Innovations in Oncology

Proteome-based individualized anticancer cell systems in neurooncology: from postgenome technologies of proteome mapping and protein profiling to modeling and peptide engineering of stem and progenitor cells

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REGENERATIVE TECHNOLOGIES IN CANCER THERAPY

REGENERATIVE TECHNOLOGIES IN CANCER THERAPY

DEVELOPMENT OF UNIVERSAL PRODUCTION TECHNOLOGY OF PERSONIFIED ANTITUMOR CELL PREPARATION AND ITS APPLICATION

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FGBU N. N. Blokhin Russian Cancer Research Center of RAMN
FGBU “V. P. Serbsky Federal Research Center of Social and Forensic Psychiatry” of the Ministry of Healthcare of the Russian Federation
FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA  of Russia,
ANO «National Institute of Regenerative Medicine”,
ZAO “NeuroVita Clinic of Interventional and Restorative Neurology and Therapy”

 «APPROVED»
At the meeting of Scientific Board of Research Institute of Experimental Diagnostics and Tumor Therapy of FGBU N.N. Blokhin Russian Cancer Research Center of RAMN

                                                                        Meeting protocol №                      dated  June 26,  2012
        «APPROVED»
At the meeting of FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA of Russia  
                                                                        Meeting protocol №                      dated June 20, 2012

PROGRAM OF COOPERATIVE SCIENTIFIC RESEARCH
«PROTEOME-BASED PERSONALIZED IMMUNOTHERAPY OF MALIGNANT BRAIN TUMORS»
                                                                      
Confidential
May not be used, divulged, published or otherwise disclosed without the written consent of FGBU N. N. Blokhin Russian Cancer Research Center of RAMN, FGBU “V. P. Serbsky Federal Research Center of Social and Forensic Psychiatry” of the Ministry of Healthcare of the Russian Federation, FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA  of Russia, ANO «National Institute of Regenerative Medicine”, ZAO “NeuroVita Clinic of Interventional and Restorative Neurology and Therapy”
                                 


Moscow 2012 
List of Participants :
Research coordinators:
Professor Bryukhovetskiy Andrey Stepanovich, PhD, MD, Director General of ZAO NeuroVita Clinic of Restorative Interventional Neurology and Therapy  
Averyanov Alexander Viacheslavovich, PhD, MD, Deputy Director of FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA  of Russia



Main Investigator:
Professor Baryshnikov Anatoliy Yurievich, PhD, MD, corresponding member of RAMN, Head of Research Institute of Experimental Diagnostics and Tumor Therapy of FGBU N.N. Blokhin Russian Cancer Research Center of RAMN


PARTICIPANTS:
 FGBU N. N. Blokhin Russian Cancer Research Center of RAMN
Director                     Prof. Mikhail Davydov, PhD, MD, member of RAN and RAMN
ANO «National Institute of Regenerative Medicine”
Director Ad Interim                                Prof. Nikita Evseev, PhD, MD
FGBU “V. P. Serbsky Federal Research Center of Social and Forensic Psychiatry” of the Ministry of Healthcare of the Russian Federation
Director Ad Interim                                 Prof. Zurab Kikelidze, PhD, MD
“Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA of Russia
Director                                     Prof. Oleg Kuzovlev, PhD, MD
ZAO “NeuroVita Clinic of Interventional and Restorative Neurology and Therapy”
Director General                                 Prof. Andrey Bryukhovetskiy, PhD, MD
                                                   



PROGRAM PASSPORT
Title      PPROTEOME-BASED PERSONALIZED IMMUNOTHERAPY OF MALIGNANT BRAIN TUMORS
Basis for project Development     Decision of Joint Scientific Board of FGBU N.N.Blokhin Russian Cancer Research Center of RAMN #16 dated September 21, 2011
Program ordered by    ANO National Institute of Regenerative Medicine
Basic sites    1.    Head organization - of FGBU N.N.Blokhin Russian Cancer Research Center of RAMN
2.    FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA  of Russia
3.    FGBU “V. P. Serbsky Federal Research Center of Social and Forensic Psychiatry” of the Ministry of Healthcare of the Russian Federation
4.    ZAO “NeuroVita Clinic of Interventional and Restorative Neurology and Therapy”
Goals and Objectives    -    development of new biotechnologies to produce and apply personalized anticancer cell immune preparations (customized vaccines and peptide-engineered preparations of immunocompetent, stem and progenitor cells) for the therapy of neurooncological disoreders with cell, genome and post-genome (proteome) and bioinformation technologies;
-    optimization of technologies of culturing, immunoseparation, proteome lysing, mapping and profiling of hematopoietic, neural, mesenchymal, stem, progenitor and stem cancer cells isolated from the tumor of specific cancer patient
-    Comparative bioinformation analysis of proteome profiles of postnatal stem and progenitor cells with analogous profiles of their cancer stem cells and development of a mathematical model to diagnose proteins, microRNA and various low-molecular combinations able to remodel proteome profiles of autologous hematopietic progenitors in peptide-engineered fashion and to restore their regulatory anticancer properties;
-    Development of peptide-engineered biotechnologies of production of proteome personalized stem/progenitor and /or immunocompetent cell preparations for experimental individualized immunotherapy in animal models of neurooncological pathology.
-    Pilot trials of proteome-personalized immunotherapy in the cases of glioblastoma and brain metastases of lung and breast cancer
-    Limited introduction of interventional post-genome technologies of neuroproteomics involving postnatal and cancer stem cells of humans into clinical practice of head medical institutions of the Ministry of Health of Russia, of FMBA of Russia, of RAMN.
-    Efficiency assessment of immunotherapy with proteome personalized immunocompetent cells in animal model and in clinical practice in neurooncological hospital and development of regulation basis for application of these peptide-engineered technologies in clinic
Implementation Period    2012-2017
Subprograms    
Implementation Periods    First stage 2012-2013
-    Analysis of the issue of personalized anticancer cell preparations in literature, the Internet and planning work in neuroproteomics, mapping and profiling proteins of postnatal regional and cancer stem cells in animal models of nerooncological disorders.
-    Development of bioinformation and mathematical models to analyze the results of proteome mapping and profiling of stem cells and diagnose biochemical agents able to provide anticancer peptide-engineering remodeling of proteome profile of progenitor systems.
-    Approval of Ethics Committees for development of cell technologies and remodeling therapeutic cell systems
-    Experimental surgeries in the therapy with proteome-personalized vaccines, cytotoxic lymphocytes, hematopoietic stem cells with remodeled proteome profile in animal models
-    Appropriate equipment of involved research clinical centers of the Ministry of Healthcare of Russia and RAMN
-    Appropriate personnel training
-    Establishing business and academic links with leading global neurooncological centers
-    Preparation of the protocols and organization of pilot trials in proteome-based personalized immunotherapy of brain cancer in clinic.

Second stage 2013-2017
-    Implementation of main events of Subprogram to apply proteome-personalized immunotherapy in experiment and clinical practice
-    Introduction of the results of experimental research into clinical practice
-    Introduction of high technologies of personalized immunotherapy of rain tumors into medical practice for patients’ therapy
List of main events    -    Providing appropriate equipment and agents to the participants
-    Appropriate personnel training
-    Development of anticancer cell technologies and methods of intervention in the therapy of mammals and humans with brain tumors
-    Information support of the participants
Co-executors     1.    GOU VPO Federal University of the Far East (Vladivostok, Russia)
2.    FGBU A.L.Polenov Russian Research Neurosurgical Institute (Saint-Petersburg, Russia)
3.    ANO National Institute of Regenerative medicine (Moscow, Russia)
Funding    The program is funded privately as well as through budget and off-budget funds of the participants
The program requires funds totaling 150 000 000 (one hundred fifty million roubles)
Expected results     -    Scientific bioengineered technology of proteome-personalized immunotherapy of brain tumors of humans
-    Introduction of proteome-based personalized immunotherapy into clinical practice
-    Improvement of life quality and survival rates in the patients with incurable neurooncological disorders
Supervision and independent expertise     Science and ethics:
Scientific Boards  and Ethics Committees of FGBU N.N. Blokhin Russian Cancer Research Centre of RAMN and FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA  of Russia

Implementation
Joint Scientific Board  of FGBU N.N. Blokhin Russian Cancer Research Centre of RAMN

Independent expertise of research
Propose to Prof. Bellur Prabhakar, (Chicago University of Illinois, USA)

Financial Expertise
Propose to supervisory Board of International Fund of Cell Technologies Development

PROBLEM BACKGROUND AND PROGRAM MEANS TO RESOLVE IT

Incidence of tumors of central nervous system (CNS) is relatively low, they constitute about 1.5% from general mortality rates in the population of Russia. Among organic diseases of central nervous system tumors incidence vary from 4.5% to 6%, in average the percentage of brain tumors is up to 5.4%, spinal cord tumors up to 4%. They are most frequent in the age group from 20 to 40 years, in males. Significance of CNS tumors is conditioned by several factors: 1) the severity of the pathology that depends not only on the grade and severity of neoplastic process, but also on its localization; 2) dependence of therapy outcomes on early diagnostics; 3) frequency of errors in diagnostics which is one of the highest related to other disorders of nervous system (Akimov G.A.; 1982). However, social meaning of “brain tumor” words is extremely depressive and consequent degree of disadaptation, virtual disability after tumor removal is tremendous.
As distinct from tumors of other organs, brain and spinal cord tumors have certain peculiarities conditioned by their location in central nervous system. They develop and grow intracranially in the skull cavity, or intravertebrally in the cavity of spinal canal, that have limited space.  Intracerebral or intracranial content is represented by cerebral tissue, blood and cerebrospinal fluid. In this respect tumor growth leads to high intracranial pressure and eventually to the development of hypertension syndrome.
Clinically brain tumors manifest in focal symptoms resulting from excitation and reduction of damaged brain compartment. As tumor grows focal symptoms deteriorate and are replaced by functional losses. Assessing focal symptoms one should keep in mind that they can intertwine with dysfunction of tumor neighboring structures due to edema, vascular disorders and other factors. Hypertension increases as well as general brain disorders (Karlov V.A., 2002).
Growing tumor interferes with CSF circulation and contributes to CSF accumulation in ventricular system, compresses blood vessels and disoreders blood supply of brain. Tumor causes destructive changes in brain tissue it compresses and consequently leads to inflammation and edema. Superficial tumors result in reactive changes in meninges and skull bones. Malignant tumors and cleavage products of brain activity intoxicate the brain. Sometimes the tumor calcifies and hemorrhages develop in the tumor tissue (Misyuk N.S. et al, 1988).
 
 Tumors of central nervous system (CNS) are divided into primary and secondary. Primary tumors develop from CNS tissue directly. Secondary tumors are metastatic, i.e. from metastases of lung cancer, stomach cancer, breast cancer and pancreatic and prostatic cancer. Primary tumors originate from neuroepithelial tissue (astrocytomas, oligodenrogliomas, epyndemomas, papillomas etc), roots of cranial and spinal nerves (schwannomas, neurofibromas) and meninges (meningiomas, meningeal sarcomas etc). Tumors of CNS can develop out of precursor cells and embryonic stem cells (germinoma, teratoma and other). Similar clinical picture can be caused by tumor-like processes, such as Rathke’s cleft cyst, epidermoid cyst (cholesteatoma), dermoid cyst, parasitar conglomerates of cysticercus, echinococcus, infectious granulomas like tuberculomas and gummas.  Apart from other CNS tumors stand tumors of sella turcica (adenomas, carcinomas and craniopharingiomas). Tumors are able to grow out of neighbouring tissues (chordomas). Benign, indolent (neurinoma, astrocytoma, teratoma, oligodendroglioma, gemangioma) and malignant undifferentiated and fast-growing tumors (glioblastoma, medulloblastoma, germinoma) are distinguished among CNS tumors. Tumors are subdivided into solitary and multiple, extra and intracerebral.
According to localization, brain tumors are classified into three big groups:  supratentorial (hemispheric, intraventricular, subcortical), subtentorial (of cerebellum, of brain stem, of IV brain ventricle etc) and hypophysial (hypophysis, sella turcica and other).
According to histological structure and differentiation degree of cell elements one distinguishes neuroectodermal, meningeal-vascular, mixed, hypophyseal, heterotopic, systemic, metastatic tumors as well as tumors histologically matching the tissues growing into cranial cavity.
Neuroactodermal tumors (gliomas) that develop from cell elements, derivatives of neuroectoderma, are the most frequent. Among them one can specify heterotypic tumors consisting of undifferentiated cells (medulloblastomas, multiformed spongioblastomas etc) with rapid infiltrative growth. Medulloblastoma is a malignant tumor found predominantly in children; it localizes in cerebellum and brainstem and is able to give metastases along SCF pathway. Multiform spongioblastoma is a tumor mostly found in middle aged and elderly people in brain hemispheres. Tumors growing out of microglia (polar spongioblastomas, astrocytomas), oligodendroglia (oligodendrogliomas), epindyma (ependymomas), villous epithelium (chorioidpapillomas), Schwann’s sheath (neurinomas).   
The diversity of neurooncological disorders is obvious from the above. We consider it expedient to concentrate on the most malignant of them, namely gliomas and glioblastomas. Experimental modeling of them, detection of patterns and peculiarities of carcinogenesis of this severe neurooncological disease will give us a clue to the development of new therapies of all neurooncological pathology through new highly technological approaches.

In recent research most experts agree that the key to carcinogenesis of neurooncological disorders lies in genetic instability and high susceptibility of neural cells to mutagenesis observed in all neurooncological disorders.
To date the specialists detected almost all key suppressor genes altered regulation of which leads to specific neoformations in neural system. Key tumor suppressor genes in leading neurooncological diseases can be systemized in the following way:
1.    Gene Р53 is a multifunctional tumor suppressor located in the short arm of chromosome 17p13, that regulates cell cycle, apoptosis, DNA reparation and secures genome integrity.
- Neurotumors associated with p53: neuroblastoma.
- Other tumors associated with 53: lung cancer, breast cancer, hepatocarcinoma, skin carcinoma, Burkitt’s lymphoma, t-cell leucosis.
2.    PTEN gene is tumor suppressor located in 10q 23.3, regulates cell cycle and stimulates apoptosis regulating PI3k-PKB/Akt signal pathway.
- Neurotumors associated with PTEN: glioma, glioblastoma, meningioma.
- Other tumors associated with PTEN: melanoma, kidney cancer, uterine cancer, breast cancer, prostate cancer, colon adenocarcinoma, lymphosarcoma, germinal tumor.
3. VHL gene is a tumor suppressor located in 3р25-26, regulates reaction towards hypoxia, inhibits angiogenesis, and suppresses VEGF expression and other hypoxia genes.
- Neurotumor associated with VHL gene: CNS hemangioblastoma.
- Other tumors associated with VHL gene: retinal angioma, pheochromacytoma, kidney carcinomas, pancreatic cancer.
4. NF1 gene/neurofibromin and NF2 gene/merlin. The former is a tumor suppressor located at 17q11.2 that inactivates active RAS genes, the latter is located at 22q11.1 and links cell membrane with cytoskeleton.
- Neurotumor associated with NF1 and NF2 genes: neurofibromatosis type I (Reclinghausen disease) and neurofibromatosis type II, neurinoma, meningioma, schwannoma.

