Effect of Vincristine on Bone Marrow Cells of Patients With Multiple Myeloma

Virchows Arch [Cell Pathol] (1981) 35 : 239-248 V' &scsA /v B �9 Springer-Verlag 1981

Effect of Vincristine on Bone Marrow Cells of Patients with Multiple Myeloma

A Cytokinetic Study*

Alberto Riccardi 1, Agostino Martinotti 1, Carlo Maurizio Montecucco 1, Giuliano Mazzini 2, and Paolo Giordano 2 1 Istituto di Patologia Medica I and 2 Centro di Studio per l'Istochimica del C.N.R., Universit~t di Pavia, Italy

Summary. The cytokinetic changes induced by Vincristine (VCR) on bone marrow erythroblasts, myeloid cells and neoplastic plasma cells have been studied in four patients with plasma cell malignancies using combined DNA cytofluorometry and in vitro tritiated thymidine cytoautoradiography. The changes observed 9 h after the administration of the drug were in accordance with its S-phase specificity. The magnitude of the stathmokinetic effect was in fact roughly proportional to the proliferative activity of the different cell lines, i.e., marked on the erythroblasts, less evident on the myeloid cells and still lower on the plasma cells. In this last cell population VCR has also blocked or partially impaired the DNA synthesis. Nine days after VCR, the plasma cells were recruited into the proliferative cycle while the regeneration of the hemopoietic cells was already exhausted. Repeated administrations of VCR spaced at about 9 day intervals are more and more effective on the plasma cell population, since the S phase specificity of the drug against the recruited plasma cells is potentiated. On the contrary, the regeneration of the hemopoietic cells is protected by this time interval.

Key words: Myeloma - Vincristine - Cytoautoradiography - DNA cytofluorometry.

Introduction

In the last few years, we have used Vincristine (VCR) monochemotherapy (2 mg/ 7-15 days for 4-5 doses) for treating patients with multiple myeloma and examined the cytomorphologic changes induced by this drug on the bone marrow cells.

* Investigation supported by C.N.R. (Consiglio Nazionale delle Ricerche - Roma - Progetto Finalizzato Controllo della Crescita Neoplastica - Grant no. 78.02857.96)

Offprint requests to: Dr. Alberto Riccardi - Istituto di Patologia Medica I - Policlinico S. Matteo, 1-27100 Pavia, Italy

0340-6075/81/0035/0239/$02.00

240 A. Riccardi et al.

The stathmokinetic effect was constantly maximal on the erythroblasts, less evident on the myeloid cells and still lower on the plasma cells (Riccardi et al. 1980 a). However the definitive effect of therapy was almost constantly a decrease in the plasma cell mass (ranging from 30 to 51% in 4 of 6 courses) which paralleled the progressive increase in the amount of bone marrow hemopoietic cells and of the number of blood leucocytes and platelets (Riccardi et al. 1980 b).

From these observations one can agree well with the conclusions drawn by Camplejohn (1980) and by Klein (1980) that the mechanism of VCR cytotoxicity does not lie entirely in its stathmokinetic effect. The metaphase block in plasma cells was, in fact, too low to justify by itself the decrease in tumor mass of the myeloma.

Our cytomorphologic studies (Riccardi et al. 1980a) have suggested that plasma cell recruitment can play a role in explaining the effectiveness of VCR. The first administration of the drug probably causes, in addition to tumor reduction, reentry of dormant neoplastic plasma cells into proliferative cycle, so that the following VCR doses spaced at 7-15 day intervals are more and more effective on the recruited tumor cells. The regeneration of normal hemopoietic cells is protected with this schedule. In experimental studies (Young 1976; Wheeler and Alexander 1978) this regeneration has in fact been rapid and is almost complete during the 2-5 days following the administration of cytostatic agents (5-fluorouracil, methotrexate and arabinosylcytosine) which, like VCR, have short blood half-lives (Calabresi and Parks 1975).

