IMMUNOTHERAPY IN PATIENTS WITH GROWING GLIOBLASTOMAS
(Study of Effectiveness: Preliminary Results)

Oliushin V.E., Tigliev G.S., Ostreiko O.V., Filatov M.B.

Polenov Neurosurgical Institute, St.Petersburg, Russia

 

According to the data on oncologic morbidity in developed countries, cerebral tumors occupy the 9th-10th place. Malignant glial neoplasms are the most frequent primary cerebral tumors in adults; their quota is equal to 35%; glioblastomas are prevalent [5].

Modern strategy of treatment of malignant gliomas is based on removal of a tumor with subsequent chemo- and radiation therapy. Achievements in the field of surgical technique and good anesthesiologic support allowed to reduce postoperative mortality (1.9% in anaplastic astrocytomas and 4.3% in glioblastomas) [3]. Radiation therapy with an integral focal radiation dose of 60 Gr prolonged survival from 4 up to 12 months [10]. Chemotherapy with drugs containing nitrozomethylurea promoted prolongation of survival in patients with malignant glial tumors. However, it concerns chemosensitive cases whose rate, according to different authors, varies from 20% up to 33.9% [3, 6, 9]. It should be kept in mind that chemotherapy did not result in a growth of an average rate of survival in the whole group of patients but only increased a number of cases who remained alive during 18 months and more [10].

Nevertheless, results of complex treatment of malignant gliomas are still poor. The average rate of survival in patients with anaplastic astrocytomas is about 24 months; as for cases with glioblastomas the situation is much worse. According to different studies, this index in the latter is seldom more than 12 months [1]; survival of 5 years makes 3-5.5% (it is equal to 60% in patients with general oncologic diseases) [4].

This situation determines striving for improvement of chemo- and radiation therapy and search for new therapeutic technologies which would help to achieve better results of treatment of patients with cerebral glioblastomas. Immunologic approaches occupy a peculiar place among such new methods as gene and virus technologies, therapy preventing a growth of vessels. If to take into account a theory of immunologic control, according to which tumor cells are detected and annihilated by the immune system, it becomes clear that immunologic approaches are rather natural. Besides, their positive features consist in possibility of combination with conventional methods and capability to compensate immunodeficiency caused by them.

The immune system is a natural barrier for malignant tumors; activation of specific and nonspecific immune responses is considered to be a potentially effective means of combating neoplasms. It is confirmed by the facts which show that immunodeficiency results in growth of a number of oncologic diseases, that tumors produce immunosuppressive substances to get over the limitations imposed on their growth by the immune system. One more confirmation is positive results of immunotherapy obtained in animals and clinical research and concerning both malignant tumors of the CNS and neoplasms with extracranial localization.

Studies in animals prove that the most effective antitumorous weapon is specific cellular immunity based on activation of cytotoxic T-lymphocytes.

Trials of different immunotherapeutic methods showed that cytokine therapy with use of IL-2 was characterized by subtherapeutic effectiveness as administration of adequate doses of interleukin resulted in complications and first of all brain edema. Low efficacy of LAK therapy, serotherapy of gliomas watched in clinical studies, as well as the latest achievements of immunology gave rise to development of new technology based on use of professional antigen-presenting cells; the most effective of them are dendritic cells. The latter express a sufficient number of costimulating molecules which are necessary for triggering an antitumorous immune response. Some researchers stick to the opinion that T-cells can become activated only when an antigen is represented by dendritic cells. They can approach tumorous cells, isolate antigens of tumor and activate T-killers (CD8) without any support of T-helpers (CD4) [8]. The results of this method used in animals were indicative of high efficacy: 60% of animals joined a group of long-livers [7].

Apperance of cytotoxic lymphocytes aimed at a tumorous target can be insufficient for an effective immune response. They should migrate into arising tumorous foci and interact with their target effectively. It is known that lymphocytes have a propensity for migration into central lymphoid organs (lymph nodes, spleen, etc.). Activated lymphocytes can migrate, in this or that way, into tissues where a meeting with an antigen is supposed to take place. Thus, cessation of activation of appropriate lymphocytes results in the following situation which is irrespective of existing immunity to some antigen: carriers of cellular immunity leave the periphery and go the central immune organs exposing the front of their struggle with a potential enemy. This circumstance leads to necessity of long-term periodic activation of lymphocytes for providing effective work of the immune system.

Patients

Specific antitumorous immunotherapy was carried out in 11 patients aged 21-61 (44 on the average). There were 6 males and 5 females with growing glioblastomas of supratentorial localization. However, the 5th female patient was not included into this study because of a short-term period of observation. Immunotherapy was preceded by craniotomy, removal of the maximum possible volume of tumor which were performed in all patients in the Polenov Neurosurgical Institute; 3, 1 and 2 of them were operated two, three and four times respectively. A multifocal growth of tumor in both hemispheres and reduction of a period of time between consecutive operations was watched in 1 patient. Presence of a tumorous node characterized by a mass effect and causing gradual increase of intracranial pressure or of neurologic deficit was an indication for repeated resection. Immunotherapy was combined with chemo- and/or radiation therapy in the majority of patients.