Studies of “candidate” genes demonstrated that epigenetic events played an important role in the pathogenesis of glioblastomas and medullblastomas most frequent in children. In order to evaluate epigenome of medulloblastoma the team of Jennifer Anderton (JA Anderton, 2008) completed genome study and determined the genes that were characterized by methylation dependant regulation. Microarray analysis of expression in medulloblastoma cells after DNA methyltransferase inhibitor treatment revealed deregulation of multiple transcripts (3%-6% probes per cell line). Eighteen independent genes demonstrated >3 fold reactivation in all tested cell lines. Bisulfite sequence analysis revealed dense CpG island methylation associated with transcriptional silencing for 12 of these genes. Extension of this analysis to primary tumors and normal cerebellum revealed three major classes of epigenetically regulated genes: (1) normally methylated genes (DAZL, ZNF157, ASN) whose methylation reflects somatic patterns observed in the cerebellum, (2) X-linked genes (MSN, POU3F4, HTR2C) that show disruption of their sex-specific methylation patterns in tumors, and (3) tumor-specific methylated genes (COL1A2, S100A10, S100A6, HTATIP2, CDH1, LXN) that display enhanced methylation levels in tumors compared with the cerebellum. Detailed analysis of COL1A2 supports a key role in medulloblastoma tumorigenesis; dense biallelic methylation associated with transcriptional silencing was observed in 46 of 60 cases (77%). Moreover, COL1A2 status distinguished infant medulloblastomas of the desmoplastic histopathological subtype, indicating that distinct molecular pathogenesis may underlie these tumors and their more favorable prognosis. These data reveal a more diverse and expansive medulloblastoma epi genome than previously understood and provide strong evidence that the methylation status of specific genes may contribute to the biological subclassification of medulloblastoma.

The other no less important component of carcinogenesis of neuroncological diseases is defective intracellular signaling of neural cells. To date, defects in intracellular signaling in main tumors of brain are almost fully described by molecular biologists. Admittedly, this direction in diagnostics and development of promising methods for brain tumor therapy is top priority in modern neurooncology.

Malignant astrocytar gliomas constitute up to 50% of all primary brain tumors with half of them being glioblastomas (GB), the most aggressive variant with the worst prognosis, when median life expectancy after diagnosing does not exceed 50 weeks. According to National Cancer Institute of US gliomas incidence rate is about 6.5 cases per 100 000 population. This is about 44% of all primary brain tumors. At the same time about 52% of all gliomas are polymorphous glioblastoma. Average age of GB patients is 57 years. Main risk factors are high dose of radiation, inborn syndromes and ageing. Last 25 years demonstrated clear tendency to malignant brain tumors incidence increase along with cancer incidence and mortality rates increase. GBs are more often detected in males, the prognosis is unfavorable, average life expectancy is 14.6 months (Stupp R, 2005) with an insignificant number of survivors over three years and more.
Usually glioblastoma is considered a sporadically developing tumor. Still, occurrence of glioblastoma is higher in the families with the history of breast cancer, colon cancer, soft tissues sarcoma and leucosis.
GB mostly localizes in brain hemispheres. Within brain hemispheres tumors locate in white matter frequently spreading into deep structures including corpus callosum, subcortical nodes and lateral ventricles.  About 10% of hemispherical GBs are located on a surface with an epicenter in the site of transition of grey matter into white. These tumors have circumscribed microscopical borders and are similar to metastases to an extent.
Progress of symptom development in glioblastoma patients varies. In 10-15% of GB cases the symptoms are preceded by a long, up to a few years, period of spastic seizures. Concurrently, patients with long-term anamnesis of paroxysms have more favorable prognosis, which might be associated with possible malignant transformation of preexisting benign astrocytoma.
It is more typical when clinical manifestations appear 3-6 months before diagnosing and are represented by enhanced intracranial hypertension and focal neurological symptoms. Fulminating development of symptoms in several weeks is observed in 15-20% of cases which is often related to the hemorrhage in tumor tissue.
The most informative diagnostic methods for brain and spinal cord are computer tomography (CT) and magnetic resonant imaging (MRI) with contrast enhancement. Heterogeneous change of density is typical feature for GB; it is mainly of hypodensive type with circular area of density enhancement which can be especially clearly seen in contrasted MRI. In a quarter of cases classical “circular” type of contrast enhancement can be absent, in such patients mixed or homogeneous type of tumor contrasting is found. Calcifications are rare in glioblastomas. Usually contrast enhanced tumor part is surrounded by perifocal edema vastly spreading through white matter of brain. The area of peritumoral edema commonly does not exceed the size of tumor and this differs GB from metastatic tumors.
 MRI displays heterogeneous tumor structure characteristic for GB. T1 mode visualizes unclearly circumscribed space-occupying lesion with mixed (iso- and hypointense) signal, central necrosis often with decreased signal intensity as compared to tumor mass.  T2 mode imaging displays mosaic of various signal intensities with hypo-, iso- and hyperintense signals from GB stroma, necrosis, cysts and hemorrhages. Small tumors are often accompanied by mass effect and white matter edema. Tumor borders can assimilate with perifocal edema. Hyperventilation enhances imaging of solid tumor.
Objectifying the tumor volume after the surgery, MRI with contrast enhancement is most informative when done first 24 hours post surgery.
Positron emission tomography (PET) is not applied to verify initial diagnosis, as the quality of imaging and resolution is inferior both to CT and MRI; however, it can be used for differential diagnostics of radiation necrosis and continued GB growth.

For the first time the term glioblastoma was used in CNS tumors classification of Bailey and Cushing, based on histological principle. The concept of tumor progression took the leading positions by the middle of 20 century; it supposed tumor emergence not from embryonic cell germs but as a consequence of stage neoplastic transformation of stem cell elements of a mature organism. Classificatory grade of St. Anne-Mayo became more popular in modern neuromorphology. The grade has four categories of astrocytar gliomas that differ according to the level of malignancy. The latter is determined according to four morphological criteria: nuclear atypia, mitosis, endothelial proliferation and necrosis. The sum of criteria grades malignancy of tumor. The fourth malignancy grade according to St. Anne-Mayo corresponds to WHO glioblastoma and is identified depending on simultaneous detection of three or all four pathohistological features. In Russian Burdenko Scientific Research Institute of Neurosurgery cytological classification was developed, that includes three types of glioblastomas: isomorphocellular, polymorphocellular and gemistocytar.
Most frequently brain metastatic tumors result from lung cancer, stomach cancer, breast cancer and pancreatic and prostatic cancer.  Experts of Blokhin Russian Cancer Research Centre of the Russian Academy of Medical Sciences Prof. Komarov and Prof. Komov (Komarov IG, Komov DV, 2002) analyzed frequency rate of brain metastases depending on the cancer type. Of 251 patients with isolated tumor, only 16 patients developed metastases to brain. In 14 of those (87.5%) malignant process was detected in the first 3 months after the onset, in 6.5% - in the period from 3 to 6 months, in the rest cases – in 12 months. Notwithstanding, the survival rate of the patients with brain metastases is quite deplorable. None of the patients survived for 3 years.

