An Assay for Monitoring Response to Therapy in Cancer Patients*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 23, No. 3 Copyright 1993, Institute for Clinical Science, Inc. An Assay for Monitoring Response to Therapy in Cancer Patients* CALVIN C. W ILH ID E, P h.d.tt and LYNDON L. LARCOM, P h.d.«department o f Oncology,t Johns Hopkins School o f Medicine, Baltimore, MD and Departments o f Microbiologyt and Physics and Astronomy Clemson University, Clemson, SC ABSTRACT Im m unosuppression is characteristic of patients with advanced m alignant neoplasms. However, none of the simple assays for immunocompetence have been found to provide results which correlate with the patients responses to therapy. The data reported here indicate that this may be because there are artifacts in data from earlier studies which have obscured such a correlation. A simple assay which removes previously unrecognized sources of error is shown to generate data which correlate well with patient responses. In this assay, lymphocytes collected from patients w ith m alignant solid tumors were stim ulated with mitogen in their autologous plasma. The am ount of radioactive thym idine incorporated during replicative deoxyribonucleic acid (DNA) synthesis was corrected to remove sources of error ignored in the previous studies. A significant improvement in mitogen-stim ulated synthesis was observed within two months for patients entering remission. For patients not responding to therapy, there was a progressive deterioration in m itogen-responsiveness. Introduction Many studies done in vivo and in vitro have d e m o n strated im m u n o su p p ress io n in p a tie n ts w ith m a lig n a n t tum ors Changes in th e c e llu la r b ran c h of th e im m une response include decreases in T cell num bers, responsiveness to phytohem ag * Address reprint requests to: Lyndon L. Larcom, Ph.D., Department of Microbiology, Clemson University, Clemson, SC glutinin (PHA) stimulation and cytotoxicity9,19,22 as well as an increase in the num ber of suppressor lymphocytes.13,17 H um oral factors are also involved in immunosuppression in these patients T he relative im portance of the cellular and humoral contrib u tio n s to im m u n o s u p p re s s io n in advanced cancer patients probably varies from patient to patient. However, it is necessary to dissect the im m unosuppressive effect as completely as possible in order to determ ine w hether or not there /93/ $01.50 Institute for Clinical Science, Inc.

2 208 WILHIDE AND LARCOM m ight b e param eters w hich could be used clinically to evaluate a p atient s responses as he undergoes therapy. Earlier studies have attem pted to use lymphocyte responses in mitogen stimulation assays for this purpose because th ey can be p erfo rm ed rap id ly and cheaply and only require periodic blood sam ples. The results of these studies have not been promising. Our data indicate that this is probably because of the way the experim ents w ere performed. The data reported here indicate that if param eters neglected in the previous studies are controlled, the responses of lymphocytes in the mitogen-stimulation assay can be indicative of w hether or not a patient is responding to a particular therapeutic protocol and might provide an early indication of his likelihood of remission. To mimic in vivo responses as closely as possible, it is necessary to culture cells in their autologous plasma. Some earlier studies have used fetal bovine serum or pooled human sera. These can contain a completely different com plem ent of biological response m odifiers from those present in vivo. In addition, if mitogen response is m onitored as the incorporation of tritium -labeled thym idine (3HdThd) into newly synthesized DNA, two a d d itio n al sources of erro r m ust be avoided: (1) different sera contain different amounts of endogenous thym idine which can compete with the labeled substrate and (2) cells from different donors have different levels of thymidine phosphorylase activity which can degrade the substrate. An assay developed in our laboratory accurately rem oves these artifacts and makes it possible to compare the m itogen-stim ulated responses of different individuals or of a particular individual at different tim es.20 The revised assay has been used to study the m itogen-responsiveness of cells from patients with solid tumors. Cells collected before therapy was begun and at two m onth intervals thereafter were mitogen-stimulated in their autologous plasm a and in plasm a collected prior to the initiation of therapy. The response to stimulation was m easured as the amount of radioactive 3H-dThd incorporated during lym phocyte DNA replication, corrected for the com plicating factors discussed previously. Patients were divided into two groups on the basis of d ise a se status as d e te rm in e d by the attending physician six months after therapy was initiated. The first group was comprised of those patients for whom the chosen therapeutic protocol was considered to be effective. T hese patients experienced remission as determ ined by the absence of detectable malignant disease. For patients in the second group, the therapy was considered to be ineffective in causing rem ission. In these cases, either the disease was progressing or there was no evidence of improvement. M ost o f th e p a tie n ts in th is group were considered term inal in the sense that they were not likely to survive for m ore than a few m ore m onths. The results indicate that the revised assay could be a valuable tool for monitoring the effectiveness of therapy for individual patients. Materials and Methods Su b j e c t s Twenty-one subjects with carcinoma of the breast, colon, or p ancreas w ere recruited from patients undergoing treatm ent at the Cancer Treatm ent Center, G reenville M em orial H ospital, G reen ville, SC. These are listed in table I. Blood samples were drawn just prior to the initial chemotherapy treatm ent and prior to treatments adm inistered at 2, 4, and 6 months after therapy was begun. H ealthy subjects w ere obtained from Redfern Health Center, Clemson University, Clemson, SC.

