Scand J Haernatol 1985;35:543-549

Key words: myeloma - interferon - in vivo - in vitro

Correlation between in vitro and in vivo sensitivity to human leucocyte interferon in patients with multiple myeloma

G. Brenning,1.2, A. Ahre3 & K. Nilsson’

’Department of Internal Medicine, *Turnour Biology Group, Department of Pathology, University Hospital, Uppsala, and Wepartment of Internal Medicine, Danderyd Hospital, Danderyd, Sweden

26 of 32 patients with multiple myeloma (MM) were successfully tested in vitro for human leucocyte interferon (IFN) sensitivity by use of the human tumour stem cell assay (HTCA) and/or jH-thymidine incorporation (LI). Altogether, 12 patients were sensitive to IFN in vitro and 14 were resistant. All patients received treatment with leucocyte IFN. 8 patients were classified as responders, 3 as partial responders and 15 as non-responders. Correlating the in vitro/in vivo results, we found that the in vitro tests of the myeloma cells reflected a true sensitivity in 9 patients (75%) and a true resistance in 12 patients (86%). Furthermore, stimulation by IFN was found in 9 in vitro tests, of which 8 were obtained from non-responsive patients. Our results show that in vitro testing for IFN sensitivity is of clinical importance in predicting response to IFN treatment. Also, a stimulating effect by IFN in vitro will imply an unfavourable response in vivo.

Accepted for publication July 20, I985

Human leucocyte interferon of a-type (IFN) has been shown to exert anti-tumoural effects in humans (1-4). The Myeloma Group of Central Sweden (MCCS) has studied the clinical effect of IFN in patients with multiple myeloma (MM) since 1978 (5, 6, 7). In one of the clinical trials, 14% of the 75 patients, mainly IgA and BJ myeloma patients, responded to 3-6 x lo6 IU IFN/d. The response rate for IgG myeloma patients was only 5 Yo (6). Recently, a pilot study was initiated with IgA and BJ myeloma patients receiving 30 x lo6 IU IFN/d. However, as the patients tolerated this dose only for a few days

the therapy protocol had to be modified such that patients received 20 x 106 IU/d for 7 d repeated every 4th week. So far, response has been noted for 8/15 (53%) patients treated with 30 x lo6 IU IFN and for 4/15 (27%) receiving 20 x lo6 IU of IFN (7). This variation in response between different clinical IFN trials and the heterogeneity in response among individual patients in any of the therapy groups appears to reflect the individuality of human myelomas with respect to IFN sensitivity. It is obvious that there is a great need for methods to predict the IFN sensitivity in the individual patient with MM.

544 BRENNING ET AL

Simultaneously with the randomized clinical trials, a few predictive in vitro tests for MM tumour cell sensitivity have been introduced.

Hamburger & Salmon have developed an in vitro assay (HTCA) for human tumour stem cells that permits cloning in soft agar of several types of human tumour cells (8, 9, 10). The predictive ability of this technique has also been investigated. A correlation between the inhibition of tumour colony formation in vitro by cytotoxic anti- tumour drugs and the therapeutic response of those drugs in some tumour forms has in fact been documented (1 1 , 12). Experience with other tech- niques to test IFN sensitivity in vitro with respect to their capacity to predict in vivo response is limited. The test for IFN-induced inhibition of growth of myeloma cells in suspension as assayed by 3H-thymidine incorporation appears, however, to have a predictive potential ( 5 ) .

We have recently tested bone marrow samples from untreated melphalan-prednisone-treated and IFN-treated patients with MM for IFN sen- sitivity in the HTCA of Hamburger & Salmon (13). The sensitivity to IFN was individually dis- tributed between the bone marrow samples. Con- centrations of 400-4 000 IU/ml usually inhibited growth. Importantly, however, lower doses of IFN (10-100 IU/ml) sometimes stimulated growth.