Currently, conventional therapy of GB implies combination of surgery, chemo and radiotherapy.
Present-day neurosurgery perceives maximal radical excision of GB to be the procedure of choice as it leads to single-step elimination of a great number of viable tumor cells, including therapeutically resistant pools, and to decrease of intracranial hypertension. Single-step removal of big mass of tumor cells activates proliferation in the remaining, allegedly rendering them more susceptible to therapeutic treatment, which is most efficient for the mitotic cells. Besides, extensive surgery is accompanied by violation of blood-brain barrier thus facilitating access of chemical agents to the site of their direct activity.
Repeated surgeries in GB are controversial: lifespan gain measured in several weeks is incomparable with risk and effort, while life time with good life quality is minimal. Indications to repeated surgery are determined by age, patient’s state and progress rate of tumor relapse.
Planning is extremely important for the forthcoming surgical operation. For it the volume of contrasted part of tumor is calculated in three dimensions, and involvement of other anatomical structures is assessed. The access is chosen to minimize cortical incision and transcortical path. The volume of removed mass should correspond with the pre-surgically detected size of neoformation.
To verify histological diagnosis stereotaxic biopsy (STB) under CT or MRI control is advisable. Indications for STB are deep-seated tumors, less than 2 cm tumors, cystic tumors and primarily multiple tumors. STB is recommended in case of radiological semiotics change, critical condition of the patient or, on the contrary, in case of minimally manifested neurological deficit.  
The most efficient therapy for GB is the surgery followed by radiotherapy. The effect of ionizing radiation on the cell level is associated with DNA molecules damage by electrons and free radicals resulting from interaction of X-ray and γ photon radiation with water molecules. One of the reasons for radioresistance of gliomas lies in lower oxygen tension as compared to adjacent brain matter. Life expectancy correlates with increase of cumulative focal dose of radiation to 70 Gray. Further increase of dose is limited by radiation necrosis. Tumor growth during radiation is a prognostically unfavorable sign. Electron acceptor compounds (Metronidazol, Misonidazol) are radiosensitizers and are able to enhance therapeutic sensitivity of anoxic tumor cells to ionizing radiation.  Interstitial brachytherapy permits direct delivery of high dose radiation to tumor through radioactive iodine, gold, iridium isotopes. Alternative to interstitial brachytherapy, stereotaxic radiosurgery is done by linear accelerator, though the results of its application in GB patients appeared to have no priority over teleirradiation. The disadvantage of the method is maximal localization of radiation dose that does not affect GB infiltration areas.
Currently the researchers attempt to enhance the efficiency of interstitional radiation combining it with chemotherapy, tissue hyperthermia, and photodynamic therapy.
Actively studied neutron therapy is a bimodal form of radiation therapy based on selective accumulation of stable boron isotopes and following neutron radiation. Energy of heavy particles releases when boron isotope interacts with neutrons and destroys cells spreading only in close vicinity (<10m) to place of isotope accumulation.
Several reasons can be given to explain for low chemotherapeutical sensitivity of GB. Primarily blood-brain barrier prevents adequate dose of preparation from accessing tumor tissue. Apart from it, drug resistance of GB can be conditioned by the reasons, common to all tumors, such as reduction of drug intracellular accumulation, inactivation of cytochromes and diaphorases enhancing chemotherapeutical influence, elevated concentration of ferments and protein substances, inactivating or destroying medications. Structural and biological heterogeneity of GB can also explain for its drug resistance.
Even though some aspects of drug resistance can be resolved through drug dose increase, such approach can lead to neurotoxicity.
Use of nitrosourea was the basic method of malignant tumors therapy. BiCNU (Carmustin) is the most efficient in the patients with progressing gliomas. Administration of nitrosourea chemotherapeutic agents differentiate depending on various types of GB: isomorphous cell tumors require such therapy in case of subtotal surgical removal of the tumor; polymorphous cell tumors need it in case of partial removal of the tumor, while the prognosis for hemistocytar type of tumor deteriorates after administration of the therapy.
Currently, Temozolomide (Temodal) in the dose of 200 mg/m2 orally is the medication of choice.
The fundamental work Principles of Neuro-Oncology published in 2005 and edited by David Schiff and Patrick O’Neill (D Schiff, 2005) first presented the cornerstone principles of practical approaches to medication of brain glial tumors on the ground of conventional as well as novel data about tumor genesis in the light of recent founds of signaling transduction pathways, that basically govern glioma growth.
Growing knowledge in molecular biology of brain tumors lead to the development of new targeted compounds. What are the prospects of conventional chemotherapy of procarbazine and CCNU used for brain tumors? Without any doubt, oncology benefited from occurred molecular revolution most of all, but still, this revolution brought additional attention to traditional drugs rather than distracted it from them. As for example, 1p chromosome deletion or simultaneous deletion of 1 p and 19q chromosomes in oligodendroglial gliomas of intermediate grade (anaplastic oligodendrogliomas) shows that the tumor is likely to be sensitive to standard therapy with alkalinizing chemotherapeutical agents. Supposedly, one or both of chromosomes have gene(s) that code protein product(s) endowing tumor cells with resistance to chemotherapy.  Its deletion results in sensitivity. It can be said that the progress in molecular biology of brain tumors is important not only for finding new targets and new agents for them, but also for enhancing efficiency of the drugs long in use: a) the drug will be chosen specifically in each case, and b) the development of resistance to traditional drugs will be more understandable. Then, neurooncologists would not only develop new medications, but use the older ones more effectively.
By now a lot of evidence has been accumulated by molecular genetic analysis of signal transduction and effector molecules, i.e. those molecules which are responsible for malignant features, such as invasiveness, motility and angiogenesis. All this must be applied to clinical practice, as it is ubiquitously known that high grade glioma is synonymous to chemotherapy resistance. Considering that oncology follows the way of identification and selective elimination of the components of biochemical pathways, it can be stated that the methods of growth regulation in high grade gliomas are an ideal platform to study available and just developing therapies giving respect to novel achievements of molecular biology.
All types of tumor cells pass through the same sequence of stages that determine their malignant phenotype. Hence, the targets for the therapy in all cases are the same and present the basis for the development of target drugs for tumor chemotherapy. Here we would like to dwell on the targets.
Signal transduction. This is the mechanism of enhancement and transferring the signal out of the cell into the one understandable at the level of nucleus and initiating expression of specific genes. Interaction between growth factors and their receptors at the cell surface activates the components of secondary messenger (as, for example Ras, PKC) that provide biochemical drainage binding components located closer to the cell membrane with its nucleus.  When they reach the nucleus, transduced signal stimulates proliferation (cell cycle) and induces the genes that impart tumor cells with malignant features. Newly emerging therapies of brain tumors mostly rely in their action on the components of signaling pathways. Theoretically, each of the stages of this pathway complex can serve as a target.
 Targeted action on the damaged pathways of signaling transduction in GB can be provided by  17-N-Allylamino-17-demethoxygeldanamycin  (17-AAG), well known as HSP90 inhibitor. Sauvageot et al. (2008) examined the properties of 17-AAG to inhibit growth of glioma cells and glioma stem cells both in vitro and in vivo and possible synergic effect in combination with radiotherapy and/or Temozolomide (Temodal) conventionally used for GB therapy. Resulting data showed that 17-AAG in vitro inhibits glioma cells and glioma stem cells, as well as affect specific proteins within these cells. Besides, 17-AAG inhibits growth of intracranial tumors and enhances radiotherapy, as shown in tissue culture and intracranial tumors. The action of Temodal was not enhanced in any of the glioma models, meaning that HSP90 inhibitors, similar to 17-AAG can have therapeutic effect on GB either as monotherapy or in combination with radiotherapy.         
2. DNA replication and cell division. Cell proliferation is the response to the signals for growth issued to the cell nucleus from outside. The process of cell division (mitosis) is a final stage, a so-called M-phase of mitosis, of a four-phase circular scheme, called a cell cycle. In its course, the cell passes through G1, S, G2 and m phases. One of inherent properties of tumor cells is their ability to repeatedly cycle without any control, i.e. to proliferate. Cycle of normal cells is strictly regulated by a checkpoint G1/S which is a point of phase transfer from g1 to S (S is DNA synthesis). Glioma cells and the cells of other tumors easily pass through this checkpoint due to mutation of one or several proteins (p53, p16, CDK4, Rb) that “guard” the checkpoint.  So,  theoretically, this situation can be corrected by the correction of genetic disorder in checkpoint components, thus restoring its functions (for example, transfer of normally functioning p16 gene, which is absent in gliomas).   Apart from the substances affecting DNA through cell cycle, there are also substances that do not depend on a cycle, such as “traditional” DNA alkalinizing agents, as platinum and nitrosourea (as BCNU).
3. Effector molecules. Signal transduction of membrane to nucleus stimulates cell cycle development thus stimulating cell proliferation, and also entails expression of genes that condition malignancy including invasiveness and angiogenesis. To illustrate such effector molecules we can name proteases as well as paracrine factors, such as VEGF that stimulates angiogenesis due to the induction of endothelial cells proliferation. As far as invasiveness and angiogenesis are inherent features of all malignant tumors, therapy addressing effector molecules can result in good clinical outcomes. Any of the elements of the cascade of events is a potential therapeutic target.
Hence, main targets and medications of brain tumor therapies can be: 1. Growth factor receptors of cell surface and targeting to growth factor receptor; 2. Neutralizing antibodies to EGFR; 2. Pharmaceutical inhibitors of EGFR and platelet growth factor; 4. Intracellular components of secondary messenger; 5. Effect of farnesyltransferase inhibitors on Ras; 6 Proteinkinase C inhibitors; 7. Agents that directly affect DNA or its replication; 8. Alkalizing agents; 9. Inhibitors of cell cycle; 10. Pharmacological therapy affecting G1/S; 11. Gene therapy to secure G1/S barrier; 12. Proteinase and integrines mediating tumor invasiveness; 13. Effector molecules for targeted action on proteinkinases, integrines, angiogenesis mediators; 14. Methods to enhance agent targeting to tumor; 15. Delivery of larger amount of the pharmaceutical to the tumor: agents increasing blood-brain barrier permeability. 16. Chemotherapy administered directly into brain parenchyma (interstitial therapy) 17. Improved distribution of a pharmaceutical in the brain (convectional approach); 15. Functional targeting of a pharmaceutical to a tumor cell.
Despite significant advances in GB therapies, survival rates remain approximately the same. Only 10% of GB cases survived over 18 months after diagnosing, and 5 year survival is almost zero. Recently the number of GB cases with survival rates over 2 years increased, but general median life expectancy did not change much. Consistent prognostic criteria that prolong life expectancy include age younger than 40, Karnofsky score higher than 70 before and after the surgery, maximal tumor reduction, time-integrated radiation dosage less than 60 Gy. Pathomorphological factors influencing life expectancy in GB cases are the sites with benign astrocytoma structure within the tumor and mononuclear and lymphoid infiltrates with no necrosis foci. The major problem of GB therapy lies in selectively aggressive tumor cells and non-selective therapeutic methods. Approaches to concentrate the agent in tumor tissue selectively should be called chemosurgery. Use of specific autologous stem cells to provide selective transport of aggressive cytolytic agents to tumor stroma is an important condition to enhance selectivity and specificity of the therapy of hyperproliferative neoformations.
As noted previously, applied anticancer therapies often lack efficiency. Besides, their action involves immunosuppression that suppresses bone-blood hematopoiesis and leads to infections as well as intestinal dysbiosis. Immune system compromised by the tumor development is exposed to another stress that weakens its activity. Hence, successful tumor therapy can be dependable on the balance between anticancer efficiency of chemical agents and immune system potential, sufficient or insufficient to cope with survived tumor cells. Therefore, several alternative/additional therapies have been offered, most of which aim to enhance anticancer activity of immune system. This new approach was named immunotherapy, and it has long demonstrated the necessity of its clinical application in the complex therapy of cancer, but so far, has not gained broad use.
The history of cancer and brain tumor immunotherapy dates back to 1892 when Dr. Coley of         Memorial Sloan-Kettering Cancer Center (USA) attempted to treat cancer patients with a brew of Streptococcus culture. In some patients growth of tumors was arrested, other died from cachexy not associated with main disease.  Supposedly, bacterial brew initiated some response that damaged the tumor. In 1962 Dr. O’Malley et al. demonstrated that hemorrhagic necrosis in tumor mice models after bacterial lipopolysaccharides (LPS) is caused by some intermediate factor developing in blood serum after LPS injection. The serum was able to eliminate tumor cells when administered to the mice that had not received LPS injection. And, finally in 1975, Dr. Carswell et al. from Memorial Sloan-Kettering Cancer Institute discovered and described mediator with cytotoxic properties towards tumor cells, developed in blood in response to LPS of Bacillus Calmette-Guerin that was called Tumor Necrosis Factor (Popovich, 2010).
In the beginning of 20-th century Prof. Mechnikov and Prof. Gamalea started discussing a viral nature of malignant tumors. In the 50-s Prof. Silber established a so-called viral-genetic concept of carcinogenesis. His studies of tumor immunology initiated research of tumor antigens and finally to discovery of specific liver alpha-fetoprotein, that led to the development of diagnostic test to liver cancer. April 1998 the National Cancer Institute (USA) launched a wide range program to examine biological approaches to cancer therapy. The decade of research resulted in 3-step method to evaluate efficiency of cancer biotherapy. Efficiency of cancer biotherapies (alpha, beta, gamma interferons, IL-1, IL-2, TNF, CSF, LAK TIL therapies, monoclonal antibodies therapy, growth factors) available at the moment was studied in detail, and new strategies to develop novel therapies were identified.
Cancer immunotherapy is based on weak immune response characteristic to many tumors and well established immunosuppressive action, the mechanisms of which are not clear yet. The state of immune depression is an important pathogenetic factor of unfavorable progress of malignant tumors, and perspectives to treat them are associated with immunotherapy methods. Basic concepts of immunotherapy are the following: 1. Membrane of tumor cells express antigens different from normal. 2. Experimental carcinogenesis and pre-cancer demonstrate immune insufficiency; 3. Clinically detectable growth of neoformations is observed when immune system is misbalanced and it is compromised by antitumor therapy; 4. Higher reactivity of immune system before and after the therapy correlates with better prognosis.
Efficiency of immunotherapy has already been well proved; however, immunotherapy does not influence cancer stem cells (CSCs) as cancer antigens are not represented on the surface membrane of CSC. Currently, it has been universally acknowledged that tumor originates from a CSC that divides either symmetrically (in two CSC or two CSC-progenitor cells) or asymmetrically (into CSC and CSC-progenitor cell). Today CSC is considered an autologous stem cell with critical mutations of genes and aneuploidies that resulted in genome (karyotype) instability, became uncontrollable by immune system regulatory mechanisms, started to continuously proliferate and divide and acquired the features of immortal cell system. The progeny of cancer stem cells (CSCs) are at different stages of differentiation and divide more vigorously as compared to analogous normal cells. Cancer cells acquire genetic mutations that result in abnormal features.  Mutations alter ability of immune system to distinguish cancer cells as abnormal, that are characterized by the absence of growth regulation dependant on growth factors. Cancer cells acquire ability to grow in suspension, they have high mitosis index, are immortal, apoptosis resistant and lost tumor suppressor gene. And certainly, surface membrane of cancer cells at various differentiation stages presents cancer antibodies that can be detected by specific oncomarkers.
Therefore, we need to know how cancer cells and CSCs intrude into immune system. Cancer cells grow faster and have down-regulated antigene expression and, accordingly, lowered immunogenicity, they release immunosuppressing factors, inhibit apoptosis, can rapidly give metastases in other tissues and organs. In response to cancer cells intervention, immune system is activated, which is manifested in cellular response (activation of white blood cells), tissue response (immune reaction of skin, of primary lymphoid organs: bone marrow, thymus; activation of secondary lymphoid organs: liver, spleen, lymph nodes), as well as in released molecules (cytokines, growth factors) of different cells, antibodies (immunoglobulines) released by B cells. As a result, cellular immunity is presented by specifc cytotoxicity of T-cells, NK-cells, macrophages and mediates cytotoxicity, releasing cytokines, while humoral immunity forms antitumor antibodies of B-cells.
Currently, immunotherapy attempts to enhance immune response of a body activating NK cells (cancer control),   macrophages (cytotoxicity), T-cells (specific cytotoxicity) and immunizing B-cells.
Immunotherapy is defined in several ways. Correction of immunity is one of the version of immunotherapy, when defect functioning is restored.  Correction of immunity is achieved through substitutive therapy or immunomodulatory (stimulating or depressive), as well as immune reconstruction. Substitutive immunotherapy replaces lacking effectors of immunity mainly due to antibodies contained in the preparation of immunoglobuline (gamma-globuline), plasma and immune serums. Use of viable donor cells in substitutive immunotherapy has extremely limited indications. Immunomodulating therapy implies influence of regulatory mechanisms on normal or disordered immunity achieved by means of immunomodulators, i.e. dose-dependable agents able stimulate or suppress immunity or activate some elements of immune system suppressing other. The agents that suppress immunity stably are named immunnosuppresants, and those stimulating immunity are called immunostimulants. Immune reconstruction is restoration of immunity usually by transplantation of viable polypotent hematopoietic stem cells of bone marrow or embryonic liver, and more rarely of central organs of immunogenesis, for example, thymus.  
Table 1 shows main types of cancer immunotherapy. As seen from the table no immunoreconstruction is included.
Different immune agents act through one or many mechanisms, namely, they 1) stimulate anticancer immune response increasing the number of effector cells (for example, lymphokines); 2) act as effector or mediator; 3) inhibit immunodeppressive influence of tumor on the body; 4) increase immunogenisity and susceptibility of cancer cells due to immunological reactions 5) improve body resistance to cytotoxic and radiotherapy.
According to Kiselevskiy (2002) effect of immunotherapy depends on basic antigene differences between normal and tumor cells. The advantage of immunotherapy lies in the suppression of cancer cells expansion sparing proliferation of normal cells. Immunotherapy was efficient in the following cancers: 1) melanoma; 2) kidney cancer; 3) non-Hodgkin lymphoma; 4) hairy cell leukemia; 5) rectal cancer; 6) ovarian cancer; 7) glioma and glioblastoma; 8) soft tissue cancer.
Active specific therapy of tumors is based on the ability to affect tumor growth. The therapy intensifies immune response of an organism to the tumor. It appeared that immune system is able to specify tumor cells and to selectively eliminate them. The task of an oncologist is to activate immune system to initiate its work against tumor.
Active immunotherapy has three basic approaches: stimulation of T-lymphocytes; stimulation of normal killers (NK), and manipulations with dendritic cells. The latter methods are mostly determined as vaccination. Sometimes used term “anti-cancer vaccine” displays misunderstanding of the idea. Vaccination is the method to form active immune system with the help of vaccines. And correct immune reaction (absence of immune deficiency, antigene presentation by immune cells) to the vaccine is prerequisite. Other term, immunization, has wider meaning, as it involves formation of both active and passive immunity, achieved in many ways, including administration of immune protection factors (antibodies, immunocompetent cells etc.). Passive immunization is often administered when the immune system of the patient is compromised.
Therefore, only methods that form specific active anticancer or antiviral immunity to carcinogenic viruses (http://www.anticancer.net/reviews/immunotherapy.php) can be considered anticancer vaccines. Anticancer vaccination is the method to initiate specific anticancer immunity by means of a vaccine containing immunogenic antigens. Depending on their composition and mechanism of immune response formation, anticancer vaccines are classified into the following: 1) Vaccines based on whole cells that can be autologous or allogeneic. 2) Antigene vaccines that can be of proteins and tumor cells proteins fragments; of DNA and RNA; recombinant viruses or anti-idiotypic vaccines; and 3) Dendritic cells vaccines.   
To obtain cell vaccines tumor cells are isolated from the patient and expanded in special conditions. Then, the cells are checked for arrested proliferation and for contamination that can potentially infect the recipient and are used for clinical needs. When cell vaccine is administered, the recipient generates immune response against tumor antigens. There are two types of cell anticancer vaccines: 1. Autologous cell vaccines of provisionally deactivated cells of the recipient; 2. Allogeneic cell vaccines of whole deactivated cells of a different person, or several persons.
Table 1.
Main types of cancer immunotherapy




Active    

Specific    Vaccines of deactivated or modified tumor cells, tumor cell extracts, purified or recombinant tumor antigens, idiotypes, viral or bacterial gene-vectors introduced in tumor. Vaccination against carcinogenic viruses, such as hepatitis D, papilloma viruses, T-cell leukemia (HTLV-1), Eppstein-Barr.   
    

Non-specific     BCG, "Imuron", Corynobacterium parvum, Cytokines (alpha, beta, gamma IFNs, Il1, Il2, Il12, TNF, Caebomoilaziridine, Levamisole, thymus hormones preparations, Glucoseminyl muramildipeptide, Terhalose dimycolate, pyran co-polymer, poly-(I:C), pyrimidines, MGN-3
    Combined     Combination of vaccines with cytokines or non-specific immunostimulators


Passive
(adoptive)    
Specific    Elimination of tumor in the body of the recipient by infused lymphocytes with specific anticancer determinants. Monoclonal antibodies, pure (Rituximab, Trastuzumab, Edrecolomab etc) pharmaceuticals, toxins or radioisotopes (Ibritumomab, Tositumomab); conjugated with plant or bacterial toxins; biospecific antibodies; T-cells, dendritic cells.  
    Non-specific
     Administration of immunocompetent donor cells to recipient: lymphokine activated killers (LAK) or tumor infiltrating lymphocytes (TIL) or inducing cytokines killer cells: LAK therapy, cytokine therapy (Il1, Il2, TNF, interferons), lectines  (Iscador), heat shock proteins
    Combined    LAK therapy combines with biospecific antibodies
      