3 MONITORING RESPONSE TO THERAPY IN CANCER PATIENTS TABLE I Characteristics of Patients Participating in the Study 209 Tumor a A B C Sex Age Therapy Carcinoma 1* F 37 Cytoxan, fluorouracil (5-FU). adriamycin Breast 2 F 42 Cytoxan, fluorouracil (5-FU), adriamycin 3 F 66 Tamoxifen 4 F 71 Carmustine (BCNU) 5 F 75 Cytoxan, fluorouracil (5-FU), adriamycin Relapse/ 6 F 51 Cytoxan, fluorouracil (5-FU), adriamycin Breast 7* F 63 Tamoxifen 8 F 73 Cytoxan, fluorouracil (5-FU), methotrexate Adjuvant/ 9* F 36 Cytoxan, fluorouracil (5-FU), methotrexate Breast 10* F 45 Cytoxan, fluorouracil (5-FU), methotrexate 11* F 46 Cytoxan, fluorouracil (5-FU), methotrexate 12* F 71 Cytoxan, fluorouracil (5-FU), adriamycin Carcinoma/ 13 M 71 Fluorouracil (5-FU) Colon 14* F 72 Fluorouracil (5-FU) Carcinoma/ 14* F 72 Flourouracil (5-FU) Lung 15* M 46 Cis platin, Etoposide (VP-16) 16 F 58 Carmustine (BCNU), cytoxan, methotrexate 17* M 66 Vincristine, adriamycin 18* M 66 Cis platin 19* M 73 Carmustine (BCNU), cytoxan, methotrexate Pancreatic/ 20* M 55 Fluorouracil (5-FU) Cancer Melanoma 21 M 48 Vincristine a Initial diagnosis: A = metastasis; 8 = metastasis to primary lymph nodes; C = no metastasis. * Remission after six months of therapy Protocols approved by the Human Subjects Committees of Clemson University and Greenville Memorial Hospital were rigorously followed. A ssays M itogen stim ulation assays w ere perform ed on lym phocytes isolated from each subject. A detailed description of the modified m itogen stimulation assay has been presented.15 Earlier assays are u n s u ita b le for stu d ie s of th is type because of the artifacts m entioned previously. Comparisons of stim ulation indices m easured for different individuals or for the same individual at different times are not reliable unless these artifacts are rem oved. T he m odified assay accom plishes this by m easuring the counts per m inute (cpm) incorporated in aliquots containing the same am ount of radioactive thymidine, but different amounts of com peting non-radioactive thym idine. T hese data are used to calculate the amount of label which would be incorporated in the absence of competing serum th y m id in e and of enzym atic dégrada-

4 210 WILHIDE AND LARCOM tion of the label. At different times after therapy was begun, cells of each subject w ere stim ulated w ith phytohem agglutinin in three different culture media: (1) medium supplem ented with autologous plasma, (2) m edium supplem ented with plasma collected from the same subject prior to the first chemotherapy treatment, and (3) medium supplem ented with fetal bovine serum (FBS). Assays w ere p erformed w ithin three hours after blood was collected from the donor. L y m p h o c y t e I s o l a t io n Fourteen ml of blood were collected and anti-coagulated with heparin. Lymphocytes were isolated on polysucrosesodium diatrozate gradients.* Seven ml of blood were layered onto 3.0 ml of Histopaque and centrifuged at 400 x g for 30 m inutes at room tem perature. After centrifugation, plasma was pipetted from the top of the tube and saved to supplem ent the culture medium. Plasma from the initial collection was frozen for use in later stim u la tio n assays. T h e cells w ere washed with phosphate-buffered saline (PBS) and adjusted to a concentration of 2.0 x 106 per ml. P r o l if e r a t io n L y m p h o c y te s w e re c u ltu r e d in Roswell Park Memorial Institute (RPMI) tissue culture m ed iu m t supplem ented with 10 percent plasma, one percent gentam ycin sulfate, one percent glutamine and 10 xg per ml of phytohemagglutinin (PHA) ph 7.30, at 37 C in an atmosphere of 5 percent C 0 2. After 48 hours, the cells were separated into 0.50 ml aliquots and pulsed with 5.0 ( Ci per ml 3H-dThd. Nonradioactive dthd was added to the various cultures to give 0, 3, * Histopaque 1077; Sigma Chemical Company, St. Louis, MO. t Gibco, Grand Island, NY. 6, or 9 times the radioactive dt hd concentration. The cultures were incubated for an additional four hours and w ere w ashed to remove unincorporated dthd. I s o l a t i o n o f DNA C ells from each aliquot w ere resu s pended in 5.0 ml PBS and lysed by the addition of an equal volume of a solution containing 2.0 percent sodium lauryl sarcosine, 0.01 M ethylenediam ine tetraacetic acid (EDTA) and 2.0 M NaOH. The suspensions were allowed to sit for one hour to assure complete lysis. To keep the DNA in single-stranded form, formaldehyde was added to give a final concentratio n of 2.0 p ercen t. T he ph was adjusted to 7.0 with HC1 as determ ined by phenol red indicator, and the DNA was separated by adding 10.0 ml PBS and filtering the solution through nitrocellulose m em brane filters (Schleicher and Schuell, BA 85). The filters were washed w ith PBS to rem ove u n b o u n d label, dried, and counted in a Beckman LS 7500 Scintillation Counter.