This hypothesis is supported by the cytokinetic data obtained during myeloma therapy using combined DNA cytofluorometry and in vitro tritiated thymidine cytoautoradiography.

Materials and Methods

We studied 3 patients suffering from multiple myeloma and one with plasma cell leukemia treated with VCR (Table 1). The criteria for diagnosis and some pertinent data on their clinical course are given elsewhere (Riccardi et al. 1980b).

Case no 1 (Cas I) was treated with VCR in order to stabilize a Peptichemio (PTC)-induced partial remission. In cases no. 2-4 (Pis L, Feb L and Fil V) VCR was administered during relapse of the disease, since cytopenia and resistance to alkylation hampered the use of such drugs.

In cases no. 1 3 VCR (2 mg) was administered i.v. every 7-15 days for a total of 5 doses. Case no. 4 received a single dose (1.5 mg) of the drug.

The cytokinetic studies were carried out on plasma cells, erythroblasts and myeloid cells from bone marrow samples obtained before and at different times (9 and 24 h and 9 days) after starting VCR therapy, as indicated in the results described.

Table 1. Clinical data of 4 patients with plasma cell malignancies treated with Vincristine (PR= partial response)

Case Name Diagnosis Time of Time of Phase of diagnosis VCR therapy disease

1 Cas I 2 Pis L 3 Feb L 4 Fil V

Multiple myeloma, IgG, 2 Jan '73 Jul '76 PR Multiple myeloma, IgG, k Jan '72 Apr '75 Relapse Plasma cell leukemia, IgA, k Apr '74 May '78 Relapse Multiple myeloma, ). Jun '75 Sep '77 Relapse

Cytokinetics of Myeloma Cells After Vincristine 241

The following parameters have been evaluated: 1) the mitotic index (MI) ; 2) the tritiated thymidine labeling index (3 H-TdR LI) and 3) the growth fraction (GF). Also evaluated on the plasma cell population were: 4) the nuclear DNA content of both the whole population and of two groups of cells, respectively,

unlabeled and labeled with 3H-TdR and 5) the median grain count (MGC) of the 3H-TdR labeled cells. The MI was determined by examining 3,000 cells for each cell type on May-Gruenwald-Giemsa

stained imprints. The bone marrow cell labeling with 3H-TdR (Radiochemical Amersham Centre, specific activity

5 Ci/mM) was studied in vitro according to Foadi et al. (1968). The incubation time was 1 h. The autoradiographs were prepared using Kodak NTB 2 nuclear track emulsion and then exposed at 4~ for one week. The L! was determined by counting 4,000 cells for each cell line. The MGC was evaluated by examining 50 labeled plasma cells.

The GF of the plasma cell population was also evaluated in vitro on the same autoradiographs according to the method of Gavosto et al. (1967) and recently confirmed by Matera and Masera (1976). This parameter was determined on the samples obtained before and 9 days after the administration of VCR alone. The changes in the LI, observed a few (9-24) hours after VCR (upon which depend those of GF) are in fact not due to percentage variations of the cycling cells but probably reflect transitory impairment of their thymidine uptake (Tarocco et al. 1968; Lampkin and Nagao 1969).

The GF of the erythroblasts and of myeloid cells was determined on morphological grounds, as the percentage of basophilic + polycromatic erythroblasts and of myeloblasts + promyelocytes + myelocytes, respectively (Killmann 1968).

The nuclear DNA content of the plasma cells was evaluated cytofluorometrically by examining 200 randomly picked cells on Feulgen stained smears by using a Leitz MPV 2 microscope photometer. The technical characteristics of the instrumentation are detailed by Prenna et al. (1974) and in our previous papers (Riccardi et al. 1977a; Silvestrini et al. 1977).

The nuclear ploidy of the plasma cells was estimated on the basis of the mean nuclear DNA content of 50 neutrophil granulocytes in the blood of healthy subjects set as diploid value (Hale and Wilson 1959; Garcia 1964) and determined by examining blood smears obtained at the same time as the bone marrow smears.