Data on patients subject to antitumorous immunotherapy are presented in the table.

N

Tumor localization

Age

Interval between operations  (months)

Removal of tumor during the last operation

Interval after diagnosis verification (months)

1.

Multifocal growth: The right frontal and occipital and the left occipital lobes

43

1

Partial

9

2.

The right parietal lobe

58

2

Total

13

3.

The right frontal and temporal lobes

54

13/9

Subtotal

38

4.

The right parietal and occipital lobes

28

27/13/4

Total

52

5.

The right frontal lobe, central gyruses

61

5

Subtotal

11

6.

Frontal lobes, the anterior 1/3 of corpus callosum

22

36/12/14

Total

74

There are certain indices allowing to estimate the efficacy of treatment in patients with growing glioblastoma of supratentorial localization. Firstly, there is constant reduction of a temporal interval from operation to operation. Secondly, survival after the last operation does not exceed a period of time between two last operations in the overwhelming majority of patients [2]. Thirdly, study of survival in numerous patients with growing glioblastomas proves that average survival after repeated operation is 6 months. It is equal to 9 months only in young patients with high Karnovsky’s index and total resection of tumor [2]. All these facts allowed us to estimate the results of treatment with taking into account not only generalized indices of the whole group of cases but also data on each individual patient.

Material and Methods

Immunotherapy is based on an original method. It was worked out in the Polenov Neurosurgical Institute together with the Konstantinov Institute of Nuclear Physics (an application for the invention entitled “A Method of Treatment of Cerebral Malignant Tumors” was presented for consideration on August 17, 2000, N 023265). Immunotherapy is carried out as follows: a piece of tumor of not less than 1 cm3 containing live tumorous cells but no healthy or necrotic tissue is taken during an operation. It is placed into sterile solution and sent for preparing an antigen material on the first day. In order to achieve it the piece of tumor is irradiated with a dose of 200 Gr and dissociated into cells. These tumorous cells are washed and destroyed by increasing PH of the medium containing them up to 11.5 with subsequent reduction of PH up to 6.5. Extracted proteins are used as an antigen for dendritic cells. Each round of treatment is preceded by taking 40-60 ml of peripheral blood of a patient into a syringe containing heparin solution. Final concentration of heparin is 30 units per 1 ml. Blood is processed in 6 hours after its taking at the latest. Monocytes are isolated and cultivated with growth factors during 7 days (a granulocyte-macrophage colony-stimulating factor and interleukin-4 in a dose of 1000 units per 1 ml) against a background of continuos control and a medium change. The antigen material prepared from the tumor of this very patient is added to dendritic cells on the 6th day. It is delivered into obtained dendritic cells with the help of an electric discharge (electoporation).

A method of taking peripheral blood for preparation of activated lymphocytes is analogous to that used for dendritic cells; but blood quantity is equal to 15-20 ml. Mononuclear leukocytes are isolated, activated for proliferation with the help of phytohemagglutinin and injected intradermally on the 4th day. The goal of this procedure is activation of the greatest possible number of T-lymphocytes in stimulation of Th-1 cellular pathway of an immune response. Dendritic cells are injected interdermally on the 8th day; a solution also contains lysate of tumorous cells of the patient. A quantity of injected cells varies from 2*105 up to 2*107. A course of treatment consists in injecting activated lymphocytes and dendritic cells on the 4th and 8th day respectively. A number of injections is equal to 6. Treatment is repeated after 2-3 months.

Thus, immunotherapy comprises three components: dendritic cells loaded with tumorous antigens, activated autologous lymphocytes and lysate of tumorous cells (Fig. 1).

Results

All the patients included into the study are alive. Fig. 2 demonstrates that a period of time which passed from beginning of specific antitumorous immonotherapy exceeds a temporal interval between two operations in 5 out of 6 patients; they are equal in one patient. This interval is characterized by six-eightfold increase in some patients. However, all the indices are characteristic of the present situation and the certain stage of the study; thus, they do not reflect survival. An average temporal interval passing from the beginning of immunotherapy is 10.5 months.

According to Karnovsky’s scale, the initial score at the beginning of immunotherapy was equal to 40-80. The analysis of the score variations showed that treatment resulted in its reduction by 10-20 points in 4 patients and its increase by 10 points in 1 case; it did not change in 1 patient. The difference between an average score before treatment and its value at the moment of analysis was 8 points (65 versus 58).

All the patients had a cutaneous reaction in the area of vaccine injection manifesting itself in hyperemia; there was local muscular pain in 5 cases. Hyperthemia (up to 39oC) was watched on the day of injection in one patient. These reactions did not demand special treatment and disappeared in 1-2 days.

We considered these reactions to be a positive factor indicative of activation of the immune system. Repeated courses of immunotherapy were characterized either by their intensification or appearance if they had been absent before.

This phenomenon is an indirect proof of necessity of repeated courses of treatment.