^ E v a l u a t io n o f t h e D a t a For each sample, there w ere four aliquots. Each aliquot contained the same am o u n t of 3H -dt hd b u t a d iffe re n t amount of unlabelled dthd. The ratio of nonradioactive to radioactive dthd was 0, 3, 6, or 9. For these, the reciprocal of cpm incorporated was plotted against ( x + 1) where ll is the ratio of nonradioactive dt hd to radioactive dt hd in a particular aliquot. A best-fit line was fit to these data using linear regression analysis. A typical plot is given in figure 1. The inverse of the slope of this line represents the amount of radioactive dthd the cells would incorporate in the absence of com peting serum dthd and of degradat Beckman Instruments, Fullerton, CA.

5 MONITORING RESPONSE TO THERAPY IN CANCER PATIENTS 211 <0 O i ' X CL O F ig u r e 1. A ty p ic a l p l o t to g e n e r a t e C m: Cm = (1/slope). (jl+ 1) tion by cellular phophorylases. This repre se n ts th e capacity of th e cells to undergo PHA-stimulated DNA synthesis and is designated Cm. For each subject, lymphocytes were stim ulated in three different media. In one, RPMI 1640 culture m edium was supplem ented with autologous plasma. In the others, the supplem ent was either plasma collected from the same patient prior to administration of chemotherapy or FBS. The mitogen responsiveness of cells collected 2, 4, or 6 months after therapy was initiated was m easured in each m edium. T hese responses w ere compared with the responses observed before therapy was begun. Results The primary aim of the study was to determ ine w hether or not results of the assay correlate w ith the response to therapy. If this should be the case, the assay could prove useful in helping to determ ine w hether or not a particular patient is responding to therapy. A second aim was to follow th e c e llu la r changes and serum changes which occur during chemotherapy. Cellular changes induced by chem otherapy w ould be d e te c te d as sig n ific a n t d iffe re n c e s betw een the mitogenic response for cells collected before therapy is begun and at different times thereafter in a particular serum supplement. Changes in response caused by changes in relative serum concentrations of the various biological response modifiers would appear when aliquots of cells collected at a particular time are cultured in sera collected at different times. To determ ine if m itogenesis as m easured by this assay might be indicative of whether or not a patient was responding to therapy, blood was drawn just prior to the first drug treatm ent and again prior to doses adm inistered at 2, 4, and 6 months later. Lymphocytes were isolated immed iately after th e sam ple was taken. Plasma from the pre-therapy sample was frozen. Sam ples taken later from the same patient were cultured both in this and in their autologous plasma. Average

6 212 WILHIDE AND LARCOM values of Cm for cells cultured in autologous plasma are given in table II for samples from the p atien ts and from the healthy donors. The average Cm for samples from healthy donors below the age of 40 is 7.4 tim es as great as for the p atien ts. T his is in ag reem en t w ith num erous earlier studies showing suppressed mitogenesis in cancer patients. More important, however, is the comparison of Cm values obtained for a given patient at different times. T he broad variability am ong individuals m akes com parisons of Cm values betw een people difficult. If the aim is to determ ine the impact of therapy on particular patients, it is most important to examine changes in Cm with time for each patient. The status of each patient, as assessed by his a tte n d in g physician after six months of therapy, was recorded. The data w e re se p a ra te d into th o se for p a tie n ts in rem issio n and those for patients