Acta Neurochir (Wien) (1989) 98: 35-4I =Acta N6urochirurgica �9 by Springer-Verlag 1989

Intratumoural Injection of Autologous Lymphocytes Plus Human Lymphoblastoid Interferon for the Treatment of Glioblastoma

J. Vaquero, R. Martinez, S. Oya, S. Coca, L. Barbolla, J. Ramiro, and F. G. Salazar

Departments of Neurosurgery and Haematology, Puerta de Hierro Clinic, Autonomous University; Departments of Cellular Biology and Pathology, Complutensis University, and Department of Neurosurgery, Gregorio Marafidn Hospital, Madrid, Spain

Summary

Preliminary experience with a clinical trial of immunotherapy for glioblastoma, by means of intratumoural injection of autologous lymphocytes (AL) mixed with low doses of human lymphoblastoid interferon (HLI) is presented.

In two of twelve patients, a transient reduction of tumoural volume was obtained. Morphological studies showed that injected lymphocytes remain within the turnout, and suggest tumoural lysis due to activity of natural killer (NK) cells.

Clinically no significant prolongation of survival time could be achieved and, as in other series, patients with additional radiation therapy survived longer. But the morphological findings suggest that immunotherapy carrying NK-cells to contact with tumoural cells might be useful in some patients with glioblastoma. Actually no explanation can be given why only two of our cases responded positively. Regarding the otherwise poor prognosis it seems justified to continue these studies.

Key'words: Immunotherapy; glioblastoma; activated lympho- cytes; interferon; natural killer cells.

Introduction

In spite of the great advances made in Neurosurgery during the las~L decades, the survival time for patients with malignant gliomas is still short. For this reason, experimentation with alternative or complementary treatments to surgery, radiotherapy, and chemother- apy, is fully justified.

Immunological treatment for malignant gliomas have been proposed since 1960, when Bloom et al.

autotransplanted glioblastoma cells into the subcuta- neous tissue, in an attempt to increase antigenicity of the tumour as well as to stimulate the immune system 2.

In 1968, when Scheinberg and Taylor emphasized the possible usefulness of immunotherapy in the treat- ment of glioblastoma 19, various clinical studies were

done, including the injection of autologous leucocytes in the tumoural area 2~ 23, 26. In these studies, some findings suggest a possible beneficial effect of the ther- apy, although the lack of follow-up with CT scans, in order to appraise the correct size and growth of the tumours, make the evaluation difficult.

On the other hand, several authors have pointed out the relationship between lymphocytie infiltration in gliomas and a longer survival time 3' 6, 15, 16. Despite the fact that these observations were controversially dis- cussed by other authors 18, there exist enough reasons to conclude that immunological phenomena play a role in the biology of malignant gliomas, and to state that manipulation of these phenomena could be useful in their treatment.

Cytotoxic activity of different lymphocytic sub- groups against tumours was well documented both by in vivo and in vitro studies, with most of this activity linked to natural killer (NK) cells 22.

Recently, Rosenberg et al. reported cancer regres- sion after systemic administration of autologous lym- phokine activated killer (LAK) cells and recombinant interleukin-2 (IL-2)17. Following these findings, other authors started to investigate the possible usefulness of LAK cells in the treatment of malignant gliomas 1, 11-13

We report our preliminary experience in the treat- ment of patients with glioblastoma by the intratu- moural injection of autologous lymphocytes (AL) mixed with human lymphoblastoid interferon (HLBI). The present study is based on the demonstrated fact that interferon may enhance the cytotoxic activity of NK cells within a lymphocytic population 7-9' 22

36

Table 1. Clinical Data o f Patients in this Series

J. Vaquero et al.: Intratumoural Injection of Autologous Lymphocytes

Case Sex Age Tumoural localization

Surgical procedure Treatment previous to immunotherapy

Intratumoural Survival after lymphocytes surgery (days) administered

1 f 62 right subtotal temporal extirpation

2 m 12 corpus stereotactic callosum biopsy

3 m 58 right stereotactic parietal biopsy

4 f 50 left partial temporal extirpation

5 m 65 right partial temporal extirpation

6 f 42 left partial frontal extirpation

7 f 45 right subtotal parietal extirpation

8 f 47 left radical parietal extirpation

9 f 39 left partial parietal extirpation

10 m 54 left sterotactic temporal biopsy

11 m 55 right radical frontal extirpation

12 m 58 left partial temporal extirpation

radiotherapy (6,000 rads)

radiotherapy (6,000 rads)

radiotherapy (6,000 rads)

