, M., et al. Cell kinetics with in vivo bromodeoxyuridine and flow cytometry: clinical significance in acute non lymphoblastic leukemia. E m . J . Cancer 1991,27,882-887. 21. Montecucco, C. M., Riccardi, A,, Merlini, G. P., et al. Plasma cell DNA content in multiple myeloma and related paraproteinemic disorders. Relationship with clinical and cytokinetic features. Eur. J. Cancer Clin. Oncol. 1984,20,81-89. 22. Chan, H. S. L., Bradley, Thorner, P. S., Haddad, G., Gallie, B. L., Ling, V. A sensitive method for immunocytochemical detection of p-glycoprotein in multidrug resistant human ovarian carcinoma cell lines. Lab. Invest. 1988,59,87&875. 23. Green, J. A., Vistica, D. T., Young, R. C., Hamilton, T. C . , Rogan, A. M., Ozols, R. F. Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion. Cancer Res. 1984,44, 5427-543 I . 24. Hayes, J. D., Wolf, C. R. Molecular mechanisms of drug resistance. Biochern. J . 1990,272,281 -295. 25. Batist, G., Tulpulc, A., Sinha, B. K., Kati, A. G., Meyers, C. E., Cowan, K. H. Overexpression of a novcl anionic glutathione s-transpherase in muitidrug resistant human breast cancer cells. J . Biol. Chern. 1986, 261,15441559. 26. Redmond, S. M. S., Joncourt, F., Buser, K., et al. Assessment of p-glycoprotein, glutathione-based detoxifying cnzymes and 06-alkylguanine-DNA alkyltransferase as potential indicators of constitutive drug resistance in human colorectal tumors. Cancer Res. 1991,51,2092-2097.

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G . UCCI E T A L .

27. Georges, E., Bradley, G., Gariepy, J., Ling, V. Detection of p-glycoprotein isoforms by gene specific monoclonal antibodies. Proc. Natl. Acad. Sci. U . S . A . 1990,87, 152-1 56. 28. Nooter, K., Sonneveld, P., Janssen, A., ei al. Expression of MDR 3 gene in prolymphocytic leukemia: association with cyclosporine A induced increase in drug accumulation. Int. J. Cancer 1990,45,626-63 1. 29. Herweijer, H., Sonneveld, P., Boos, F., Nooter, K. Expression of MDR 1 and MDR 3 multidrug resistance genes in human acute and chronic leukemias and association with stimulation of drug accumulation by cyclosporine. J. Natl. Cancer Inst. 1990,82, 1133-1 140. 30. Sato, H., Preisler, H., Day, R., ei al. MDRl transcript levels as an indication of resistant disease in acute myelogenous leukemia. Br. J. Haematol. 1990,75,34&345. 31. Musto, P., Melillo, L., Lombardi, G., Matera, R., DiGiorgio, G., Carotenuto, M. High risk of early resistant relapse for leukemic patients with presence of multidrug resistance associated P-glycoprotein positive cells in complete remission. Br. J. Haematol. 1991,77,50-53. 32. Riccardi, A., Giordano, M., Girino, M. Treatment of adult acute non lymphoblastic leukemia: a computer-aided analysis. Haematologica 1987,72,71-88. 33. Belch, A., Shelley, W., Bergsagel, D., et al. A randomized trial of maintenance versus no maintenance melphalan and prednisone in responding multiple myeloma patients. Er. J. Cancer 1988,57,94-99. 34. Chabner, B. A., Wilson, W. Reversal of multidrug resistance. J. Clin. Oncol. 1991,9,4-6. 35. Salmon, S. E., Dalton, W. S., Grogan, T. M., et al. Multidrug resistant myeloma: laboratory and clinical effects of verapamil as chemosensitizer. Blood 1991,78,44-50. 36. Gianni, A. M., Siena, S., Bregni, M., et al. Granulocyte macrophage colony stimulating factor to harvest circulating hemopoietic stem cells for autotransplantation. Lancet 1989, ii, 580-585.