whose condition had worsened or rem ained unchanged. For each subject, the ratio of Cm measured in autologous plasma at time t to that m easured in autologous plasm a before therapy was begun was calculated. Averages of these values for patients in the two groups for t = 2, 4, and 6 months are given in table III. It is clear that the average response for patients in rem ission was significantly TABLE II Mitogen Responsiveness for Healthy Individuals and for Cancer Patients Prior to Therapy Donor No. of Subjects Age (Years) Mean Value of CrrP ± S.E.M.b (x 10-6) Healthy ±0.34 Healthy ±0.22 Patients ±0.11 acm for cells cultured in their autologous plasma. ^Standard error of the mean. TABLE III Changes in Immunocompetence of Cancer Patients During Therapy* Remission Terminal 2 Months 4 Months 6 Months (Mean ratios of Cm ± S.E.M.p ± ± ± ± ±0.01 Average of ratios of Cm to Cm measured prior to treatment. aratio of Cm for cells collected at timet to Cm for cells collected prior to treatment. Cm values are for cells stimulated in their autologous plasma. greater after therapy was begun. There was a dramatic increase in response for sam ples co lle cte d two m onths after therapy was begun. This decreased at later times, indicating that the two-month value may rep re sen t an overshoot effect typical of many biological systems. For patients not responding to therapy, there was a continual decrease in m itogen responsiveness. Average values of Cm are given in table IV. Although the same trends are obvious, changes in the m ean value of Cm are much smaller than changes in the ratios given in table III. Nevertheless, the mean Cm for patients who responded to therapy increased by a factor of 2.5 after six months and that for patients not responding decreased by a factor of 5. In both cases, the direction of the change was obvious by the second month. The ratios given in table III overemphasize the magnitude of the changes because in severely immunosuppressed patients there was almost no response to PHA stimulation initially. However, it is the ratios w hich indicate actual responsiveness. Each patient s Cm is compared to his pre-therapy Cm value. An increase in the ratio indicates enhancem ent of the mitogenic response during therapy. This is what would be monitored in the clinical evaluation of individual patients. Clearly, these data indicate that Cm val-

7 M ONITORING RESPONSE TO THERAPY IN CANCER PATIENTS TABLE IV Average Cm Values for Patients Grouped According to Their After the Sixth Month of Therapy 213 Initial 2 Months 4 Months 6 Months Sample (Mean values of Cm ± S.E.M.a) (x 10-6) Remission 0.39 ± ± ± ± subjects Terminal 0.25 ± ± ± ± subjects 'Standard error of the mean. ues m ight be of significant predictive value in determ ining early w hether a particular therapeutic regim en should be abandoned in favor of an alternative therapy. It is known that serum factors in cancer patients suppress m itogen-stim ulated lymphocyte proliferation. To analyze the m echanism s responsible for changes observed as patients progressed through therapy, the proliferative response was dissected by stim ulating lym phocytes from each patient at different times not only in their autologous plasma, but also in plasma collected before therapy was begun. The values in table V are average ratios of the Cm m easured for cells collected at tim e t in th e ir autologous plasma to the Cm for the same cells in plasm a collected before therapy was begun (both measurements made in the same experiment). Both for patients who w ere diagnosed as entering rem ission after six m onths of therapy and those diagnosed as term inal, cells collected after two months of therapy responded better to m itogen stim ulation in their autologous plasm a than in plasm a collected prior to initiation of therapy. After four or six months of therapy, there was continued improvement of the response in autologous plasma relative to that in pre-treatm ent plasma for patients going into rem ission (by a factor of more than 10). However, for patients not responding to therapy, this ratio decreased in an equally dram atic way. T h ese results imply that the extent to which serum factors suppress the mitogenic response in advanced cancer p atien ts is d irectly related to either the stage of the disease or its