Antigene vaccines do not contain whole cells and only antigens of tumor cells. A tumor can be represented with a wide range of antigens. Some of them are found in all tumors of specific type, others are unique and found only in this very case. Antigene can be introduced into the composition in many ways. Proteins or tumor cell protein fragments are introduced into the organism as a vaccine directly. Genetic material coding these proteins (DNA and RNA vaccines) is administered and the virus can be used as vehicle. Such viruses are called viral vectors and do not initiate disease. These viruses infect human cells in vitro and become carriers of tumor antigens. The virus is able to infect only small number of the cells of organism, which is enough to generate immune response, but insufficient to cause disease.
The methods of gene engineering permit usage of viruses to release cytokines or incorporate proteins at the surface of the virus activating immunocompetent cells. Hence, modified viruses can be introduced to the recipient independently or in combination with vaccine to enhance immune response.
Antibodies can be used as antigens in vaccine. Antibodies to tumor antigens are administered to the recipient, B-lymphocytes release antibodies to these antibodies that also recognize tumor cells. These vaccines are called “anti-idiotypic” and differ from passive therapy with antibodies.
Antigene presenting cell (APC) vaccines are based on APCs that maximally activate T-lymphocytes for elimination of tumor cells. Most frequently, dendritic cells are used. Anticancer vaccines contain dendritic cells that are susceptible to primary action of antigene, or grow in its presence. Antigen (or other APCs) susceptible dendritic cells carry tumor antigens on their surface and activate T-lymphocytes proliferation and their elimination of tumor cells in the body of the recipient.
Anticancer vaccines are predominantly experimental type of therapy so far. Currently, a lot of vaccines pass clinical trials. April 8, 2008 Antigenics announced that Oncophage® (HSPPC-96) was approved in the Russian Federation to treat kidney cancer cases with high risk of recurrence. The vaccine is a heat shock protein isolated from specific tumor and targeted specifically on it. Oncophage was tried as the vaccine to prevent kidney cancer relapse, namely hypernephrome. Antigenics report  that Oncophage prolonged period of remission by 45% in kidney cancer cases with low risk of relapse, in average 1.8 years, as compared to control group.   
April 25, 2002 the clinical trial to test anticancer vaccine Onyvax (anti-idiotypic vaccine based on monoclonal antibodies 105AD7) for late stages of colorectal adenocarcinoma) was started in St. George's Hospital, London, UK. The vaccine is administered intradermally together with BCG vaccine or intramuscularly together with adjuvant aluminum hydrate.
Cancer VAX (melanoma multivalent vaccine) is used along with surgical intervention for 3 stage of melanoma in three medical centers of Australia, in France, Israel and 25 US hospitals since February 17, 2000. To enhance cell immune response it is administered simultaneously with BCG.
From March 1, 2001, University of Texas, USA performs clinical trial to treat HLA-A2+ patients with carcinoembryonic antigene (CEA) producing adenocarcinomas of gastrointestinal (GI) tract origin by peptide fragments of this antigen of seven aminoacids (carcinoembryonic antigen (CEA) peptide cap 1-6D) (http://clinicaltrials.gov/show/NCT00012246).
Since April 2, 2001 in Herbert Irving Comprehensive Cancer Center, New York, the peptide vaccine NY-ESO-1 is administered subcutaneously for II-IV stage sarcomas of soft tissues, if the tumor releases antigens NY-ESO-1, LAGE NY-ESO-1 or LAGE. Together with vaccine granulocyte-macrophage colony stimulating factor - GM-CSF is injected subcutaneously for 5 days beginning 2 days prior to vaccine (http://clinicaltrials.gov/show/NCT00027911).
Hoag Memorial Hospital Presbyterian, Newport Beach, California performs the study of autologous treated tumor and dendritic cells suspended in GM-CSF for stage IV renal cell carcinoma from November 8, 2001 (http://clinicaltrials.gov/show/NCT00014131).
The vaccine based on prostate specific membrane antigen for hormone resisting Metastatic Prostate Cancer is being tried in the University of Chicago Cancer Research Center, USA from November 9, 2001. To suppress angiogenesis in metastases IL-12 is administered (http://clinicaltrials.gov/show/NCT00015977).  
Vaccine ALVAC-CEA/B7.1 for colorectal cancer is a deactivated viral strain, the antigene structure of which greatly repeats antigens expressed by colorectal tumors. The vaccine is on trial in several medical centers of the USA. It is administered after the diagnosis is established along with chemotherapy, and first results seem to be very promising with minimal side effects.  
The vaccine VG-1000 acts against protective mechanisms of malignant cells. It is most efficient for carcinoma and melanoma, as well as successfully applied for some types of sarcoma and leukemia. The vaccine is applied when immunity is not depressed by chemo or radiotherapy. VG-1000 can be recommended as the first line drug for newly diagnosed cancer, and to prevent relapses. The method is currently used in two hospitals, the Immune Augmentative Therapy Clinic, Freeport, Grand Bahamas, and in CHIPSA, Center for Integrative Medicine in Tijuana, Baja California, Mexico.
New-York scientists consider the so-called heat shock proteins to be the shortest way to cancer vaccines.
Clinical trial phase II of Tricom vaccine to evaluate efficiency of locally recurrent prostate cancer treatment was started in Maryland. Tricom is an abbreviated combination of three molecule enhancing T-cell response (B7-1, ICAM and LFA-3).The first phase of the trial demonstrated safety of the vaccine.
Summing up the above said, immunization of the patient with tumor antigens, development of anticancer dendritic autocellular vaccines, antigen-specific cytotoxic T-lymphocytes obtained from ex vivo lymphocute activation with tumor antigens seems to be the most promising avenues for immunotherapy.  The methods of autovaccines production on the basis of dendritic cells (DCs) to activate specific antitumor immunity are being vigorously developed. DCs display high potential to present tumor antigens to T-killers and rendering them immune to tumor cells. DCs are generated by peripheral stem cells or bone marrow being cultured with colony-stimulating factors (GM-CSF, IL-4 etc.). DCs are incubated with tumor antigens, administered to the patient and activate specific anticancer immunity in vitro. The procedure improves detection of tumor cells, characterized by low immunogenicity, cytotoxic T-lymphocytes. The patients with extensive cancers susceptible to immunotherapy (melanoma, kidney and colorectal cancers) are included into clinical trials. Also vaccines can be used to treat ovarian cancer. Obviously, it should be used after maximal elimination of the tumor, i.e. after surgical intervention and standard chemotherapy. Besides, surgical intervention will provide tissue sample, which antigens will be presented by DCs (Kiselevskiy, M.V., 2010).
Another promising way of using immunotherapy for neurooncology is targeted application of graft versus host disease (GVH). Podoltseva E.I. (2003) in her article “Graft versus host disease is a promising method of malignant neoformations immunotherapy” (http://www.practical- oncology.ru/arh015/07.pdf) demonstrated that modern immunotherapy can be practical alternative to the chemotherapy resistant patients.  Despite certain achievements in chemotherapy of cancers, such patients remain a problem. Increase of cytostatic drugs doses to submaximal for eradication of leftover cells of tumor clone resulted in introduction of the method of allogeneic or autologous hematopoietic stem cells (HSCs) transplantation. Traditionally, high dose chemotherapy is considered  basic component in HSCs transplantation and administered as substitutive therapy to the patients who received lethal doses of chemotherapy. However, long-term follow up showed relapse in some patients, and the rate of recurrence was higher in the patients after autologous or syngenic HSCs transplantation, as compared to allogeneic. The lowest rates were observed in the cases of allogeneic transplantation with manifestations of graft-versus-host disease.
This fact led to the hypothesis that these patients had immune-mediated of graft-versus-leukemia (GVL) effect that played the leads in elimination of the residual malignant cells surviving chemo- and radiotherapy. This way, the concept of transplant-versus-leukemia or transplant-versus-tumor effect induction by means of administration of donor lymphocytes after HSCs transplantation was developed.
Hypothesis that allogeneic lymphocytes in HSCs transplantation can eliminate leucosis cells due to immune-mediated effect, i.e. GVL, relied on the results of experimental and clinical HSCs transplantations. Direct correlation of acute and chronic GVH disease and reduction of leucosis relapses rate in clinical practice was first described by P. Weiden et al. the graft-versus-tumor (GVT) effect similar to GVL, was initially described in mice model of spontaneous sarcoma, and then in the humans with breast cancer. Further immune mediated GVL effect during HSCs transplantation was confirmed by multiple observations that relapses developed after cyclosporine A therapy (CAT) had been arrested after its withdrawal. It was also found that relapses rate reduced in the cases of lower CSA doses.  These data show that in allogeneic HSCs transplantation donor T-lymphocytes can react against residual tumor cells. Therefore, the advantage of HSCs transplantation as compared to standard chemotherapy lies in combined effect of myeloablative dose of chemo- or radiotherapy in pre-transplantation period and ability of immunocompetent allogeneic donor T-lymphocytes to eliminate residual tumor cells resulting from GVT or GVL effect. Hence, it was supposed that infusion of allogeneic lymphocytes in post-transplantation period can be used for therapy and prevention of relapses in the patients with high risks. For the first time donor lymphocytes (DL) were successfully transplanted to a two-year old child with acute pre-B lymphoblast leucosis in Jerusalem in 1986. Afterwards, this method to treat relapses after HSCs transplantation was widely used in the cases of acute lymphoblast and myeloblast leucosis, chronic myeloleucosis and myelodysplastic syndrome. Relying on experimental data recombinant Il-2 activated DL infusions are used to treat relapses in some hospitals.
The evidence on the efficiency of DL infusions in the cases with “minimal residual disease” led to the recommendations to regularly monitor the patients at high risk of relapse for chimerism (donor and recipient DNA markers). Along with T- lymphocytes, other cells are involved into induction and realization of GVL effect.
NK cells, monocytes, macrophages produce antitumor effect independent from histocompatibility antigens expression. At the same time many tumor cells, in particular metastatic, do not express molecules of major histocompatibility complex.
As seen from the research in mice and in humans, alloreactive NK cells can induce GVT and GVL without graft-versus-host disease initiation. Hence, available clinical and experimental data demonstrated possibility to completely eradicate tumor cells in DL infusion post-transplantation period in malignant diseases of blood and in solid tumors. An important role in the therapy of malignant diseases of blood belongs to alloreactive T-lymphocytes.
Original method of glial tumors immunotherapy was proposed by the researchers of Saint-Petersburg (Olyushin V.E., Tigliev G.S., Ostreyko O.V., Filatov M.V.; 2009) following the technology developed in Polenov Russian Research Institute of Neurosurgery and Konstantinov Petersburg Institute of Nuclear Physics (application for invention of “Method of therapy of brain malignant tumors” dated 17.08.2000, № 023265). Six patients with recurrent glioblastoma received the following immunotherapy: tumor sample of no less than 1cm3 size containing only viable tumor cells was isolated during the surgery. The sample was stored in sterile solution and antigene material was prepared of it within first 24 hours. For that, the sample is radiumized at 200 Grays and the cells were isolated. Obtained tumor cells were washed and eliminated with medium pH increase to 11.5 and further decrease to 6.5. The received protein extract was used as antigen for dendritic cells. Before every therapeutic session, from 40 to 60 ml of peripheral blood of the recipient were taken and stored in syringe with heparin, with final concentration of 30 units per 1ml. No later than 6 hours after blood sampling, monocytes are isolated from the sample, cultured for 7 days with growth factors (granulocyte macrophage colony stimulating factor and interleukin-4, 1000 units per 1 ml) under constant control and with regular medium change. At day 6, antigen material obtained from recipient’s tumor was added to dendritic cells and introduced inside them with electroporation. From 15 to 20 ml of peripheral blood to prepare activated lymphocytes was sampled similarly to that for dendritic cells. Proliferation was induced in mononuclear white blood cells with phytohemagglutinin and at day 4 they were injected subcutaneously in order to activate as many T-lymphocytes as possible predominantly stimulating Th-1 cell pathway of immune response. At day 8 dendritic cells were subcutaneously administered in the solution with tumor cells lysate. The number of injected cells varied from 2x105 to 107. Therapy course consisted of three injections of activated lymphocytes and three injections of dendritic cells. The interval between the courses was about 2 or 3 months. Thus, immunotherapy had three components: dendritic cells loaded with tumor antigens, activated autologous lymphocytes and tumor cell lysate. All study participants survived. The interval from the beginning of immunospecific therapy exceeded the interval between the last surgeries in 5 out 6 cases, in some cases in 6-8 times; in one case the intervals were equal. Average time period from the beginning of immunotherapy to control examination made 10.2+3.1 months. Baseline Karnofsky index at the beginning of immunotherapy varied from 40 to 80 points. Analysis of the score changes showed that during the therapy the score decreased by 10-20 points in 4 cases, increased by 10 in 1 case and did not change in 1 case.
Similar research in immunotherapy of solid tumors with brain metastases were performed by John R. Yannelli of the University of Kentucky. Since 2004 Yannelli studies application of immunotherapy in non-small cell lung cancer that often gives metastases to brain. Non-small lung cancer is the disease with poor prognosis and low susceptibility to conventional anticancer therapy, so the opportunities of immunotherapy of this disease are of special interest. The trial consisted of 16 cases of I-III stage non-small cell lung cancer. Before the therapy all patients received conventional therapy (surgical or multimodal treatment). The scientist also works with immunized dendritic cells. Yanelli tried to enhance immune response to vaccine and co-cultured dendritic cells of the patient with “dead” tumor cells. After that dendritic cells stimulate proliferation and differentiation of antigen-specific T-lymphocytes more efficiently, initiating vigorous immune response to the tumor. The researchers used cell line 1650 as inducing tumor cells, as this line produces high number of tumor antigens  (Her2/neu, CEA, WT1, Mage2, and survivin). Tumor cells were processed with UV and cesium-137 to induce apoptosis. Preparation of dying tumor cells was added to the culture of the patient’s dendritic cells in the ratio of 1:1 for their stimulation and “training”.
“Trained” dendritic cells were injected to the patients subcutaneously 2 times with 1 month interval. The researchers evaluated specific immunological reaction: 5 patients showed no reaction, 5 showed non-specific to tumor antigens reaction, and 6 demonstrated specific immunological responses. Further research of the protocol to specify groups with high susceptibility and to evaluate long-term results are required. Overall clinical protocol of dendritic cells therapy seems extremely promising as it has few side effects and does not influence life quality.
Similar data were obtained by Prof. Mentkevich G.L. and Prof. Dolgopolov I.S. (2010) when they used dendritic vaccines and systemic intrathecal admisnitration of allogeneic stem cells for glial tumors of brain in 8 children. Survival was extended to 3.5 years in three patients. These results received universal approval of international oncologic community.
Combination of different approaches can ameliorate expected efficiency of immunotherapy. For example, the group of 65 patients with glioblastoma and anaplastic astrocytoma of III stage received intrathecal injections of autologous mononuclears incubated with tumor cells with IL-2 followed by subcutaneous injections of cytokine complex. Two year survival improved from 25.9% in control group to 505 in the main group. In the main group the number of patients who stayed in remission for 3 years doubled (Oleshko M.I. et al, 2009).
Hence, the principle of immunotherapy of neurooncological diseases lies in the therapy with native/activated immunocompetent cells of the patient/their haploidentical donor combined with various cytokines, as well as production of autovaccines by co-culturing immune cells of the patient with their tumor cells or proteins. Co-culturing T-lymphocytes with the tumor cells awards immune cells with new anticancer immune features and ability to suppress tumor cells growth. For the last decade it has been proved that immunological interaction between donor T-lymphocytes and tumor cells of the recipient play an important role in the therapy of hematological malignant diseases. By now it became clear that recurrent leucosis after allogeneic transplantation can be cured by donor leucocytes. These facts led to the proposal that graft-versus-tumor reaction is the main therapeutic factor in allogeneic transplantation and not high doses of chemotherapy combined or not with radiotherapy, included into conditioning regimen. When haploidentic immunocompetent cells are used, the GVT effect is realized with the help of T-lymphocytes and NK cells. The difference between low rates of relapses in haploidentical transplantation in comparison with HLS compatible transplantations can be explained by recent discoveries in NK functioning and their role in anticancer immunity. In partially compatible transplantations HALC variant of determinants (KIR2D receptors) is detected by donor NK cells and direct cytotoxic affect is initiated.
Summing up contemporary conventional approaches to brain tumor therapies we can note that some positive effect have been achieved recently, but that did not exert much influence on the survival rates of the patients. To date it has been proved that healthy immune system is able to fight tumor cells in case their number does not exceed 500 000. However, most researchers are convinced that this number of the cells in a healthy body is conditioned not only by anticancer lytic properties of immune system but also by its regulatory effect on cancer stem cells. The researches have multiply demonstrated that proliferative properties of cancer stem cells were controlled by immune system through HSC and hematopoiesis precursors. Disorder of CSC regulation and control by HSC and hematopoietic precursor cells (HPC) promote tumors or tumor relapses. Imbalance of immune system regulatory control of CSC niche cells conditions expansion of new tumor cells.
Therapy of cancers in general and of neurooncological diseases in particular is exceptionally complex and, as seen from the data, rather churlish. No protocol of malignant brain tumors demonstrated patients’ survival for over 2 years. Obviously, recent achievements in the tumor therapy cannot be neglected, when in the US the survival in cancer cases increased by 24.2% in the past decade due to administration of contemporary targeted chemotherapy. Novel approaches involving fluorescence-guided surgery of brain tumor permit maximal reduction of tumor cell mass to 109 neoplastic cells. New methods of radiotherapy, radiosurgery and chemotherapy reduce the number of tumor cells to 107 (Chernykh E.G., Stupak V.V., Centner M.I. et al, 2004). Sanogenetic abilities of human immune system in oncological process and therapeutic opportunities of conventional anticancer therapy differ by two orders of magnitude more approximately (from 105 cells to 107 cells). Many research teams lay their hopes on immune therapy (Chernykh E.G. et al, 2006). In certain cases it is realizable and currently, immunotherapy leads to efficient immune response in 30-34.2% cases and reduces the number of tumor cells to 103 thus extending lives of the patients.
However, immunotherapy is not a cure-all solution in neurooncolongy and has significant limitations in generalized tumor process and is almost inactive when total tumor cell mass exceeds 107.
However, cancer stem cells (CSCs) are observed in the body of a cancer patient even after maximal reduction of tumor cells number resulting from efficient immunotherapy (less than 103-104) being the source of all tumor cells of specific neoplastic formation.  In favorable conditions CSCs are able to restore critical mass of neoplastic cells very rapidly. Regulatory functions of autologous HSCs and hematopoietic progenitors are significantly limited under the conditions of tumor manifestation, and are unable to maintain mutated SC proliferative processes adequately, i.e. to regulate CSCs. Few available protocols of cancer immunotherapy imply therapeutic use of the patient’s immune system cells to affect CSCs. Mostly, therapeutic action is aimed at total elimination of CSCs. However, so far total elimination of CSCs has not lead to any significant results and will hardly ever do, as the “stemness’ of a somatic cell is an optimal and universal form of cell survival under unfavorable conditions. This concept is applicable to SCSs. Potential of immune system targetedly affecting tumor CSCs can be explained by the fact that HSC as parental cells of immune system must perform regulatory effect on effector functions (mitosis, division, migration etc) of CSCs and control CSCs proliferative processes (Bryukhovetskiy A.S. et al., 2011). However, these anticancer functions of HSCs and hematopoiesis progenitors are inhibited in the environment of malignant tumor development considerably. As the basis of the protocol we used preclinical research of FGBU V.P.Serbski State Research Center of Social and Forensic Psychiatry as well as prior clinical research performed by Prof. George Mentkevich of FGBU N.N. Blokhin Russian Cancer Research Center of the Russian Academy of Medical Sciences (I. Dolgopolov et al., 2010) demonstrating that immunotherapy with dendritic vaccines obtained from brain tumor (glioblastomas, astrocytomas, gliomas) cell bioptates accompanied by intrathecal infusions of allogeneic haploidentical HSCs led to 3 year remission in glioblsatoma in 30% of cases and increase of survival rates. According to I. Dolgopolov et al.  (2010) PET showed that malignant tumor is not eliminated completely. It still persists as a small tumor focus, but its proliferative and metastatic potential is restricted. Presumably, allogeneic haploidentical allogeneic HSC and hematopoietic precursors affect and regulate CSCs of the tumors.
Obviously, all conventional therapeutic approaches and novel therapies on their basis are necessary and grounded in planning contemporary anticancer therapy, as they have their limited and frequently fixed level of therapeutic efficacy reducing general number of tumor cells to critical concentrations.
Probably, they should be used in every protocol with the consideration of their proven therapeutic resource. Available achievements of anticancer therapy cannot be neglected. This would methodologically incorrect and theoretically unreasonable. New protocols of anticancer therapy must be consequent, expedient and successive in programmed quantitative elimination of TCs due to anticancer treatment. Every protocol of cancer therapy must include 1) conventional surgical intervention to provide maximal elimination of main tumor mass (to 109 TCs), 2) morphological, histological, immunochemical, genome and post-genome tests, 3) standard radiotherapy up to 60 Grays, 4) up-to-date  chemotherapy that provides cytostatic and cytotoxic effect on TCs located beyond resection area. This guarantees reduction of TCs number 107-106. Immunotherapy should become natural and logical continuation of compulsory anticancer measures as it completes the process of quantitative reduction of TCs in the patient’s body to physiologically acceptable level (<105 TCs). It is also clear that conventional immunotherapy does not permit application of standard approaches of active and passive (adoptive) immunotherapy at concentration of TCs lower than physiological level < 105. Main task of immune system at physiological concentrations of TCs in the body is regulatory, i.e. maintaining and controlling level of TCs. However, this fact does not exclude contemporary methods of immunotherapy for TCs regulation at these concentration levels. Immunotherapy in a broad sense is a capacious scientific and clinical term involving more than mere understanding it as immunostimulation, immunomodulation or immunosuppression of the number and/or functions of immunocompetent cells. New concept of immunotherapy of tumors implies partial or complete restoration of anticancer regulatory functions and features of a whole immune system.  Methods of active and passive (adoptive) immunotherapy or substitutive transfusion of autologous mononuclear and stem cells or mobilization of HSC to peripheral blood using granulocyte colony stimulating factor are capable to restore these functions of immune system partially. Our research demonstrated that single application of human SC and precursors therapeutically activates inherent anticancer features of cell systems and offers opportunity to suspend neoplastic growth of brain glial tumor in animal models and humans significantly (to 30-50%) (Bryukhovetskiy A.S., 2011). It is possible that systemic immunotherapeutic intrathecal administrations of human SCs and precursors to glioblastoma patients are able to enhance the potential of brain anticancer system, evoke specific cell response and arrest or delay dissemination of neoplastic process. Probably, they are able to trigger other anticancer immune mechanism “graft-versus-tumor” (Mentkevich G.L. et al, 2010).
Another way to achieve partial restoration of anticancer features of immune system is genetic modification of autologous immunocompetent stem cells to obtain induced pluripotent stem cells (iPSCs) or to obtain iPSCs from the umbilical blood stem cells. Though the source of iPSCs is autologous SCs, to date it has been proved that genetic transformation violate mechanisms of histocompatibility in these cells and they are perceived as foreign cells by the recipient’s immune system. They initiate typical reaction of cell transplant rejection, so the opportunity to use these technologies in modern neurooncology is quite limited.  
Complete restoration of anticancer functions can be achieved in replacing immune system cells of the patient with HLA-compatible donor (allogeneic or haploidentical) cells of immune system that form due to donor bone marrow stromal stem cells engraftment after high dose chemotherapy and consequent transplantation of bone marrow  cells (TBMC) or mobilized allogeneic or haploidentic donor HSCs. Also, the literature describes successful cases of complete replacement of the recipient’s immune system with that of a donor through transplantation of umbilical blood cells.
Complete restoration of anticancer immune system by it global replacement or partial replacing of its system elements (using induced progenitor stem cells – iPSC) is understandable and methodologically justified. Obviously, simple change of cell elements in the patient’s immune system (immunostimulation, immunomodulation or immunosuppression) will not result in regulation of TCs effector functions and control of their quantity. Qualitative and not quantitative modification of autologous SCs is necessary to regulate TCS by means of immune system cells.
Apparently, fundamental provisions of tumor immunology demand adjustment due to newly detected innovations and details of protein mapping and proteome profiling (PP) of tumor cells and immune system cells at various differentiation stages in cancer cases: from SC to highly differentiated immunocompetent cells. The features detected at mapping and profiling proteins of immunocompetent and tumor cells offer a new viewpoint on tumor immunology issue (Bryukhovetskiy A.S., 2012). Detection of patterns of clinical oncoproteomics of tumor cells led to theoretical prerequisites that formed the concept of restorative immunotherapy. The patterns of TCs PP are the following: 1) Cell surface of tumor cells express antigens different from normal; the number of tumor antigens in TCs PP structure varies from 95 to 60%, their representation on membrane depends on the degree of TCs differentiation directly: the higher differentiation is, the more explicit is antigene representation of oncospecific proteins; 2) The membrane of cancer SCs contains very few tumor-specific antigens; hence, they are inaccessible to immunologic control of immune system; 3) The number of tumor-specific antigens at TCs membrane reduces under unfavorable conditions (toxic, mechanic, ionizing effects) due to activated evolutionary survival through TCs dedifferentiation, development of stem features (loss of cell surface markers, TCs membrane rounding, suppression of main effector functions of TCs, activation of intracellular reparative processes and reproductive potential) that considerably decreases anticancer regulation with autologous regional stem and immunocompetent cells; 4) immunodeficiency observed in experimental carcinogenesis and precancer diseases in humans is conditioned by proteomic incompatibility of regulatory immune cell systems with tumor cells which is the basic reason for the loss of control over TCs number and effector functions by immunocompetent cells; 5) Growth of neoformations can be detected due to immunotherapeutic measures being inadequate to real immunologic processes in the organism of a cancer patient; 6) Immunosuppression, immunomodulation, as the methods of tumor immunotherapy can be efficient only at early stages of cancer, while in pro-gradient process main mechanisms of immunocorrection must be oriented towards regulation of functional state of cancer stem and progenitor cells through intact pathways of signal transduction; higher reactivity of immune system during the therapy will depend on target orientation of effect on CSCs and correct choice of targets, proteome antigens, for immunocorrection and immunoregulation. We offer additional and partially alternative methodology of immunotherapy of CNS tumors, based on fundamental principles of sanogenesis and cell mechanisms to enhance local anticancer immunity of brain: restorative immunotherapy.