The aim of this study was to investigate the predictive ability of the HTCA and the 3H-thy- midine incorporation technique measuring pro- fileration for patients with untreated MM enter- ing the clinical trials conducted by MGCS. In particular we wanted to see how the 2 in vitro methods for measuring IFN sensitivity/resistance compared in predictive precision. We also want- ed to investigate whether the previously observed

TABLE I Clinical data of 26 patients successfully tested f o r IFN sensitivity/resistance in vitro

Patients Age Sex Type Stage

I804 1888 1892 1983 1947 1

18614 18632 18673 I999 I994 2006 20002 1984 2010 2030 2034 2037 2040 2099 2108 2124 2129 2140 2192 2194 2196

59 70 78 60 41 58 62 72 56 79 82 80 71 66 66 80 62 39 69 62 50 43 82 82 71 64

M M F F F M F F M M F F M F F M F M F M M F M M M M

BJ A G A A BJ A G BJ A A A BJ A A A A A BJ A BJ A A A A A

111 A I1 A

111 A I11 A 11 A

111 A I 1 A I A

111 B I A I A I A

111 A 111 A

I A I11 A 111 A 111 A 111 A 111 A 111 A I1 A

I11 A 111 A I1 A I A

IFN treatment protocol

Time to clinical Clinical

response response (weeks)

A A A A A A A A B B B B C C C C C C C C C C C C C C

7 156

9 I 1 12 4

13 16 30 14 12 20 16 2 8 2 2

48 20 12

5 12 12 12 10 I2

NR PR NR PR NU NR NR NR NR NR R NR NU PR R R R NR NR R R NR NR R R NR

Numerals indicate patients who were tested during or after withdrawal of IFN treatment: 1 1 month; 22 months; 3 3 months; 1 month after withdrawal of IFN. Column for IFN treatment protocol see Methods.

MYELOMA IFN-a SENSITIVITY IN VITRO/IN VIVO 545

growth stimulation by IFN in vitro was associ- ated with non-response in vivo.

Material and methods Patients 26 of 32 patients with MM were successfully assayed for IFN sensitivity in vitro (Table 1). For the diagnosis of multiple myeloma, at least 2 of the following criteria had to be fulfilled (14):

I ) Monoclonal immunoglobulin (lg) in serum and/or urine with subnormal serum concentrations of at least one non-monoclonal immunoglobulin class; 2) > 10% plasma cells in bone marrow smears; 3) osteolytic and/or osteoporotic bone lesions compatible with MM.

The mean age of the patients was 65.5 yr (range 39- 82). The male to female ratio was 14/12. 3 patients had IgG, 18 IgA and 5 had BJ myeloma. Patients were staged clinically according to Durie & Salmon (15). 6 were in stage 1, 5 in stage I1 and 15 in stage I l l .

21 patients were tested before treatment and 5 patients after 1-3 months of IFN treatment or after withdrawal of IFN (Table 1).

The patients received human leucocyte IFN follow- ing 3 different therapy protocols. According to pro- tocol A, patients received 3-6 x 106 IU IFN/d. Higher IFN doses were given in treatment group B, i.e. 30 x lo6 IU IFN/d. However, the patients could tolerate this dose for only a few d. The dose was therefore changed to 20 x 106 1U/d. Following protocol C, 20 x 106 IU of IFN were given daily for 7 d repeated every 4th wk. Therapy was continued until progression of the disease (6) . Patients were evaluated for response every 4-6 wk in treatment protocol A and every 2-4 wk in treatment protocols B and C.

Clinical response was defined as follows: a) Responders (R) had a > 5 0 % reduction of the

M-component, a decrease of urinary Ig light chain excretion to less than 2 g/24 h and a decrease in tumour cell mass exceeding 50 To. In addition, patients were considered responders only if the Hb level was 2 90 g/l, the S-Albumin 2 30 g/l and the S-Calcium 5 2.6 mmol/l.

b) Partial responders (PR) had a reduction of the M-component in the serum or of the urinary Ig light chain excretion by at least 25% combined with improved physical condition such as a reduction or disappearance of bone pain.

c) Non-responders (NR) were all those patients not fulfilling the criteria for R or PR.

8 patients were classified as R, 3 as PR and 15 NR. Further clinical results are reported elsewhere ( 6 , 7).