For comparison of the DNA values obtained from bone marrow smears in various experiments, they were transformed as a function of a normal theoretic diploid value set equal to 10, according to the formula x=lO.c/Cm, where x is the transformed value, c is the experimental value and Cm is the arithmetic mean of the DNA values found for each experiment in the control neutrophils (Perugini et al. 1957).

The variability of the nuclear DNA content of the neoplastic plasma cells was then estimated on the basis of the frequency of the values included in the following classes: diploid (2n) values, within 10• 2 SD, where SD was the standard deviation relative to the normal neutrophils (which ranged from 0.28 to 0.37); tetraploid (4n) values, within 2 . (10• SD) and values between the 2n and 4n modes, considered as typical of cells in the DNA synthesis phase (2n-4n cells).

The DNA content of the 3H-TdR unlabeled and labeled plasma cells was evaluated by measuring 100 elements of each of the two groups identified on a photographic map according to Ernst and Killmann (1970). The percentage of cells in the early and late S phase was determined from the incidence of DNA values before and after the midpoint of the above determined 2n-4n interval.

The significance of the MGC variations was evaluated using the Student's t test after logarithmic transformation of the values obtained for every labeled cell. The significance of the variations of the other examined parameters was evaluated using the Z ~ method.

Results

The results obtained are summarized in Tables 2-4 and in Figs. 1-3.

Neoplas t ic P l a s m a Cells. T a b l e 2 s h o w s t h a t b e f o r e t h e r a p y the p r o l i f e r a t i v e

a c t i v i t y o f t he n e o p l a s t i c p l a s m a cells , as e x p r e s s e d b y t he v a l u e s o f t h e i r M I ,


Table 2. Mitotic index (MI), percentage incidence of the classes of DNA values, labeling index (LI), growth fraction (GF) and median grain count (MGC) in the plasma cell population of bone marrow of four patients with plasma cell malignancies treated with VCR (2n=diploid and 4n = tetraploid values)

Case Time MI Classes of DNA LI GF MGC ~ o values % %

2n 2 n ~ n 4n

l(Cas I) Before therapy 5.0 68 20 12 14.4 48 41 9 h after the 1st VCR dose 15.6" 51 32* 17" 8.2* - 22*

2(Pis L) Before therapy 3.6 95 5 0 3.5 19 42 9 h after the 1st VCR dose 20.4* 73 24* 3* 4.9* - 32* 9daysafter the l s tVCRdose 6.7* 88 9** 3** 7.1 ' 36* 50

3(Feb L) Before therapy 0 95 3 2 2.60 16 51 9 h after the 1st VCR dose 10.2" 90 7 3 2.55 - 19' 9 h after the 2nd VCR dose 14.3" 90 7 3 2.50 - 22*

24 h after the 4th VCR dose 8.3* 88 4 8 2.31 - 25* 9 days after the 5th VCR dose 2.7" 92 4 4 3.70" 64" 46

4(Fil V) Before therapy 0.8 93 5 2 4.3 19 51 9 days after the 1st VCR dose 1.7"* 89 8 3 6.8* 38* 40**

* P<0.01 and ** P<0.05 (as compared with the pretreatment value)

Table 3. Mitotic index (MI), labeling index (LI) and growth fraction (GF) in the bone marrow erythroblasts of four patients with plasma cell malignancies treated with VCR

Case Time MI LI GF ~ % %

l(Cas I) Before therapy 14.6 19.6 70 9 h after the 1st VCR dose 158.0" 16.2' -

2(Pis L) Before therapy 23.6 21.0 77 9 h after the 1st VCR dose 121.4" 18.2' - 9 days after the 1st VCR dose 25.6 21.2 65*

3(Feb L) Before therapy 11.2 23.5 70 9 h after the 1st VCR dose 160.5" 20.8* - 9 h after the 2nd VCR dose 181.3" 17.1 * -

24 h after the 4th VCR dose 225.7* 19.5" - 9 days after the 5th VCR dose 13.3 25.0 61 *

4(Fil V) Before therapy 18.9 25.3 66 9 days after the 1st VCR dose 19.9 27.2 54*

* P < 0.01 (as compared with the pretreatment values)

L I a n d G F , a n d b y t he p e r c e n t a g e o f 2 n - 4 n cells, w a s h i g h e s t in case no . 1

a n d m o r e m o d e s t in cases no . 2 -4 .