                                                                                                                            Table 2.
                     Main and experimental immunotherapies of tumors



Active    
Specific    Vaccines of deactivated or modified tumor cells, tumor cell extracts, purified or recombinant tumor antigens, idiotypes, viral or bacterial gene-vectors introduced in tumor. Vaccination against carcinogenic viruses, such as hepatitis D, papilloma viruses, T-cell leukemia (HTLV-1), Eppstein-Barr.   
    
Non-specific     BCG, "Imuron", Corynobacterium parvum, Cytokines (alpha, beta, gamma IFNs, Il1, Il2, Il12, TNF, Caebomoilaziridine, Levamisole, thymus hormones preparations, Glucoseminyl muramildipeptide, Terhalose dimycolate, pyran co-polymer, poly-(I:C), pyrimidines, MGN-3
    Combined     Combination of vaccines with cytokines or non-specific immunostimulators



Passive
(adoptive)    

Specific    Elimination of tumor in the body of the recipient by infused lymphocytes with specific anticancer determinants. Monoclonal antibodies, pure (Rituximab, Trastuzumab, Edrecolomab etc) pharmaceuticals, toxins or radioisotopes (Ibritumomab, Tositumomab); conjugated with plant or bacterial toxins; biospecific antibodies; T-cells, dendritic cells.  
    
Non-specific
     Administration of immunocompetent donor cells to recipient: lymphokine activated killers (LAK) or tumor infiltrating lymphocytes (TIL) or inducing cytokines killer cells:LAK therapy, cytokine therapy (Il1, Il2, TNF, interferons), lectines  (Iscador), heat shock proteins
    Combined    LAK therapy combines with biospecific antibodies




Restorative
(regulatory)    
Specific    Restoration of specific and regulatory characteristics of immune cells in the recipient’s body: stem cells, progenitor cells and immunocompetent cells through remodeling their proteome or transcriptome profile with chemical induction, genetic or molecular engineering
    
Non-specific
    Restoration of immune system of a cancer patient replacing its basic cell components: transplantation of autologous and allogeneic bone marrow, transplantation of mobilized hematopoietic stem cells, transfusions of autologous mesenchymal and hematopoietic stem and mononuclear cells after general radiotherapy or high dose chemotherapy of bone marrow.
    Combined    Transplantation of bone marrow and vaccine-therapy with antigene-presenting cell systems, bone marrow transplantation and intrathecal transplantation of hematopoietic stem and progenitor cells along with cytokine therapy



This type of immunotherapy is used in clinical practice for over 60 years, though viewed not from the angle of tumor immunology, but transplantology, and in particular, bone marrow transplantation. It seems that summarizing table of immunotherapies should take into account restorative approaches and they are shown at Table 2.
Obviously, large volume of tumor cell mass (>107 TCs) prevents manifestation of necessary anticancer cytolytic effect from immunotherapy. It is appropriate to view immunotherapy as compulsory and logical continuation of specific anticancer immunotherapy, and not as independent type of anticancer therapy. That is any course of anticancer surgical treatment, radio and chemotherapy must end with compulsory immunotherapy. To enhance efficiency and individualization of immunotherapy, it will be practical to involve the results of mapping and profiling of TCs and CSCs proteins isolated from the patients’ tumor. Such characteristics as specificity, individuality and concentration of detected protein changes in proteomic profiles of autologous TCs and CSCs permits detection of the most therapeutically meaningful antigene chains and targets, as well as to find neoplastically non-modified membrane proteins, signal pathways proteins and gene expressed proteins that can provide regulation of effector functions and CSCs and TCs number.
Diagnostics of the most individually meaningful immunogeneic cancer-specific proteins and target proteins of the patient’s CSCs that are able to respond to targeted regulatory effect, is achieved through comparative mapping and profiling of a whole range of proteins of the analyzed tumor cell and hematopoiesis progenitors systems. Further, this permits proteome-based individualized modifications of phenotype of all immunocompetent cells (dendritic cell, HSCs, cytotoxic T-lymphocytes) used for immunotherapy by loading them with additional individually meaningful highly specific cancer-antigenes or regulatory proteins. Proteome diagnostics of TCs, HSCs and CSCs is able to reveal target proteins in TCs and CSCs whose effect can regulate effector functions and functional state of TCS and CSCs. Basic proteins able to affect target proteins of CSCs and TCs are detected according to the available databases of protein-protein interaction. Loading HSC or regional progenitor cells of the patient with individually detected by proteome profiling proteins, we obtain autologous cell systems with remodeled proteome profile capable to affect the patient’s CSCs regulatorily.  As far as we use autologous cell system transplantation with unmodified gene to regulate CSCs and TCs, these cells will home in tumor and in preset period of time (from 2 to 7 days) release proteins into tumor intercellular space performing target regulatory effect on target proteins of CSCs and TCs. Hence, proteome based immunotherapy can be the following: 1. Active specific immunotherapy with autologous dendritic cells vaccine loaded with non-differentiated tumor-specific recombinant proteins and individually tailored cancer specific recombinant proteins, evoking more efficient immune response to administered antigens and contributing to development of target cytotoxic lymphocytes; 2. Individually tailored passive (adoptive) specific immunotherapy with cytotoxic lymphocytes demonstrating specific anticancer determinants and causing tumor elimination in the organism of the patient. 3. Personalized specific restorative immunotherapy with allogeneic haploidentical or autologous SC with specifically remodeled proteome profile. The method of proteome profile remodeling is patented in the Russian Federation (the application is filed).
The main goal of specific restorative immunotherapy is to restore anticancer regulatory functions of autologous SCs (anti-proliferative, anti-mitotic, anti-angiogenic, anti-migrational functions etc.) in the patient’s immune system and to properly affect CSCs functional effector activity.
These considerations were taken as the basis for the protocol of research of multi-vector immune effect on brain neoformations in the cases of malignant glial tumors and secondary solid tumors metastasing to brain in humans.
GOALS AND OBJECTIVES

Main goal of the program:  Development and introduction of novel biotechnology of proteome-personalized immunotherapy of neurooncological disorders by means of production and application of individualized autologous cell preparations (dendritic vaccines, cytotoxic lymphocytes, postnatal stem and progenitor cells with remodeled profile) produced through peptide engineering of postnatal hematopoietic progenitor cell systems on the basis of post-genome research of comparative proteome mapping and profiling of immunocompetent, stem and tumor cells of the patient into clinical practice.  
Local goals:

- To develop new paradigm of personalized immunotherapy of malignant neurooncological diseases, base on proteome mapping and profiling of proteins of the patient’s cell systems and to scientifically validate its necessity and practicability for clinical neurooncology;
- To develop the transition mechanism of malignant cancer process in the brain from acute and lethal into non-lethal and chronic;
           - To develop science-based technology of proteome personalized immunotherapy of neurooncological diseases and to establish scientific organizational and technical solutions to improve basic indicants of population health, improve survival rates and life quality of the patients with severe and incurable neurooncological diseases;
            -To develop and introduce contemporary post-genome, peptide engineering and cell technologies into medical practice of neurooncological diagnostics and therapy of brain tumors into clinical practice;   
  -To establish basic universal algorithm for clinical application of low-invasive methods of radiosurgery, endovideosurgery, cell transplantology for distant regional restoration of brain pathological structure and disordered functions in neurosurgical patients;
 -To develop and introduce the technologies of mapping and profiling of various cell systems and biological fluids in neurooncological cases into neurooncological practice, as well as to develop technologies of anticancer peptide engineering of hematopoiesis progenitors;
-To work out recommendations on introduction of peptide engineered and cell biotechnologies into clinical practice.