Preparation of human myeloma cells The myeloma cells from bone marrow aspirations (1-2 ml) were separated as detailed (13). Briefly, single cell suspensions were prepared by forcing the marrow sam- ples through 25-gauge needles. The mononuclear cells were then isolated by Ficoll-Isopaque density gradient centrifugation (16), washed 3 times in phosphate buff- ered saline (PBS), examined for viability by the trypan blue exclusion method and counted in a Biirker cham- ber. The viability was 85-100%. Smears prepared by means of a Shandon cytocentrifuge were fixed and stained with Giemsa. The smears revealed that the cell population after separation consisted of 35-90 % plas- ma cells.

Interferon Partially purified leucocyte IFN with a specific activity of approximately 1 x lo6 IU/mg protein was supplied by Dr K. Cantell, Helsinki, Finland. The original IFN preparation contained 3 x 106 IU/ml. This stock solu- tion was further diluted with PBS and 10% FCS to suitable concentrations.

Soft agar techniques Cells were cultured in agar according to Hamburger & Salmon (8, 13, 17). Prior to the plating of 5 x 10’ cells, IFN was added at different final concentrations (40, 400, 4000 IU/ml). Triplicates were made for each concentration and for controls without interferon. The plates were examined with an inverted microscope immediately after plating to assure that a single cell suspension had been achieved. Cultures were then incu- bated at 37°C in 5 % CO, in humidified air. Final counts of colonies and clusters were made on d 12-14. Colonies were defined as aggregates of 30 or more cells and clusters as aggregates of 16-29 cells (13). The identification of the cells was performed as previously described (13). The mean value of the sum of colonies and clusters was recorded for the 3 control plates and for the plates with different final concentrations of interferon. At least 30 colonies-clusters/plate were required in the control plates to assure an adequate range for measurements of INF effect. Sensitivity to IFN was defined as a > 50% reduction of colony/ cluster growth in IFN containing plates compared to the control plates (17, 18). The definition of in vitro resistance was set when 5 50% reduction of colony/ cluster growth was found (19).

546 BRENNING ET AL

3H-thymidine labelling Tubes containing 5 x lo' bone marrow cells suspended in 1 ml RPMI 1640 + 10% FCS were cultured for 72 h without or with IFN (40, 400, 4000 IU/ml). Duplicates were made for controls and for each IFN con- centration. The cells were then incubated for 1 h at 37°C with tritiated thymidine (1 pC/ml. Specific activity 5 Ci/mmol, Amersham lnternational plk, Unit- ed Kingdom). Cytocentrifuge smears were thereafter prepared and autoradiography was performed (20). The percentage of labelled plasma cells (LI) was calcu- lated after counting 1000 plasma cells. Sensitivity to IFN was defined as a depression of LI by > 50%. Accordingly, when LI was less reduced the plasma cells were considered to be resistant.

Evaluation of in vivolin vitro sensitivity resistance In vitro sensitivity/resistance was evaluated at different concentrations of IFN in the agar culture system, and in the LI studies, depending on the in vivo dose given

to the patient from whom the bone marrow sample was obtained. For patients receiving 3-6 x 1 8 1U IFN (Group A), the equivalent in vitro dose was set at 400 IU and patients receiving 20-30 x 1 8 IU (Group B + C) were evaluated in vitro at 4000 IU/ml of IFN. The same dose limits were set for measuring the effect of IFN on LI.

Results

Soft agar cultures Myeloma cell colony formation was found in assays of 23/32 patients (72%). 10 assays con- tained < 30 colonies/clusters per control plate. Thus, IFN sensitivity could be evaluated in 13/32 HTCA assays (41 070). These cultures contained a median of 74 colonies/clusters (range 30- 164). 8 of the cultures showed IFN sensitivity. Resis-

TABLE 2 In vitro/in vivo sensitivity for 26 patients tested with HTCA and LI-measurements

In vitro response

Sensitive Resistant

Correlation in vitro/in vivo Patients Clinical

response H T C A LI H T C A LI H T C A LI

1983 1947 1867 2030 2037 2040 2099 2108 2192 1999 I994 2006 2000

1804 1888 I892 1861 1863 1984 2010 2034 2124 2129 2140 2194 2196

P R NR NR R R NR NR R R NR NR R NR

NR P R NR NR NR NR P R R R NR NR R NR

+ N T

+

+ +

+ N T

+

+ + + +

+ + + I + ' + + I

+ I

+

+

N T

+ I

+ ' + ' + I

+

+

+

+ N T + +

+ + + +

N T

N T +

+ + .t + + + +

+

+ + +

1 In vitro tests showing stimulation of growth by lower doses of IFN. N T not tested.

MYELOMA IFN-a SENSITIVITY 1N VITRO/IN VIVO 547

tance to IFN was found in 5 cultures, one of these showing stimulation of growth at 40 IU/ ml.