0.32 x 108 106

0.17 x 108 46

0.3 x 108 38

0.27 x 108 31

0.5 x 108 I67

1.3 x 109 492 1.35 x 108 2.17x 109

2.2 x 109 162

2.7 x 109 315

0.13 x 108 341

0.53 x 108 259 1.2 x 109

1.6 x 109 218

2.3 x 109 122

Clinical Material and Methods

The present series inciudes twelve patients with glioblastoma multiforme who were treated by immunotherapy with AL-HLBI, and whose cases were followed up until the patients died. Nine patients were diagnosed after surgical procedures where resection of the turnout was macroscopically considered as total in two cases, subtotal in another two cases, and partial in five cases. Three patients underwent stereotactic biopsies for diagnosis, and they were not operated on later. Three patients in the series were irradiated after surgery.

Table 1 summarizes the clinical data of the patients included in this series. The patients diagnosed by stereotactic biopsies were treated with immunotherapy within the first week after diagnosis. The group of six patients operated on and not irradiated, underwent immunotherapy in the next month after the operation, when on CT scan it was possible to evaluate the magnitude of ~umoural resection without postsurgical artifacts. The three patients operated on, and irradiated were treated by immunotherapy within six months after surgery, when postirradiation CT scan showed growth of the tu- moural remnants. Immunotherapy treatment consisted of injections of AL and HLBI either intratumourally or within the tumoural bed.

Injections were done under stereotactic conditions through a burr- hole of the craniotomy or after doing a small drill hole. In two cases, a previously implanted Ommaya reservoir was used. Lymphocytic extraction from peripheral blood was done as previously described 24. The bloody suspension rich in lymphocytes was purified on Ficoll- Hypaque gradients, and the cells were suspended on RPMI-1640 medium (Microbiological Associates, Walkersville, Md). Cell counts were performed in a haemocytometer Coulter 2B-1. A volume of 4cm 3 of suspension was obtained from each extraction, with lym- phocyte concentration that ranged from 0.13 x 108 to 2.7 x 109 (mean 9.2 x 108).

In each case, 3 x 106IU of purified human iymphoblastoid in- terferon (Wellferon, The Wellcome Foundation Ltd, London) were added to the suspension. A final volume of 5 cm 3 was obtained and maintained at 37 degree centigrade until the mixture was injected, within the next two hours.

CT scans were done, in every case, immediately after injection, and every 2-4 months, until the patient's death.

Steroids were withdrawn during immunotherapy and in the fol- low-up, but in some cases they were transitorily administered, mainly in the final stages of the disease.

Ten cases of the series underwent one injection of AL-HLBI. In

J. Vaquero et al.: Intratumoural Injection of Autologous Lymphocytes 37

two patients (cases 6 and 10) who showed response to treatment, the procedure was repeated every 2~4 months until a rapid tumour growth appeared.

With exception of patients with the Ommaya reservoirs, biopsies were taken in each case, previously to intratumoural injection, in order to assess the correct location of the stereotactic cannula, and in three patients (cases 7, 1 i, and 12) tumoural biopsies were taken also at 30 days after the injection of AL-HLBI. In addition to stan- dard examination with light and electron microscopy, imnmnostain for phenotypic identification of NK cells was performed on frozen samples (Leu-7 antigen) using monoclonal antibody. The avidin- biotin-peroxidase complex (ABC) technique was employed.

For identification of Leu-7, murine monoclonal antibody (Bec- ton-Dickinson, Sunnyvale, CA) was used. Secondary biotinylated horse antibody against mouse immunoglobulins (Vector Lab., Bur- lingame, CA) and avidin-peroxidase complex (ABC reagent, Vecta- stain, Vector Lab., Burlingame, CA) were used. The peroxidase sites were visualized with 3.3 diaminobenzidine (DAB) in 0.3% hydrogen peroxidase/phosphate-buffered saline (PBS); the latter agent was enhanced with 0.5% cooper sulphate. Counterstaining was per- formed with haematoxylin, and Canada balsam was used for mount- ing.