Main objectives:

1. To develop and introduce basic algorithm of poly-vector immune cytolytic and regulatory effect on neoplastic focus in the brain and validate mechanisms of control of cancer stem cells in pathological area of brain;
2. To develop technology of “small” production and application of individually tailored peptide-engineered dendritic vaccines, cytotoxic T-lymphocytes and progenitors of hematopoiesis,  based on comparative neurooncoproteomics of different postnatal progenitor cell systems of the brain cancer patients;
3. To develop the mechanism of detection dominating oncospecific, regulatory and key proteins in the isolated patient’s CSC;
4. To develop the method of application of cell preparations for proteome-individualized immunotherapy of brain tumors;
5. To develop methods of peptide-engineering remodeling of proteomic profile of dendritic vaccines, cytotoxic lymphocytes and postnatal hematopoietic stem cells;
6. To determine indications and contraindications to proteome-individualized immunotherapy of brain tumors, to study possible complications and methods of their prevention and arrest.
7. To perform mathematical modeling of proteomic profiles of hematopoiesis progenitors to detect remodeling agents able to restore regulatory anticancer features in autologous progenitors of hematopoiesis of the patient;
8. To prepare system project and to develop special mathematical and program software for the main stages of biotechnology with consequent estimation of introduction efficiency.
9. To develop recommendations on introduction of the technology into clinical practice of the Russian healthcare system.
Solution of these tasks will lead to significant decrease of neurooncological diseases incidence and mortality rates in the Russian population.

IMPLEMENTATION PERIOD
The program will be implemented from 2012 to 2017 in two stages.
The first stage (2012-2013) implies experimental treatment of animal models of neurooncological diseases according to main protocols of immunotherapy of this program, development of infrastructure of participating academic and clinical institutions of healthcare, training of medical and technical personnel, development of main activities in data management of participating institutions.
The second stage (2014-2017) includes multicentre research of experimental therapy and introduction of proteome-individualized technologies into clinical practice of neurooncological hospitals, equipment of leading medical centers of the Russian healthcare system, training of highly qualified experts in the sphere of post-genome proteome technologies including opening of specialized department and training courses of the Institute of Advanced Training of the Ministry of Healthcare, introduction of proteome-based immunotherapeutic medical technologies.
- Neurooncological assistance to adult population with severe brain pathologies incurable with conventional neurological and neurosurgical methods.
- Combination immunotherapeutic technologies with intraarterial magistral and microcirculatory remodeling of brain vessels (balloon angioplasty, microthrombosing of brain tumor etc), as well as modern low-invasive (sparing) methods of microendoscopic and endovascular radiosurgery, microsurgery and functional neurosurgery.
-  Universal bank of stem cell cultures.
Competition in this sphere is almost absent in the Russian Federation as well as globally. In Russia such studies are performed as experimental in the research work and dissertation study and only in single research centers of Moscow and Saint-Petersburg.
Meanwhile, according to the reports at the XX World Congress of Neurology (Morocco, 2011) post-genome and cell technologies, as well as peptide engineering are the most progressive for the therapy of nervous diseases and in particular neurooncological diseases. Available market capacity for such services in the world, and in Russia, allows for prognosis that potential number of those requiring medical assistance will considerably exceed productivity of developing clinical base even in the case of full scope international works according to the project.
LIST OF MAIN EVENTS

The system of program activities implies execution of exact objectives interconnected and agreed according to schedule, resources and executors and regulated by tendencies of healthcare development, regulatory documents, business environment and results of research.
The program implies step-by-step development of peptide-engineered technologies to restore human postnatal progenitor cell using novel achievements of cell transplantology and consecutive introduction of post-genome technologies into clinical practice. The first stage implies development of organization and establishment, improvement of facilities and equipment, academic and human resourcing, so that the second stage will provide valid results and their introduction into clinical practice.
FUNDING

The program is financed from non-budgetary sources received from the ordering party for research and therapy of national and international patients. Planned expenses for the Program implementation make 150 000 000 (one hundred fifty million) of non-budget funds. The expenses for the program implementation are specified annually as appropriate.

MECHANISM OF THE PROGRAM IMPLEMENTATION

The Program is implemented under supervision of the head institution on the basis of typical agreements on research and developmental products between head institution, Blokhin Russian Cancer Research Centre of the Russian Academy of Medical Sciences, and  ordering parties. To this effect, Blokhin Russian Cancer Research Centre of the Russian Academy of Medical Sciences transfers the functions of research coordination to the scientific Research Institute of Tumor Experimental Diagnostics and Therapies, and organization and finance functions to ANO “National Institute of Regenerative Medicine” hereinafter referred to as the Institute.
The Institute coordinates main executing bodies, controls targeted and efficient funding and performance of planned activities, forms consolidated request to the Ordering Party together with main executing bodies specifying finance allocated to the consecutive implementation of the Program activities naming specific procedures in cost and physical indicators, as well as regularly reports on performed research and evaluates its efficiency. The Institute takes measures to provide full finance of the Program; organizes and controls implementation of the Program activities.
Main executing bodies of the Subprogram sign agreements on joint activity with the head institution and the Institute.
The Institute agrees annual plans of the executing bodies with the Ordering Party, hires research teams or experts to perform specific research. The role and place of the Institute in the structure of medical services market can be corrected for better quality of the Program implementation.
Executing bodies of certain parts of the program, the organizations and experts, are specified on the basis of competitive bidding according to the Regulation of Procurement of Goods, Works and Services for State Requirements ratified by the RF Presidential Edict №305 dated April 8, 1997 “On Immediate Measures to Prevent Corruption and Reduce Budget Expenses in Procurement for State Requirements”.
In the course of Program implementation The Institute will take measures to sign agreements with international organizations in order to attract additional funds for further development of the Program, improve facilities and equipment of the executing parties and to promote beneficial cooperation. The Institute will represent the interests of head organization in other countries, provide information support and present the Program abroad to attract investments on a mutually rewarding parity basis.

PROGRAM MANAGEMENT AND SUPERVISION
The structure of the program management consists of three levels: general management, coordination and scientific and methodological support.
Coordination of the implementation of the program projects, congruence with adjacent programs “National Technological Base”, “Medicine of High Technologies”, “New Cell Technologies to Medicine” etc.
Management of project implementation as the basic element of the Program, congruence with adjacent projects in the sphere of transplantology and artificial organs.
Generally, the program implementation is managed by the ordering party who determined requirements to the content of the Program and projects, those, who provided resources and supervise Program implementation, and research supervisor responsible for the content, scientific and technical level of the Program and the projects.
Together with the Russian Academy of Medical Sciences, the ordering parties provide the conditions for the Institute to function as directorate in the program implementation, coordinator and auditor of activities under the Program.
Direct scientific supervision of the program is delegated to the   authorized representative of the Russian Academy of Medical Sciences, director of Blokhin Russian Cancer Research Centre of the Russian Academy of Medical Sciences Prof. Mikhail I. Davydov. The Ministry of Health controls implementation of the program through annual reports at the meetings of Scientific Board of FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA of Russia.
Executive directors of the Program are Prof. Bryukhovetskiy A.S. and Prof. Averyanov A.V. who develop the program concept, its structure, guidelines, coordinate scientific supervisors, prepare conferences and meetings under the program.
The implementation of the Program is supervised according to the Order of development and implementation of the federal target programs and international target programs with participation of the Russian Federation.

EXPECTED RESULTS AND EFFICIENCY EVALUATION

-    Science-driven, organ sparing, peptide-engineered, low-invasive, ecologically clean technology of proteome-personalized immunotherapy of brain rumors will be developed and introduced into clinical practice on the theoretical and experimental basis of post-genome approach in medicine, as well as scientific methodological and experimental baselines of introduction this novel biotechnology into neurooncology.

-    Science-based technical solutions as specific software permitting management of biotechnological activities, system engineering and analysis of clinical and diagnostic criteria, surgical intervention and evaluation of efficiency of all stages of diagnostic and therapeutic neurooncological process will be developed for the first time.

-    Individualized predictive and antigen specific immunotherapy brain tumors based on mathematically assessed oncospecific and key regulatory proteins of proteome profile of the patients’ cell systems will be implemented for the first time in the clinical practice of global healthcare.

-    For the first time the technology will develop a universal peptide engineered method of peptide modification of the phenotype of basic hematopoietic cell elements of bone marrow tissue with remodeling their proteome biochemical agent detected in the analysis and comparison of proteome and transcriptome information database of cell systems with the data of proteome mapping and profiling of tumor and cancer stem cells of the patient for cell restoration;
   
-    National priority will be secured in the peptide engineered restoration of anticancer properties of hematopoietic progenitors and restoration of disordered brain functions by means of the developed technology with high economic effect of its application in the central medical institutions of the Russian healthcare.

-    The first national bank of stem progenitor cell cultures will be established for proteome-individualized immunotherapy of neurooncological disorders.

-    The methodology to transfer acute and lethal neurooncological process into chronic and non-lethal will be mathematically substantiated and implemented in the clinic for the first time.

-    The recommendations on introduction of scientific practical results into the practice of management organs of highly technological and specialized medical institutions of the Russian Federation will be developed.

MAIN GROUPS OF NEUROONCOLOGICAL CASES RECOMMENDED FOR THE PROGRAM

1.    Cases of recurrent glioblastoma of brain;
2.    Cases of anaplastic astrocytoma of brain;  
3.    Cases of lung cancer (small cell, non-small cell and adenocarcinoma) with brain metastases;  
4.    Cases of breast cancer with brain metastases;  

MAIN CONTRAINDICATIONS FOR THE PROGRAM THERAPY
1.    Decompensated  severe chronic cardiovascular disease
2.    Severe chronic kidney impairment
3.    Acute and chronic blood diseases
4.    Severe forms of multi-organ and pluri-glandular deficiency
5.    Critical level of neurospecific proteins and their antibodies in the blood serum and cerebrospinal fluid
Specialized rehabilitation after proteome-individualized therapy will be given in the neurorehabilitation department of ZAO NeuroVita Clinic and FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA of Russia according to individual specialized programs of rehabilitation and therapeutic exercise.
The patients are followed up for 3 years after the completion of the course every three months.
REVIEW OF THE RESEARCH PLAN, ITS ORDER AND THERAPEUTIC REGIMEN
The research involves three clinical centers: the Department of Neurosurgery of the Research Institute of Clinical Oncology of Blokhin Russian Cancer Research Centre of the Russian Academy of Medical Sciences, hospital of ZAO “ NeuroVita Clinic of Restorative Interventional Neurology and Therapy” and the Department of Neurosurgery of FGBU “ Federal Research And Clinical Centre For Specialized Medical Assistance And Medical Technologies” of the FMBA of Russia. The research will follow three protocols: Protocol №1 GBM “Proteome-personalized immunotherapy of brain glioblastomas”, Protocol №2 CrLu «Proteome-personalized immunotherapy of lung cancer metastases to brain”, Protocol №3 BrCr “Proteome-personalized immunotherapy of breast cancer metastases to brain”. Production of personalized anticancer cell preparations for immunotherapy will follow the Protocol of experimental biological research № 4 BioTECH «Biotechnology of production of anticancer cell preparations for proteome-personalized immunotherapy of brain tumors”.
General schedule of tests and procedures for every clinical protocol is shown at table 3.  
Screening examination and inclusion into trial. Screening examination cannot be done without prior Informed Consent. If the patient is excluded from the trial, the reason must be stated.
Screening
Medical history including prior and current therapy
Table 3. Examination schedule for the main period of the trial

Procedures    Screening    Baseline examination    Research Phase

        Day 1    Week 8    Week 16    Week 24
Written Informed Consent    X                
Inclusion/exclusion criteria
    X    
            
Medical history    X    X            
Physical examination    X    X    X    X    X
General condition according to KPS    X    X    X    X    X
Contrast CT    X    X            
Neurological evaluation (рубрифицировано)    X    X    X    X    X
Contrast MRI        X    X    X    X
Laboratory tests     X    X    X    х X    X
Randomization        X            

•    Physical examination
•    Neurological evaluation specifying intensity of: 1) impairment of consciousness,  2) focal symptoms of functional loss, 3) focal symptoms of excitation, 4) hypertension and dislocation symptoms
•    Integral evaluation of clinical condition according WHO scale and Karnofsky score (Table 4 and 5)
•    Contrast enhanced X-ray CT one time at baseline and repeatedly if intratumoral bleeding is suspected
•    Contrast enhanced MRI at least once in two months
•    Ophthalmologic examination
•    General clinical tests
Hematologic test – hemoglobin, hematocrit, red blood cells, white blood cells and differential, platelets
Biochemical blood serum test – total protein, bilirubin, AST, creatinine, blood urea nitrogen, alkaline phosphatase
Coagulogram
Clinical urine test
Chest and paranasal sinuses X-ray           
ECG, functional respiratory tests, abdominal ultrasonography
Stereotaxic biopsy of tumor node is done to all patients followed by cytological and histological tests in the Department of Pathomorphology of the Blokhin Cancer Research Centre. If doubted, the samples are tested repeatedly in N.N. Burdenko Research Institute of Neurosurgery. Part of the sample is forwarded to the laboratory of Immunochemistry of the Serbsky Institute of Social and Forensic Psychiatry for culturing and isolation of cancer stem cells (gliomaspheres). Hematopoietic stem cells are mobilized in all patients, and olfactory sheath of a nose is sampled endoscopically. Isolated hematopoietic stem cells are standardized and certified in the Bone Marrow bank of the Blokhin Cancer Research Center. Similarly, neural stem cells are standardized and certified in the Serbsky Institute of Social and Forensic Psychiatry. The appropriate protocols are provided.
Isolated from glioblastoma cancer stem cells (gliomaspheres) are divided into two parts. The proteome of the cancer stem cells of one part is analyzed in the FGU of V.A. Orekhovich Research Institute of Biochemical Medicine or in the Laboratory of Proteome Analysis of the Research Institute of Carcinogenesis Blokhin Cancer Research Center. The cultures of healthy hematopoietic stem cells are transferred there too. The protocol of comparative proteome analysis of the cells is provided in the section Biomaterial Analysis.
The other part of gliomasphere culture, as well as Cultures of healthy hematopoietic CD34+ stem cells and neural stem cells of the patient, are transferred to the Laboratory of Immunochemistry of the Serbsky Institute of Social and Forensic Psychiatry, where the cells are cultured, immunosorted, and the resulting lysate is transferred to the Laboratory of Oncoproteomics.
When randomized the patients are stratified according to:
Karnofsky Performance scale (less than 70 points – 70 and over)                                  
Involvement of brainstem/diencephalic formations (Yes/No)
Maximal size of tumor node (less than 4cm - 4cm and over)
Cytoreductive surgical intervention (yes – no)
 General condition is evaluated through WHO and Karnofsky Performance scales during screening, at baseline and every month during the trial.
At baseline and during follow-up period the following estimates are registered:
 Disease progress  
 Stable disease
 Therapeutic effect
 Not established.
 General condition evaluation
General condition of the patients is evaluated by the researcher at screening, baseline and every visit with 4-week interval till the end of the trial. The score for each condition is evaluated according to 5-point WHO Perfomance Score and a 100-point Karnofsky score.
Table 4. WHO Performance Score
Score    Condition
0    Fully active, able to carry on all predisease activities without restriction
1    Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature.
2    Ambulatory and capable of all self care but unable to carry out any work activities. Up and about more than 50% of waking hours
3    Capable of only limited self-care, confined to bed or chair 50% or more of waking hours
4    Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair
Table 5. Karnofsky score
Description    Score
Normal, no complaints, no signs of disease    100
Capable of normal activity, few symptoms or signs of disease    90
Normal activity with some difficulty, some symptoms or signs    80
Caring for self, not capable of normal activity or work    70
Requiring some help, can take care of most personal requirements    60
Requires help often, requires frequent medical care    50
Disabled, requires special care and help    40
Severely disabled, hospital admission indicated but no risk of death    30
Very ill, urgently requiring admission, requires supportive measures or treatment    20
Moribund, rapidly progressive fatal disease processes    10
Trial and Randomization Order
The patients are randomized into main and control groups to assess efficiency. During randomization the patients are stratified as said above.
The trial is open, so no blinding procedure is performed.
ANALYSIS PLAN
 Basic variables of the trial
•    Size of tumor node and perifocal zone
•    Intensity of intracranial hypertension
•    Integral evaluation of patient’s performance
•    Life quality
•    Daily dose of dexamethasone
Efficiency Analysis
A whole pool of the randomized patients is analyzed according to baseline data and thereafter at month 3, 6 and 12 of the trial.
Efficiency criteria:
Stabilized process,
Slowed tumor growth – survival exceeds 50 weeks from tumor verification date
General clinical improvement – Karnofsky score over 70.
The trial includes patients with glioblastoma involving hemispheres of brain verified by contrast enhanced MRI and CT and confirmed by histological test.
Before inclusion into trial the following indicants are evaluated that serve as criteria for subdivision into 4 subgroups:
1.    Clinical status evaluation  - Karnofsky score 70;
2.    Maximal tumor node size - 4 cm
3.    Involvement (dissemination, growth source) of brainstem and diencephalic formations
4.    Prior cytoreductive surgery.