Here are some clinical examples:

  1. Patient T., aged 28, underwent 4 operation for glioblastoma of the right parietal and occipital lobes. The interval between them was equal to 27, 13 and 4 months. Specific immuniotherapy was started after the 4th operation. The results of MRI before and after the forth operation are given in Fig. 3. Fig. 4 illustrates the results of MRI examination 4 and 6 months after the beginning of immunotherapy. They are indicative of absence of tumor growth 6 months after immunotherapy. The interval between the last two operations is exceeded by 2 months. The patient is alive; the score of social adaptation according to Karnovsky’s scale has been increased by 10 points and is equal to 80.
  2. Female patient G., aged 58, was operated for glioblastoma of the right parietal lobe. There was total resection. Chemo- and radiation therapy resulted in worsening of the state 2 months after the operation. Growing glioblastoma was diagnosed (Fig. 5). The second operation and tumor removal was followed by specific antitumorous immunotherapy. CT examination carried out in 3 and 7 months demonstrated no growth of tumor (Fig. 6). The patient is alive. The score according to Karnovsky’s scale remained unchanged and is equal to 60 points.

Conclusions

We have failed to find any available reports which would describe treatment of patients with malignant gliomas with the help of dendritic cells. The results of the present study show that specific antitumorous immunotherapy prolongs a period of survival which exceeds an interval between the last two operations. This phenomenon has not been watched before application of immunotherapy (it is marked to the utmost in patients operated more than two times). Specific immunotherapy has a curative potential whose maximum efficacy is to be clarified as the terms of observation are not sufficient for making certain conclusions. We have not watched any serious complications in patients treated by our method. The positive results obtained by us in application of technology of dendritic cells in cases with growing malignant gliomas allow to recommend this method for treatment of patients with other malignant tumors of the nervous system.

Larger groups of patients and further observation will dot the “i’s” and cross the “t’s”.

References

  1. Anin E.A., Shcheglov V.I., Osinsky S.P. On expediency of operative treatment of malignant gliomas and prospects of intraarterial chemotherapy//Ukraiinsky Zhurnal Maloinvasivny i Endoscopicheski Khirurgii.-1998.-N2;4.-P.50-53 (Rus.).
  2. Golanov A.V. Glioblastomas of cerebral hemispheres: Results of combined treatment and factors effecting prognosis. Author’s abstract of thesis for a degree of Doctor of Medical Science.- Moscow, 1999 (Rus.).
  3. Marchenko S.V. Complex treatment of malignant gliomas of cerebral hemispheres. Author’s thesis for a degree of a Candidate of Medical Science.- Saint Petersburg, 1997 (Rus.).
  4. Trapeznikov N.N., Aklsel E.M. et al. Statistics of malignant neoplasms and a state of oncologic care in countries of the CIS. The II Congress of Oncologists of Countries of the CIS, Ukraine, Kiev, May 2000 (Rus.).
  5. Ulitin A.Yu. Epidemiology of primary cerebral tumors in a population of a large city and ways of improvement of medical care rendered to patients with this pathology (Saint Petersburg model). Author’s abstract of thesis for a degree of a Candidate of Medical Science.- Saint Petersburg, 1997 (Rus.).
  6. Fine H.A., Dear K.B. et al. Metaanalysis of radiation therapy with and without adjuvant chemotherapy for malignant gliomas in adults. Cancer 71:2585-2597, 1993.
  7. Liau L. et al. Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens. J. Neurosurg. Vol.90, p.1115-1124, 1999.
  8. Parney I. et al. Glioma immunology and immunotherapy. Neurosurg. Vol.46, N 4, 778-792, 2000.
  9. Takakura K., Abe H. et al. Effects of ACNU and radiation therapy on malignant glioma. J. Neurosurg. N 64, p.53-57, 1986.
  10. Walker M.D., Alexander E. Jr., Hunt W.E. et al. Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas: A cooperative clinical trial. J. Neurosurg 49:333-343, 1978.

Fig 1.
Fig. 1. A diagram of components of specific antitumorous immunotherapy.

Fig 2.
Fig. 2. A diagram of correlation of intervals between preceding operations (the first columns) and an interval passing from the beginning of immunotherapy (the last columns).

Fig 3a.Fig 3b.
Fig. 3. MRI examination of patient T. before the forth operation: 1 – a tumorous node (glioblastoma) in the right parietal and occipital lobes, 2 – postoperative examination – total resection , a tumor bed is filled with liquor.

Fig 4a.Fig 4b.
Fig. 4. MRI examination of patient T. 4 and 6 months after the beginning of immunotherapy: there is no evidence of tumor growth.

Fig 5a.Fig 5b.
Fig. 5. CT examination of patient G. before the operation: 1 – glioblastoma of the right parietal lobe, 2 – results of CT two months after the operation and chemo- and radiation therapy. There is further growth of glioblastoma.

Fig 6a.Fig 6b.Fig 6c.
Fig. 6. CT examination of patient G. 3 and 7 months after repeated operation for growing glioblastoma of the right parietal lobe and the beginning of specific antitumorous immunotherapy. There is no evidence of further growth of glioblastoma.