The tissue specimens was embedded in Optimal Cooling Tem- perature (OCT) compound (Miles Lab., Illinois), placed into liquid nitrogen (--170 ~ for 10 minutes, and stored at 70 ~ Frozen sections were made at 10 g thickness at - -20 ~ placed into acetone at 0 ~ for 5 seconds, and allowed to dry. The slides were stored at

20 ~ in a closed container. Staining, which was performed within 1 week of processing, began by refixation of the slides in acetone for t0 minutes, followed by washing and rehydration in PBS for 10 minutes. Normal horse serum (3%) was applied for 20 minutes to decrease nonspecific background staining, followed by incubation with primary antibody for 30 minutes in a humidified chamber. This was followed by washing in PBS for l 5 minutes, repeated three times. Secondary antibody was then applied for 20 minutes, followed by a 45-minute PBS wash. Avidin-peroxidase was added for an addi- tional 20 minutes, followed by washing for 60 minutes in four 15- minute intervals. The DAB was added for 5 minutes, followed by 5 minutes of cooper sulphate after a 5-minute wash, and finally hae- matoxylin was added for 5 minutes. The slides were mounted and observed under the, microscope. Sections of normal peripheral nerve served as positive controls.

In the follow-up of the patients, laboratory studies included parameters of haematological and renal function.

The immunotl~.erapy protocol employed was approved by the Commission of Clinical Trials of the Puerta de Hierro Clinic. All the patients and their relatives were informed about the aims and possible complications, and written consent was obtained.

tumour notwithstanding the immunotherapy. In two cases (cases 6 and 10) control CT scan after intratu- moural injection of AL-HLBI showed tumoural re- gression. These cases are described in detail.

Case 6

This 42-year-old woman, with right hemiparesis and dysphasia, underwent partial resection of a left frontal partially cystic glioblastoma, in May 1986. An Ore- maya reservoir was left within the cavity of the tu- moural cyst. After surgery, she was irradiated with a 6,000 rads dose over the region of the tumour. In July 1986, the patient was referred for immunotherapy, when evidence of tumour regrowth appeared in the follow-up CT scans. At this time, she complained of headache and right hemiparesis, and an AL-HLBI sus- pension was injected through the reservoir. In October 1986, when this procedure was repeated, the patient was symptom-free, and CT scan showed disappearance of the previously enhanced tumoural nodule (Fig. 1). In January 1987 there were no changes on CT scan, and a new injection of AL-HLBI was performed. In March 1987, the patient started with headache, severe dysphasia and right hemiparesis. CT scan showed clear tumoural progression. Dexamethasone was adminis- tered, but death occurred in August 1987.

Case 10

This 54-year-old man was admitted in May 1987 with a two months history of headache, dysphasia and right hemiparesis. CT scan showed a lesion in the left hemisphere which suggested a ma- lignant glioma. A solid glioblastoma was diagnosed by stereotactic

Results

Clinical Data

The 12 patients of the present series underwent 15 intratumoural injections of AL-HLBI. All the injec- tions were well tolerated without any side effect. There were no changes in the renal or haematological param- eters related to the procedure. The mean survival rate in the patients of the series was 6 months. In ten cases, follow-up CT scans showed progressive growth of the

Fig. 1. Case 6. Left: CT scan after contrast administration, showing a cystic glioblastoma, with an enhanced tumoural nodule. Right: CT scan after contrast administration, three months after intratu- moural injection of 1.3 x l09 autologous lymphocytes and 3 x 106 IU of human lymphoblastoid interferon. The disappearance of the en- hanced nodule can be seen

38 J. Vaquero et al. : Intratumoural Injection of Autologous Lymphocytes

biopsies, and an intratumoural injection of AL-HLBI was performed. During the following weeks the patient improved, and CT scans performed 2 and 4 months after the procedure showed a progressive reduction of tumoural volume (Fig. 2). At this time, a new intra- tumoural injection of AL-HLBI was performed after stereotactic biopsies were done on the enhanced nodule, and surrounding zones. The clinical picture did not change until October 1987, when the patient developed further clinical deterioration, and CT scan showed important tumoural growth, with corpus callosum involvement. Lit- tle improvement was obtained with steroids, and death occurred in January, 1988.

Fig. 2. Case 10. Solid glioblastoma histologically confirmed by ster- eotactic biopsies, a) CT scan, without contrast administration, pre- vious to histological diagnosis, b) CT scan, after contrast adminis- tration, five hours after biopsy procedure and intratumoural injection of 0.53 x l0 s autologous lymphocytes and 3 x 10 6 IU of human lym- phoblastoid interferon, c) CT scan after contrast administration, two months later, showing tumoural regression, d) CT scan, after contrast administration four months after histological diagnosis, showing a 50% reduction of initial lesion, and normalization of middle line

Morphological Data

In four cases of the series, biopsies after i mmuno-

therapy were performed in order to check the presence

of injected lymphocytes within the turnout . In case 10,

who responded to t reatment , stereotactic biopsies

showed tissue necrosis where biopsies taken four

mon ths before showed tumoura l tissue. At the level of

the enhanced nodule, tumoura l cells appeared with

swollen cytoplasm, and electron microscope showed in

these ceils a great a m o u n t of gliofilaments and lipids.