Formation of trial group implies equal randomization according to the criteria:
- ways of administration AHSC with apoptosis inducing НК – intratumor or intraventricular (in case glioblastoma grows from or into lateral ventricle),
- ways of administration AHSC with apoptosis inducing НК:
а) intrathecal administration into CSF system
б) System intravenous administration  to the patient’s blood
Dexamethasone in the daily dose from 8 to 24ng can be administered to the randomized patients prior to the therapy.
The patients are stratified depending on clinical status evaluation and tumor node size.
After the trial the patients can continue course treatment with this protocol of immunotherapy if wanted.
                 APPLICATION OF CELL PREPARATIONS
А. Preparation of cell suspension for transfusion and its application. Thawing of stem cells.
The cells are thawed before transplantation in 37-400C water bath. Then, the cells are centrifuged at 1500 revolutions per minute and sedimented. Supernatant is pumped out and 1ml of 0.9% of saline NaCl is added.  The procedure is repeated twice. Thawed autologous stem cells can be applied within next 6 hours. If the cells are not used, they must be eliminated.
B. Transfusion of thawed autologous hematopoietic stem cells.
Intrathecal administration of allogeneic HSCs, MSCs and NSCs with remodeled proteome profile (RPP).
Intrathecal administration of RPP hematopoietic SC must be performed only in neurologic intensive care unit. Autologous HSC are administered through standard lumbar puncture (L3-l4) under local anesthesia with 1% lidocaine solution. 3ml of CSF are mixed with 3mcl of cell suspension (maximally to 1ml), resuspended and slowly injected into subarachnoid space. Aseptic dressing is applied. If necessary, intrathecal administrations of SC are repeated in two or three weeks. To correct possible allergic reactions single intramuscular injection of corticosteroids (4mg of dexamethasone) is given.
Intraventricular administration of allogeneic HSCs and autologous RPP HSC and NSCs.
According to standard method intraventricular port is installed into lateral ventricles of brain in the course of neurosurgical intervention. RPP HSCs or NSCs are infused in medical treatment room of a neurosurgical in-patient department. After port implantation, absence of blood in brain ventricles is checked by transcutaneous puncture of the port. One or two milliliters of CSF are taken and mixed with 1mcl of cell preparation, resuspended and slowly injected into intraventricular space. RPP HSCs can be repeatedly injected only after complete sanation of CSF confirmed by laboratory tests.
                     EARLY PERIOD AFTER STEM CELLS ADMINISTRATION
After cell administration the patient is followed up 24 hours for 7 days. An intensivist and an assisting doctor control condition of the patient to evaluate possible risk of complications. Besides, regular monitoring of neurosurgeon or neurologist is recommended in cooperation with hematologist, immunologist and laboratory doctors.  
Main efficiency criteria are improvement of neurological symptoms. The period of expectation is individual for every patient and depends on the size of brain tumor, onset time and compensation of disordered functions. Expected efficiency of the therapy varies from 30 days to 12-15 months after NK SCs and is evaluated by clinical indexes (WHO and Karnofsky) and neurophysiological tests (cerebral mapping, transcranial magnetic stimulation, somatosensory evoked potentials and electroneuromiography).

       POSSIBLE COMPLICATIONS AND THEIR MANAGEMENT

Possible side effects demand to perform intrathecal and intraventricular manipulations only under the conditions of Intensive care unit of Neurosurgical department. Possible complications, their prevention, diagnostics and management are summed up in Table 1. In case of side effects it is necessary to exclude:
1. Local inflammation
2. Intracranial hypertension (eye ground, Echo-EG, neuro-imaging).
3. Coagulopathy, thrombocytopenia, heparin injections 6 hours prior to puncture
4. Side effects and abnormal laboratory data
Clinical side effects
A side effect is any negative clinical presentation observed in the trial participant or a patient who receives a pharmaceutical and not necessarily associated with the therapy. Therefore, any unwanted and unfavorable symptoms (including deviation from standard laboratory values) and diseases emerging simultaneously with the administration of the studied pharmaceutical that and possibly unassociated with the pharmaceutical. Clinical conditions that were observed before the trial and exacerbated during its course are also registered as side effects.
 Intensity of side effects is measured by a four-point scale (weak, moderate, severe, life-threatening)  
•    Weak – causes discomfort but does not interfere with daily activity.
•    Moderate – discomfort that decreases daily activity or influences it in a negative way.
•    Severe – disability or inability to perform routine daily actions.
•    Life-threatening – presenting direct threat to life
It is necessary to mark association of the event with the therapy.
Abnormal laboratory tests
Abnormal laboratory value is not registered as adverse event unless it is associated with clinically important condition for which the combined therapy has been administered or a previous therapy has been adjusted, if it is not a serious side effect entailing therapy cessation.
Considering specific trial group, progress of main disorder can be expected in some cases. The patients can be taken to hospital in case disease progress is not associated with the therapy, or for radiotherapy or surgical intervention to prevent brain compression not associated with the therapy.
Side effects, specifically those labeled as not associated with cell immunoliposomal chemotherapy, are followed up till stabilized or restored condition at baseline.

 Pregnancy
Female participants of the trial must be warned about the necessity to cease trial pharmaceuticals administration and immediate notification of the researcher in case of pregnancy. The researcher must consult the patient on possible hazard of pregnancy continuation and possible effects on fetus.
Criteria of early withdrawal from the trial
The patients have the right to withdraw from the trial at any time for any reason. The researcher has the right to withdraw the patient from the trial in case of intercurrent disorder, side effects, inefficiency of administered therapy, violation of protocol, for administrative or other reasons. Intensive withdrawal can influence the results of the trial, hence, it is recommended to withdraw the patient only for serious reasons. After withdrawal from the trial, complete final examination must be performed and the reason of withdrawal registered.
Special recommendations and warnings
For the time of the protocol approval no special recommendations and/or warnings are available concerning cell immunoliposomal radioisotopic diagnostics and chemotherapy.
Additional therapy
Additional pharmaceuticals/non-pharmaceutical therapy will be provided in the list.
Evaluation criteria
General neurological response will be clinically evaluated according to the efficiency criteria and registered in the Patient’s Report (PR).
Neurological status is evaluated at baseline and according to the visit schedule. The researchers will evaluate neurological response according to points 3.5.2. and 3.5.3 after baseline evaluation.
The schemes of neurological responses will be reviewed. After the review and MRI to evaluate morphological changes the neurological response will be evaluated.
Primary efficiency outcomes:
A) Proportion of “objective” responses:
The number will be calculated by division of the number of patients demonstrating “objective” reaction to the implant in MRI by the total number of the patients in the population. For every patient the best result ever observed during the trial will be registered.
B) Proportion of ‘clinical” responses:
The number will be calculated by division of the number of patients demonstrating “clinical” reaction by the total number of the patients in the population. The patients are considered to have clinical reaction if they have objective reaction or they meet at least ONE of the following criteria:
А) Unambiguous improvement
Б) Unambiguous signs of tumor reduction or elimination on MRI
В) Unambiguous improvement in paraclinical criteria (EEG, blood and CSF immuinochemistry, etc)
Confidence intervals (binomial, 95%, Pearson-Clopper method) will be calculated for respondents’ data.
Secondary efficiency outcomes:
A) General survival
It is calculated from the first day of trial to the day of death from any reason.
B) Functional independence conversion indicator.
It will be calculated from the first trial day to the day of registered neurological improvement or day of death for any reason.
Survival patterns for general survival will be presented (Kaplan-Meier survival curve) and disease free interval (actuarial method).
 Safety evaluation
•    Side effect
Safety evaluation is mostly based on frequency of side effects, especially those leading to therapy termination and laboratory values deviations.  
Side effects will be summed up as general figure and percentage of the patients with side effects, their type and intensity. Side effects, initiating death, trial withdrawal or classified as demanding dose reduction, will be shown separately.
•    Laboratory tests
Laboratory data will be summed according to NCI/NIH criteria (see Appendix 6).
•    Neurological status/body weight
Neurological changes as compared to baseline and changes in body weight will be summed at appropriate intervals. Statistical tables will be provided at appropriate intervals.
•    Pain
Changes in combined pain scores and pain interference scores will be calculated at appropriate intervals. Statistical tables will be provided at appropriate intervals.
Administration of pain relievers
Tables of interchangeability will be presented to sum changes in administration of pain relievers as compared to baseline at appropriate intervals.

Therapy administration
Cell preparation is administered in the course of medical procedure by the research doctor, head investigator or one of their colleagues. Individual records should be kept for every patient. The number of cell preparation units that were received, distributed and returned must be determined and registered.  The data are kept for 5 years after the end of the therapy.
Schedule of visits and assessment
 Visit schedule
The patients must be followed in the Research Center according to the schedule showed at Table 5.  


For all trial groups the examination consists of two phases: screening, administration of ASC with NK and dynamic follow-up of the therapy for 12 weeks. Assessment at screening stage is performed within 30 days before trial therapy.

Table 5. Visits and assessment schedule
Phase    Screening (l)    Therapy week
Week    -30/20 days    0    1    2    3    4    5    6    7    8    9    1
0    1 1    12(2)    16(5)    20(5)    24(5)    26
Visit /Report No.    1    2    3    4    5    6    7    8    9    1 0    1
I    1
2    1
3    14    15    16    17    15/18
Transplantation of cell precursors of NK         X                                                                
Written Informed Consent

 Согласие    X                                                                    
Inclusion/exclusion criteria    X                                                                    
Pregnancy test (if applicable)     X    X                                                                X
Demographic data/relevant medical case history/current medical history case    X                                                                    X
Blood sampling and storage




    X                                                                    
Isolation of olfactory glia and cryopreservation  (3)    X                                                                    
Isolation of CD34+ stem cells and cryopreservation (4)    X                                                                    
Administration report  (5)        X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    
Neurological assessment    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X
Eyeground examination    X                    X                X                X                X
Somatosensory and evoked motor potentials    X            X        X        X        X        X        X    X    X    X    X
Blood and CSF immunochemistry    X                X            X            X            X    X    X    X    X
ECG    X                X            X            X            X    X    X    X    X
Echocardiogram    X                            X                        X                X
CSF test    X    X    X    X    X            X            X            X    X    X    X    X
Physical/vital functions assessment    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X
Pain/analgesics assessment    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X
Paramagnetic, T1+T2 MRI of brain and spinal cord (5)    X            X        X        X        X        X        X    X    X    X    X
Neurospecific Ag and Ab    X                X            X            X            X    X    X    X    X
Hematology    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X
Blood biochemistry    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X    X
Serology    X                X            X            X            X    X    X    X    X
Side effects    Continuous data collection
Additional administrations of pharmaceuticals/therapies    X    Continuous data collection
Comments    X    Continuous data collection
Research completion            x(2)            x(5)    

(1) screening assessments can be done 30 days prior to the therapy beginning. (2) Therapy completion for groups 1 and 2; completion of stay in rehabilitation centre. Can be completed any time in case of terminated research. (3) MRI must be done within 30 days before the beginning of therapy. MRI must be done to the brain and spinal cord, with and without paramagnetic enhancement and with spectroscopic sequences.