N i n e h o u r s a f t e r t he a d m i n i s t r a t i o n o f V C R , t he M I i n c r e a s e d to a s i m i l a r

e x t e n t in t h r e e o f t h e cases (no. 1 -3) e x a m i n e d .

T h e L I d e c r e a s e d in case no . 1, w a s a u g m e n t e d in case no . 2 a n d d i d n o t

c h a n g e in case no . 3. I n all cases t he a l i q u o t o f 2 n - 4 n p l a s m a cel ls i n c r e a s e d


Table 4. Mitotic index (MI), labeling index (LI) and growth fraction (GF) in the bone marrow myeloid cells of four patients with plasma cell malignancies treated with VCR

Case Time MI LI GF

~ % %

l (Cas I) Before therapy 8.3 9.2 51 9 h after the 1st VCR dose 33.6* 8.8 -

2(Pis L) Before therapy 7.3 8.0 51 9 h after the 1st VCR dose 29.6* 8.2 - 9 days after the 1st VCR dose 8.1 6.6** 46*

3(Feb L) Before therapy 8.9 8.8 80 9 h after the 1st VCR dose 26.7* 9.0 - 9 h after the 2nd VCR dose 29.8* 8.2 -

24 h after the 4th VCR dose 23.5* 13.0 ' - 9 days after the 5th VCR dose 8.3 8.1 63*

4(Fil V) Before therapy 9.3 12.1 70 9 days after the 1st VCR dose 10.1 13.3 60*

* P<0 .01 and ** P < 0 . 0 5 (as compared with the pretreatment values)

Fig. 1. Distribution of the nuclear D N A content of 3H-TdR unlabeled (D) and labeled ( i ) bone marrow plasma cells in case no. 1 (Cas I) (A = before and B = 9 h after the 1 st VCR dose ; 2n = diploid and 4n = tetraploid value)

Fig. 2. Distribution of the nuclear D N A content of 3H-TdR unlabeled (~) and labeled (m) bone marrow plasma cells in case no. 2 (Pis L) (A=before therapy; B = 9 h and C = 9 days after the 1st VCR dose; 2n = diploid and 4n = tetraploid value)

markedly, so that the difference between this parameter and the LI was augmented. Tetraploid nuclei were also more frequently observed than before thera-

PY. Figures 1-3 demonstrate the increased incidence of 3H-TdR unlabeled 2nMn

plasma cells (U-like cells) after VCR. The 3H-TdR labeled cells, which before


Fig. 3. Distribution of the nuclear DNA content of 3H-TdR unlabeled (n) and labeled (m) bone marrow plasma cells in case no. 3 (Feb L) (A=before therapy; B=9 h after the 1st VCR dose; C=9 h after the 2rid VCR dose; D=24 h after the 4th VCR dose; E=9 days after the 5th VCR dose; 2n= diploid and 4n= tetraploid value)

therapy were fairly evenly distributed in the 2n-4n interval, were much more frequent in the late S phase.

Similar findings were also observed 24 h after VCR (in case no. 3) (Fig. 3). At 24 h the incidence of U-like cells was however slightly lower than at 9 h after VCR.

Table 2 demonstrates that 9 days after the administration of VCR the proliferative activity was higher than before starting therapy in cases no. 2-4. The values of the MI and of the aliquot of 2n-4n cells, although lower than 9-24 h after VCR, were in fact increased as compared to the pretreatment values. The LI and the GF were similarly augmented. The U-like cells were almost absent and most of the 3H-TdR labeled cells were in the early S phase.