aggressiveness. To assess changes which m ight have occurred in the cells during therapy, the ratio calculated was: Cm for cells collected at 2, 4, or 6 months and mitogenstim ulated in plasm a collected before therapy was begun to Cm for cells taken from the same patient prior to the initia- Remission Terminal TABLE V Effects of Serum Changes on Mitogen Responsiveness* 2 Months 4 Months 6 Months (Mean ratios of Cm values ± S.E.M.) 2.73 ± ± ± ± ± ±0.52 Values given are ratios of Cm measured for cells collected at time t and stimulated in their auologous plasma to Cm for the same cells stimulated in plasma collected prior to therapy. amean ratio of Cm for lymphocytes collected at time t and stimulated In their autologous plasma to Cm for the same cells stimulated in plasma collected from the same patient prior to administration of therapy.

8 214 WILHIDE AND LARCOM tion of therapy and stim ulated in their autologous plasm a. T h ese ratios are given in table VI. For patients entering remission, the response of cells collected after two m onths of therapy and stim u lated in pre-therapy plasma was almost four-fold greater than that of cells collected prior to beginning therapy and stim u lated in the sam e plasm a. For patients who did not respond to therapy, this response at this time was 50 percent smaller. There was no significant change over the next four months for patients with a good prognosis. For patients with progressing disease, the response at four months im proved, but deteriorated again. M any laboratories perform m itogen stimulation assays in fetal bovine serum (FBS). For each of over 60 healthy subjects studied in this laboratory, the proliferative response of lymphocytes stim u lated in th eir autologous plasm a was significantly greater than for the same cells stim ulated in FBS. The present authors also attem pted to assess changes in the cells of the cancer patients by stim ulating cells collected at different times in FBS. All experiments were performed with FBS from the same lot. For each patient, Cm was m easured in FBS for cells collected prior to his beginning TABLE VI Changes In Mitogen Responsiveness Owing to Cellular Changes* 2 Months 4 Months 6 Months (Mean ratios of Cm values ± S.E.M.p Remission ± 1.13 Terminal 0.46 ± ± ± 0.32 Values given are the mean ratios of Cm for cells collected at different times after initiation of therapy and stimulated in plasma collected prior to beginning therapy to Cm for cells collected prior to beginning therapy in their autologous serum. avalues given are Cm (cells collected at time t In pre-therapy plasma) / Cm (cells collected prior to therapy in pre-therapy plasma). TABLE VII Changes in Mitogen Responsiveness Owing to Cellular Changes when Stimulations were Performed in the Presence of Fetal Bovine Serum* Remission Terminal 2 Months 4 Months 6 Months (Mean ratios of Cm values ± S.E.M.) 2.20 ± ± ± ± ± ± 0.07 Values greater than 1 represent enhanced stimulation compared to cells collected prior to therapy. athe same ratio as in table VI, except that all proliferations were performed in fetal bovine serum. therapy and again for cells collected after 2, 4, or 6 months of therapy. The ratio of Cm for cells collected at time t to Cm prior to beginning therapy was calculated. The averages of these ratios for all patients are given in table VII. Again, for patients responding to therapy, better proliferation was observed for cells collected after th e ra p y w as b e g u n. F o r th o se n o t responding, deterioration in the proliferative response was observed. Discussion The results presented here indicate that for patients with solid tumors, m itogen-stim ulated proliferation of lym phocytes collected periodically after therapy is begun could be of significant prognostic value. If the lym phocytes are stim u lated in their autologous plasm a, com parison of the proliferative index for cells collected after a few weeks of treatm ent w ith the response of cells co llected before therapy is begun can indicate whether or not the therapeutic protocol being em ployed is effective. Im provem ent in responses of patients entering rem ission seem to be prim arily attributable to changes in serum factors, but changes in the m ononuclear cells them selves also occur.

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