Labelling index studies 10 of the bone marrow samples with evaluable in vitro colony growth were also studied with 3H- thymidine incorporation. 13 additional bone marrow biopsies could be studied with LI mea- surements only. The median LI in the control cultures was 6.1 To (range 1.0-15.8). Taken together, in 10 of the 23 cases sensitivity to IFN (400-4000 IU/ml) was found. 13 samples show- ed IFN resistance, and, in 9 of these, IFN (40- 4000 IU/ml) stimulated growth measured as an increase in LI.

HTCA and LI measurements - correlation and combined results When comparing the 2 different in vitro methods a good correlation was found. At 9 out of 10 test occasions the same results with respect to sen- sitivity/resistance were obtained (Table 2). The exceptional bone marrow sample (2099) was sen- sitive to IFN in the soft agar system but resistant as measured by 3H-thymidine incorporation. Taken together, the 2 different methods provided evaluable in vitro results in 26/32 (81 Yo) of the tested patients. 12 patients were sensitive to IFN in vitro and 14 were resistant. Successful in vitro tests were obtained in 8/11 group A patients, 417 group B and 14/14 group C patients.

Correlation between in vitro and in vivo response The patients were clinically evaluated as 8 responders, 15 non-responders and 3 partial

responders (Tables 1 and 2). Patients with partial response were grouped together with responders. These 3 patients all had bone marrow cultures showing a reduction of colonies/cluster growth or depression of LI of between 26-76'7'0 (data not shown).

In non-responsive patients, not only in vitro resistance to IFN but also stimulation by IFN was found (Table 2). 8 non-responding patients had an in vitro assay showing stimulation at lower doses of IFN. However, an additional patient (2037) with in vitro stimulation measured by HTCA and LI responded in vivo to IFN treatment. This patient received 20 x I@ units of IFN/d for 1 wk. 1 wk later clinical response was evaluated and the patient was classified as a responder. She received treatment for 1 addi- tional wk and died of pneumonia 1 wk later. Bone marrow from this patient was tested before treatment and after 22 d. 4000 IU IFN reduced colony/cluster growth by 32% in the 1st and 88 Yo in the 2nd assay. On both occasions, labell- ing index was higher in cultures with IFN (all concentrations) than without.

Serial in vitro evaluation was performed in 3 non-responding patients before and during IFN treatment (2040, 1999, 1994). The bone marrow sample from patient 2040 tested before IFN treatment showed IFN stimulation measured by LI while the 2nd exhibited stimulation in both in vitro tests. The 1st assay from patient 1999 was moderately inhibited but the 2nd was stimulated by IFN. The 3rd patient (1994) was on both occasions sensitive in vitro but not responsive in vivo (data not shown).

The overall in vitro/in vivo correlation is shown in Table 3. In vitro sensitivity/resistance

TABLE 3 Overall in vitro/in vivo correlations in 26 patients with MM

ss SR RS RR

Sensitive both in vitro Sensitive in vitro Resistant in vitro Resistant both in vitro and in vivo but resistant in vivo but sensitive in vivo and in vivo

(true positive) (false positive) (false negative) (true negative)

Number of patients 9 3 2 12 Percent 75 % (43-96) 25 % 14% 86% (57-98)

~ ~~

95 To confidence limits within parentheses.

548 BRENNING ET AL

is presented utilizing a combination of the 2 techniques. True positive in vitro sensitivity was found in 75 To (9/12) and false in vitro sensitivity was found in 25% (3/12) of the patients. Resi- stance both in vitro and in vivo was found in 86% (12/14) of the patients, whereas 2 patients were false-negative in vitro (14%). One of the patients in the true negative group had deviating in vitro results by the 2 techniques and resistance in vivo correlated to resistance in vitro measured by LI. Nonetheless, 75 Yo sensitivity and 86 Yo resistance prediction allowed an association between in vitro and in vivo findings.