Erythroblasts. Table 3 shows that before therapy the mitotic activity of the erythroblasts was much higher than that of the neoplastic plasma cells.

The rise of the MI observed 9-24 h after VCR was distinctly more marked in erythroblasts than in the plasma cells. The LI of the erythroblasts was slightly decreased.

Nine days after VCR the values of the MI, LI and GF indicated a proliferative activity roughly similar to before therapy.


Myeloid Cells. Table 4 shows that before therapy the proliferative activity of the myeloid cells was intermediate between that of the plasma cells which was lower and that of the erythroblasts which was higher.

The increase in the MI observed 9-24 h after VCR was also intermediate between that observed in the other cell types. The changes in the LI were modest, as in the case of the erythroblasts.

Nine days after VCR the proliferative activity of the myeloid cells was similar to before treatment.

Discussion

The findings obtained 9 h after VCR firstly support the conclusion drawn by Frei et al. (1964) from a study of the bone marrow erythroblasts and myeloid cells, that the stathmokinetic effect of the drug on cell populations is roughly proportional to their proliferative activity. The increase in MI, due to the stathmokinetic effect (Riccardi et al. 1980a), has been in fact marked in the erythroblasts, more modest in the myeloid cells and still lower in the plasma cells, according to the difference in the size of their pretreatment LI. In cytologic studies (Riccardi et al. 1980 a) the magnitude of the stathmokinetic effect corre- lated well, in turn, with the cytotoxicity. The percentage of the bone marrow erythroblasts was in fact sharply decreased while the effect of the drug on the myeloid and plasma cells was less evident.

The data presented demonstrate, however, that VCR exerts two other effects on plasma cells, besides the stathmokinetic one. The drug completely inhibits or slows their DNA synthesis and recruits them into the proliferative cycle. The phase S-specificity (Wilkoff et al. 1968, Mauro and Madoc-Jones 1970) of VCR is therefore potentiated against the recruited plasma cells by spacing the single doses of the drug at about 9 days. On the contrary this treatment schedule spares the hemopoietic cells, whose regeneration is exhausted in this lag time.

The inhibition and slowing of DNA synthesis in the plasma cell population results from the findings observed 9-24 h after VCR. They show in fact (Figs. 1-3) increased frequency of U-like cells, that is, cells whose DNA synthesis is completely blocked. This accounts for the augmented difference between the aliquot of 2m4n cells and the LI after VCR already reported by Fried et al. (1976) in a case of histiocytic lymphoma, and by ourselves (Riccardi et al. 1977b) in three patients with non-lymphoblastic acute leukemias. Slowing of DNA synthesis in other plasma cells is suggested by the building up in the late S phase of labeled nuclei showing decreased MGC. This last parameter is not a clear cut measure of the DNA synthesis rate (Nicolini 1975) but can be accepted as indicative of its impairment in the context of the other findings (Shackney 1976). A cytotoxic effect of VCR on S phase cells has been recently documented also in C3H/He spontaneous mammary tumor (Schiffer et al. 1976; Braunschweiger and Schiffer 1978).

It is only a matter of showing that in the different patients the LI of the whole population can decrease, remain the same or increase according to the


relative aliquots of U-like cells (unlabeled) and of cells with slowed DNA synthesis (labeled). This was observed in our cases and in other (Brook et al. 1975; Pileri et al. 1976) cases of plasmocytoma treated with VCR.

The importance of the S phase damage on the overall cytocidal effect of VCR does not seem great. This action appears in fact limited to the cells which were engaged in DNA synthesis at the time of VCR administration. The scarcity, after VCR, of cells in early S phase (Figs. 1-3) indicates in fact that the transit G1/o-S is inhibited as already suggested by Tarocco et al. (1968) and by Lampkin and Nagao (1969) in acute lymphoblastic leukemia. It is therefore improbable that VCR damages the DNA synthesis of cells which were diploid (G1/0) at the time of the drug administration. If one considers finally that a part of the S phase cells reach mitosis, where they are killed by metaphase block, the number of plasma cells which are damaged directly in S phase is further reduced.