A significant quant i ty of lymphocytes was not en-

countered in these biopsies, but immunos t a in ing dis-

covered several mononuc lea r cells with N K phenotype

between tumoura l cells (Fig~ 3).

In another three cases, biopsies of the tu rnou t were

taken 30 days after A L - H L B I injection. In all samples

there was a significant a m o u n t of lymphocytes among

the tumoura l cells. When peripheral zones of the tu-

m o u r was studied, lymphocytes formed a limit between

neural and tumoura l tissue (Fig. 4).

Fig. 3. Case 10. Tumoural tissue four months after initial immunotherapy. Glioblastoma cells show a swollen cytoplasm. H.E. technique, x250. Inset: Mononuclear cells with Leu-7 immunoreactivity can be seen between tumoural ceils. ABC technique, x 250

J. Vaquero et al.: Intratumoural Injection of Autologous Lymphocytes 39

Fig.4. Case 11. Tumoural pic- ture, 30 days after immunother- apy, showing extensive lympho- cytic infiltration both in the pe- riphery of tumour (left) and in deep zones (right), B: normal brain tissue. H.E. technique, x 75

Ultrastructural studies disclosed conjugate bindings between tumoural cells and large granular lymphocytes (NK cells). These lymphocytes protruded among cy- toplasmatic foldings of the tumoural cells that show

vacuolization and degenerative changes, evidencing a mechanism of cellular killing (Fig. 5).

Surface antigens of NK-cell (Leu-7) were identified

in some lymphocytes between tumoural cells, but the number of stained cells was greater in the periphery of the tumour (Fig. 6).

Fig, 5. Case 12. Tumoural tissue obtained 30 days after AL-HLBI injection. Electron microscope showed an NK cell (NK) killing a tumoural cell (7). P: NK ceil cytoplasmatic protrusion among cy- toplasmatic enfolding of the glioblastoma cell. x 12,000

Discussion

It is generally accepted that most of the cytotoxic activity of the immune system against tumours is due to one fraction of lymphocytes defined as NK-cells;

nowadays we know many features of their morpho- logical identification and their mechanism of cellular lysis 4, 5, 22.

In our study, biopsies after intratumoural injection of AL-HLBI showed the usefulness of the procedure leaving effective cells with cytotoxic capacity within the tumour. Ultrastructural and immunostaining data showed persistence of N K cells between tumoural cells after injection, and suggest that lysis of glioblastoma cells is possible with this procedure.

40 J. Vaquero et al. : Intratumoural Injection of Autologous Lymphocytes

Fig. 6. Case 11. Strong immunoreac- tivity for Leu-7 antigen over injected lymphocytes. Tissue sample obtained 30 days after intratumoural injection of AL-HLBI. ABC technique, x 250

In 1977 Young et al. suggested a beneficial effect in patients with glioblastoma during intratumoural injec- tion of AL. However it has proved difficult to relate morphological changes seen in tumours due to im- munotherapy rather than to radiotherapy or chemo- therapy 26. In our cases, morphological changes in tu- mours after immunotherapy are due to the above de- scribed procedure, since the patients studied were not treated by any other procedures.

On the other hand, our treatment differs from that of Young et al. 26, because we injected the AL mixed with low doses of HLBI. We chose this combination based on the fact that a brief pre-incubation of HLBI with peripheral blood mononuclear leucocytes induced a rapid and strong potentiation of cytotoxic cell activ- ities 9. It is possible that NK cell activation induced by interferon would be mediated by IL-2 22. Moreover, interplay between interferon and IL-2 is complex, be- cause interferon can induce IL-2 receptors on NK cells 14, and IL-2 can induce growth and maturation of NK cells 21. These observations, and the scarcity of ultastructural data on lysis of tumoural cells by LAK cells ~0, suggest that cytotoxicity attributed to LAK cells in some studies would be due to NK cells.

Clinically, the analysis of our preliminary results does not show a prolongation of survival time after immunotherapy. And as in other series 25, glioblasto- mas with additional radiation therapy survived longer. Nevertheless, we obtained evidence of transient tumour regression in two cases. This fact encourages us to perform further studies in order to improve the efficacy

of this therapy and to find the reasons why only two of our cases responded positively to this form of im- munotherapy.

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Correspondence and Reprints: J. Vaquero, M.D., Department of Neurosurgery, Puerta de Hierro Clinic, San Martin de Porres, 4, E-28035 Madrid, Spain.