Discussion The HTCA has been shown to be useful for prediction of in vivo sensitivity to cytotoxic anti- tumour drugs for some different forms of human tumours including MM (11, 12). However, the frequency of valuable results is low and the test is time-consuming. The use of a radiolabelled thymidine incorporation assay for myeloma cells growing in suspension and exposed to IFN in vitro for 72 h was found to represent an additio- nal method to determine the cytostatic/cytotoxic effects of IFN ( 5 ) . Bone marrow samples show- ing no growth in HTCA could, by this method, be successfully evaluated in vitro.

We found a good overall correlation between the 2 investigated methods. However, one of the bone marrows showed differing results and in this case resistance meaured by LI corresponded to the clinical response. 41 070 of the patients could be successfully tested for IFN sensitivity in vitro by use of the soft agar culture and, utilizing the combination of the 2 methods, a total of 81 Yo of the patients could be evaluated in vitro.

Our definition of in vitro sensitivity to IFN was set to a more than 50% reduction of clono- genic growth and/or a corresponding depression of LI. Clinical response was furthermore defined mainly as a more than 50% reduction of the M-component. These definitions are in agree- ment with other reports concerning the effect of cytotoxic drugs and cloned human leucocyte IFN on tumour cells tested by the HTCA (14, 17, 19).

The definition of clinical response based on the reduction of the M-component is commonly used in clinical practice evaluating the effect of cytotoxic anti-tumour drugs. However, the mechanism(s) for the anti-tumour effect of IFN is presently unclear, demanding further investiga- tion of the biological effects of IFN on human myeloma cells.

In vitro sensitivity/resistance was evaluated at 400 IU/ml for patients from treatment group A and at 4000 IU/ml for patients from treatment group B + C. These concentrations were chosen based on determined serum IFN levels obtained from some of the investigated patients. Thus, the highest serum IFN concentration-yielded titre was 400 IU/ml from group A and 1600 IU/ml from group B + C (unpublished data). The in vitro dose limit of 4000 IU could be considered too high but the fact that 15 of 36 in vitro assays showed resistance at this level indicates that even at this concentration a heterogeneity of IFN sen- sitivity was present.

Importantly, stimulation by IFN was found in a total of 10 of the in vitro studies. A similar in vitro stimulating activity of IFN has been reported previously (13, 17, 21, 22). Our present results indicate that stimulation in vitro occurs mainly in non-responding patients, suggesting that in vitro stimulation by IFN could predict an unfavourable stimulatory in vivo response.

9 of our patients were sensitive to IFN in vitro and responded clinically to IFN treatment, implying a true predictive sensitivity in vitro of 75 Yo. The corresponding true resistance was 86%. Our material is small but our results are in agreement with the results on patients with MM presented by Durie et a1 (18). Using HTCA, the true predictive cytotoxic drug sensitivity was 73% and the true resistance was 83%. Similar results have been reported for various other tumours tested by the colony assay (19).

In conclusion, it was possible to evaluate in vitro IFN sensitivity in 81 Vo of the patients using either the HTCA or LI assays or a combination of the 2 methods. In 75% of the patients, a favourable response, and in 86% a poor clinical response could be predicted in vitro. Further-

MYELOMA IFN-a SENSITIVITY IN VITROIIN VlVO 549

more, stimulation by IFN in vitro indicated an unfavourable in vivo response. Our results imply that in vitro testing is of importance in clinical practice. Unfortunately, in vitro tests do not provide valuable results in all patients and they are time-consuming. Only by improving the in vitro methods and by further investigation of the biological anti-tumour effects of IFN can clinical trials be accurately performed in the future.

Acknowledgements This study, a report from the Myeloma Group of Central Sweden (MGCS), was supported by grants from the Swedish Cancer Society. We thank Aino Ruusala for excellent technical assistance.

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Correspondence to: Gunilla Brenning, M.D. Department of Internal Medicine University Hospital S-751 85 Uppsala Sweden