The findings observed 9 days after VCR demonstrate that the mitotic activity of the plasma cells was higher than before therapy while that of the hemopoietic cells was not modified. In cytologic researches (Riccardi et al. 1980a) we observed that in this time interval the bone marrow plasmocytomatous infiltrate was decreased, while the aliquot of hemopoietic cells was restored, having being reduced 9 h after VCR.

It can be deduced that plasma cells are recruited into the proliferative cycle following VCR-induced tumor mass reduction, as also documented after alkylating therapy (Pileri et al. 1976; Riccardi et al. 1978). On the contrary the regeneration of the hemopoietic cells is exhausted after having repopulated the bone marrow. In experimental studies (Young 1976; Wheeler and Alexander 1978) this has in fact occurred 2-5 days after the administration of cytostatic drugs (5-fluorouracil, methotrexate, arabinosylcytosine) which have short half life in the blood like VCR (Calabresi and Parks 1975). It is only necessary to note that the different chronology of the regenerative phenomena of the plasma cells on one hand and of hemopoietic cells on the other is accounted for by the different mean generation times of these cell populations. They are in fact long (48-192 h) in the plasma cells (Killmann et al. 1962; Harris and Hoelzer 1971; Pileri and Tarocco 1974; Drewinko and Alexanian 1977) and distinctly shorter (20-30 h) in the erythroblasts (Lajtha and Oliver 1960) and in myeloid cells (Cronkite 1968).

The above cytokinetic differences justify the concept that repeated VCR administrations spaced at about 9 days are more and more effective on the recruited plasma cells, since the drug is phase S-specific (Wilkoff et al. 1968; Mauro and Madoc-Jones 1970). This time lag on the contrary hampers hemopoietic cell damage during the phase of more intense regeneration. The progressive reduction of the bone marrow plasmocytomatous infiltrate allows the hemopoietic cell pool to increase over the pretreatment level.

This interpretation of the cytokinetic findings is strengthened by both clinical results - which demonstrate progressive reduction of the myeloma mass and improvement of the hematological conditions of the treated patients (Riccardi et al. 1980b), and by the cytologic data obtained in case no. 3, who experienced a progressive decrease in the bone marrow neoplastic infiltrate concomitant


wi th i nc rea s ing n u m b e r s o f e r y t h r o b l a s t s a n d m y e l o i d cel ls d u r i n g V C R m o n o -

c h e m o t h e r a p y ( R i c c a r d i et al. 1980 a).

W e c a n also a d d tha t an in t e rva l o f 7 -10 days b e t w e e n the s ingle V C R

a d m i n i s t r a t i o n s a p p e a r s to be c l in ica l ly a d v a n t a g e o u s as c o m p a r e d wi th l o n g e r

in tervals . W i t h this V C R schedule we a c h i e v e d pos i t ive resul ts in 3 o f 4 pa t i en t s

t r e a t ed d u r i n g re lapse o f the m y e l o m a , whi le the f o u r t h one , to w h o m V C R

was a d m i n i s t e r e d a t 15 day in te rva ls , e x p e r i e n c e d p r o g r e s s i o n o f the d isease

a f te r ini t ia l f a v o u r a b l e response . I t is c o n c e i v a b l e tha t in the first th ree cases

the p l a s m a cell r e c r u i t m e n t has p o t e n t i a t e d the e f fec t iveness o f the t h e r a p y

whi le in the f o u r t h o n e it was e x h a u s t e d at the t ime o f V C R a d m i n i s t r a t i o n .

Acknowledgments. We wish to thank Prof. Dante G. Scarpelli and Guido Goggi for helpful criticism and Mr. Giorgio Michelazzo for expert technical assistance.

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Received November 5, 1980 / Accepted November 27, 1980

Effect of Vincristine on Bone Marrow Cells of Patients With Multiple Myeloma

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