Screening assessments
Prior to any medical procedures, the Informed Consent must be received from the patient.
Laboratory screening tests, physical examination, including neurologic assessment involving international scores. Vital functions assessment, body weight must be registered 30 days prior to the beginning of the research.
For females of productive age the serum must be tested for pregnancy prior to peripheral blood sampling.
All results must be registered in the Patient Report (PR) (visit#1).
Transplant under study is delivered to the patient during transplantation procedure.
EFFICIENCY, SAFETY AND SIDE EFFECTS EVALUATION  
Parameters of primary efficiency – neurological progress
Neurological assessment
Neurological assessment is done at standard neurological examination.
It is preferable that screening visits and visits at the therapy phase will be done by different specialists. Preceding clinical reports will be blinded.
Neurological response will be evaluated every week at the examination by neurologist. Neurological response will be registered according to international scales in progress during a year.  
 Safety assessment
Safety assessment consists in evaluation of side effects (SE) and serious side effects (SSE), laboratory data including hematology, biochemistry, neurospecific antigens and antibodies. Vital functions, physical and neurologic examinations. Registration of all additional pharmaceuticals and/or therapies. Toxicity is evaluated according to Common Toxicity Criteria of NCI, version 2.0 (Appendix 1).  
Side effects
Information on all side effects provided by the patient, detected by the researcher during interrogation, physical examination, laboratory testing or other methods, is registered in the report on side effects (SE) and appropriately followed. Side effects are any adverse sign, symptom or medical condition appeared after the therapy beginning even if the event is not considered to be associated with administered therapy.  
Medical conditions observed prior to the therapy beginning are considered side effects only in case of their deterioration after the beginning of the therapy.  If observed after the beginning of the therapy and after signing of the Informed Consent, side effects (but not SSE) are registered in the Case history/Current condition report only in case the patient receives the therapy according to the research. Deviations in laboratory data are considered a side effect only if they lead to clinical symptoms or signs or demand therapy, when they are registered in Side Effects Report as signs, symptoms or associated diagnoses.
Description of every side effect must include the following, as far as it is possible:
1.    Duration (date of onset and end, or continuation at the time of final examination)   
2.    Degree of severity 1-4 according to NCI/NIH Common Toxicity Criteria (Appendix 1)
3.    Association with transplant (supposed/not supposed)
4.    Measures taken
Any SE appeared during the research (within 12 weeks after transplantation) must be registered at SE page of PR.
Serious Side Effects
Information on serious side effects must be registered in Serious Side Effects (SSE). For safety guarantee every serious side effects must be reported to Main Researcher and NeuroVita Clinic, during 24 hours after it is known. Serious side effect on the whole is an adverse event which is:
1.    Mortal
2.    Life-threatening
3.    Demands hospitalization or duration of hospital stay
4.    Leading to significant or long-term disability or incapacitation.
5.    An inborn anomaly or inborn malformation

The events are not considered Serious Side Effects, when they demand hospitalization for the following reasons:
•    Routine therapy or monitoring of investigated value, not associated with any condition deterioration
•    Optional or planned therapy for previous disorder not associated with investigated disease.
•    Admission to hospital or other institution, not associated with any deterioration.
•    Urgent ambulatory medical care for the event that does not fall into the category of SSE and does not lead to hospitalization.
As distinct from usual safety evaluation, SSE must be observed constantly and must meet special demands to reporting; see section 9.1.  
Any SSE developed after signing Informed Consent and within four weeks after research completion, must be registered and reported. All side effects must be treated appropriately. The treatment may include changes in the investigated therapy, including possible pause or arrest of the therapy, administration or withdrawal of additional pharmaceuticals, hospitalization or any other medical intervention. When the side effect is detected, it must be followed till resolution during every visit, or if necessary more often. Any changes must be evaluated according to severity, supposed association with investigated therapy, necessary interventions aiming at management and outcome.
Information on standard side effects associated with investigated transplant can be found in the Investigator’s brochure. The information must be enclosed to the Informed Consent of the Patient and discussed with the patient in the course of investigation, if necessary.
Pregnancy
Any pregnancy or fatherhood happening during ASC with NK administration and in the course of 26 weeks after transplantation must be registered in PR.
Laboratory tests
Laboratory tests will be done according to Visit Schedule (Fig. 1). The copy of laboratory certificate and tabulation of normal ranges must be provided. In case the tests will be done in a vendor laboratory, the copy of certificate and tabulation of normal ranges for that laboratory must be provided as well.
Any time during investigation, clinically relevant (leading to clinical signs or symptoms or demanding medical intervention) deviation in laboratory values must be registered at appropriate page of the patient’s report, notwithstanding specific protocol demands and aside from appropriate page of laboratory tests.  In case deviations of laboratory values or tests constitute side effect (for example, lead to clinical signs/symptoms or demand therapy), they must be reflected in the Patient’s Report on Side Effects.
Peripheral blood
•    Hematology
Hematoglobulin, RBC plasma ratio, RBC, WBC, platelets. Differential count, including neutrophils, T and B lymphocytes, eosinophils.
Analysis of time of bleeding, platelet aggregation, PT and APPT. During screening visit the patients’ blood group and Rh factor.
Any deviation must be registered in the Patient’s Report on Side Effects. Besides, hematological safety tests must be done during 6 hours after peripheral blood harvest. These tests are registered in PR every visit.
•    Biochemistry
Glucose, sodium, potassium, calcium, iron, osmolarity, blood urea and urea nitrogen, creatinine, total protein, albumin, SPE, bilirubin (total, dire ct, indirect), alkaline phosphatase, GGT, AST, ALT and LDH.  
Besides, biochemical safety tests must be done during 6 hours after peripheral blood harvest. These tests are registered in PR every visit.
•    Serology
Neurospecific antigens (GFAP, NSE, MBP). Neurospecific antibodies (Anti-GFAP, anti-NSE, anti- MBP).
Infection agents (CMV, HSV, VZV, EBV, VDRL, HIV-1-2, HTLV-1-2, Hepatitis  В and С, toxoplasma, listeria)
Wassermann’s test, PCR for infections, anti-HBV, anti -HCV, anti -HIV1-2 antibodies.
Immunophenotypes: CD3, CD4, CD8, CD4/CD45RO, CD3-CD16/CD56+, CD19, CD14, IgG, IgA, IgM.
Serological test results will be compared to the screening results
Cerebrospinal Fluid
Glucose, proteins, cell number, PCR for infections
Neurospecific antigens (GFAP, NSE, MBP).
Neurospecific antibodies (Anti-GFAP, anti-NSE, anti- MBP).
 Urine
Glucose, protein, blood, bilirubin, ketone bodies, nitrites, pH, osmolarity, culture.
Physical and vital functions assessment
Physical and vital functions assessment is performed according to the Visit and Assessment schedule. Information on physical examination and vital functions must be registered in original documents at the trial site. Meaningful findings detected at baseline before administration of investigated preparation must be registered in the section Relevant Case History/Current Medical Conditions of PR. Meaningful findings observed after investigation was begun, must be registered in the section for Side Effects of PR. PR Registration of normal findings during physical and vital functions examination is not supposed.
Body weight
Body weight is measured according to the Visit schedule (Fig. 1). It is recommended that the patients measure their weight themselves and report any change of 2kg or more as compared to baseline to the investigator.
Pain/analgesics assessment
Pain is assessed according to pain values using Brief Pain Inventory (BPI) (Appendix 4). BPI specifies three pain values: pain severity, composite index and pain interference. In this investigation composite index of pain will be used, i.e. average scores of the points 3, 4, 5 and 6 and pain interference.
Use of analgesics is assessed according to pain relieving indexes (Appendix zz).
Registration of administered doses
The weight, volume, number of stem cells or immunocompetent cells of administered cell preparation is registered.
8. Amendments to protocols/changes in investigation procedure
Any changes or additions (excluding administrative) in the protocols must be executed as written amendments to protocol. Amendments that considerably influence safety of the patient, scope or scientific quality of investigation demand additional agreement with the investigators and Neurovita Clinic, for example:
1.    Considerable changes in the investigation design
2.    Increase the number of invasive procedures
3.    Increase or reduction of testing procedures necessary for safety monitoring.
These demands of agreement must not interfere with any actions of the Investigators or NeuroVita Clinic for the patients’ safety. In case the main Investigator demands urgent changes in the protocol, and these changes are introduced for the safety of the patients, NeuroVita Clinic must be notified of that immediately. Amendments that influence only administrative aspects of investigation do not require formal amendments of the protocol. Examples of administrative changes not requiring formal amendments of the protocol:
1.    Changes in the personnel of administration (e.g. NeuroVita Clinic)
2.    Small changes in criteria of inclusion/exclusion used for selection of the patient
3.    Small changes in the package or labeling of the investigated transplant.

9. Data Processing
Will be performed by the representative of NeuroVita Clinic.
9.1. Data Gathering
The investigators must record the necessary appropriate information. Details of neurologic response must be documented in the case history of the patient.
9.2.    Data base processing/quality control

PR data are recorded in the investigation data base using separate data input.
Further, information from data base is regularly supervised by the main Investigator.
Additional pharmaceuticals entered into data base are coded according to the list of medicines of WHO based on ATC code. Concomitant diseases and side effects will be coded according to MedDRA.
  STATISTIC METHODS  
Statistic methods
 It is an open clinical trial. Design of standard trial of I/IIa phase is inapplicable to this trial, as well as statistic rules with predefined coefficient of error rates for graft retention/rejection of studied transplant, based on predefined coefficient of success/failures. Moreover, there is no opportunity to modulate inclusion of the patients basing the observed results due to a long period necessary for clinical response.
The candidates are included into the trial prospectively, receive their treatment, are followed according to the protocol and the results are regularly renewed and controlled by toxicity and efficiency.  
Populations
Population of analyzed safety (PAS) includes everyone who received at least one dose of the investigated therapy.
Population intending to be treated (PIT) includes the patients who were included into the trial and received the treatment. The patients withdrawn from the trial for SE or toxicity before key reaction assessment are considered treatment failures.
Population of analyzed efficiency (PAE) will consist of the patients who:
•    Comply with criteria of inclusion/exclusion
and
•    Have completed the treatment phase or were withdrawn from the trial for death
Or
•    Were withdrawn from the trial for toxicity (SE associated with cell preparations) and passed at least one assessment of key reaction.
As far as the primary goal is to evaluate treatment efficiency, primary analysis of safety will be performed in PAS.  
As the secondary goal is to evaluate treatment induced restoration of neurologic functions, primary analysis of safety will be performed in PAE and confirmed in PIT, all secondary analyses of efficiency will be performed in PIT population only.  
It is not supposed to substitute the patients that do not enter PAE group. The patients can be substituted in particular cases after discussion with the investigators and NeuroVita Clinic if the patient is supposed to lack information to assess safety and efficiency of the transplant.
Demographic and biographic data
Demographic data (age, sex, and nationality), diagnosis, history and original characteristics (activity status) will be summed up for all included patients.
Other data for included patients will be enlisted.
Investigated cell preparation  
Dosage information will be enlisted in PR.
 Type of treatment
      -  Intrathecal administration of allogeneic  haploidentic HLA-compatible HSCs.
     -  Intramuscular or subcutaneous administration of proteome-personalized dendritic vaccines
     -  Intrathecal administration of autologous peptide modified HSC preparation
     - Administration of individualized  cytotoxic T-lymphocytes in projections of lymphatic nodes collectors
 Audit procedures
                 ZAO NeuroVita Clinic, FGBU RONC RAMN, FGBU FNKC SMP and MT FMBA of Russia will provide all cell preparations to the Main Investigator.  The cell preparation is stored in appropriate safe place (e.g. locked cabinet) according to the conditions set in the protocol. The researchers must provide exact registration of transporting and distribution of the investigated transplant in report form on cell preparation.  Accurate records on date and quantity of cell preparation distributed for every patient must be available for supervision every day. All implantation material must be used only for this protocol and not for any other purposes.
The investigators must not eliminate any labels from containers with cell preparations or partially used or unused material. The trial being completed and if necessary in the course of trial, the investigators store (FROZEN IN A SREPARATE CONTAINER) all used and unused cell preparation containers  with tubes labels and copy of the record of preparation distribution.
Results Publication
          Any formal presentation or publication of the data gathered in the course of the investigation will be considered joint publication of the investigator(s) and corresponding personnel of ZAO NeuroVita Clinic and ANO National Institute of Regenerative Medicine.
Public Disclosure and Confidentiality
Signing the protocol the investigator agrees to keep all information on the investigation in secrecy and demand similar confidentiality from their personnel and Ethic Committee (protocols, investigator’s brochure, RP and other).
Termination
Investigation can be terminated for clinical or other reasons at any time.
Ethic Committee
           Prior to investigation beginning, the protocol, proposed Informed Consent and other information for patients must be approved by the Ethic Committee (EC) of FGBU FNKC SVMPi MT FMBA of Russia. Before investigation, the signed and dated claim that the protocol and Informed Consent was approved by the Ethic Committee (EC) of FGBU FNKC SVMPi MT FMBA of Russia. The names and positions of   the chair and members of the EC must be presented to ZAO NeuroVita Clinic. The committee must give permissions to any changes in the protocol but for administrative.
Informed Consent
          The Investigator must explain to every patient (or authorized representative) the nature of investigation, its goal, planned procedures and expected discomfort. Every patient must be aware that participation in the trial is volitional, and they are free to withdraw from the trial anytime and withdrawal of appropriate Informed Consent will not influence further therapy or attitude of attending doctor.
        The Informed Consent must be presented as a standard written notice in a non-technical style. In case written consent is impossible, an oral consent is acceptable if witnessed by two non-participants of the study and stated reason for the patient’s inability to sign.  None of the patients can participate in the study before signing the Informed Consent.
       The Informed Consent is a part of the protocol and must be filed together with the protocol for the EC approval (Appendix 2).
WMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects
 Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964
and amended by the:
29th WMA General Assembly, Tokyo, Japan, October 1975
35th WMA General Assembly, Venice, Italy, October 1983
41st WMA General Assembly, Hong Kong, September 1989
48th WMA General Assembly, Somerset West, South Africa, October 1996
52nd WMA General Assembly, Edinburgh, Scotland, October 2000
53rd WMA General Assembly, Washington, DC, USA, October 2002
(Note of Clarification on paragraph 29 added)
55th WMA General Assembly, Tokyo, Japan, October 2004
(Note of Clarification on Paragraph 30 added)
59th WMA General Assembly, Seoul, Korea, October 2008
A.  INTRODUCTION
1. The World Medical Association (WMA) has developed the Declaration of Helsinki as a statement of ethical principles for medical research involving human subjects, including research on identifiable human material and data.
    The Declaration is intended to be read as a whole and each of its constituent paragraphs should not be applied without consideration of all other relevant paragraphs.
2. Although the Declaration is addressed primarily to physicians, the WMA encourages other participants in medical research involving human subjects to adopt these principles.
3. It is the duty of the physician to promote and safeguard the health of patients, including those who are involved in medical research. The physician's knowledge and conscience are dedicated to the fulfilment of this duty.
4. The Declaration of Geneva of the WMA binds the physician with the words, "The health of my patient will be my first consideration," and the International Code of Medical Ethics declares that, "A physician shall act in the patient's best interest when providing medical care."
5. Medical progress is based on research that ultimately must include studies involving human subjects. Populations that are underrepresented in medical research should be provided appropriate access to participation in research.
6. In medical research involving human subjects, the well-being of the individual research subject must take precedence over all other interests.
7. The primary purpose of medical research involving human subjects is to understand the causes, development and effects of diseases and improve preventive, diagnostic and therapeutic interventions (methods, procedures and treatments). Even the best current interventions must be evaluated continually through research for their safety, effectiveness, efficiency, accessibility and quality.
8. In medical practice and in medical research, most interventions involve risks and burdens.
9. Medical research is subject to ethical standards that promote respect for all human subjects and protect their health and rights. Some research populations are particularly vulnerable and need special protection. These include those who cannot give or refuse consent for themselves and those who may be vulnerable to coercion or undue influence.
10. Physicians should consider the ethical, legal and regulatory norms and standards for research involving human subjects in their own countries as well as applicable international norms and standards. No national or international ethical, legal or regulatory requirement should reduce or eliminate any of the protections for research subjects set forth in this Declaration.
B.  PRINCIPLES FOR ALL MEDICAL RESEARCH
11. It is the duty of physicians who participate in medical research to protect the life, health, dignity, integrity, right to self-determination, privacy, and confidentiality of personal information of research subjects.
12. Medical research involving human subjects must conform to generally accepted scientific principles, be based on a thorough knowledge of the scientific literature, other relevant sources of information, and adequate laboratory and, as appropriate, animal experimentation. The welfare of animals used for research must be respected.
13. Appropriate caution must be exercised in the conduct of medical research that may harm the environment.
14. The design and performance of each research study involving human subjects must be clearly described in a research protocol. The protocol should contain a statement of the ethical considerations involved and should indicate how the principles in this Declaration have been addressed. The protocol should include information regarding funding, sponsors, institutional affiliations, other potential conflicts of interest, incentives for subjects and provisions for treating and/or compensating subjects who are harmed as a consequence of participation in the research study. The protocol should describe arrangements for post-study access by study subjects to interventions identified as beneficial in the study or access to other appropriate care or benefits.
15. The research protocol must be submitted for consideration, comment, guidance and approval to a research ethics committee before the study begins. This committee must be independent of the researcher, the sponsor and any other undue influence. It must take into consideration the laws and regulations of the country or countries in which the research is to be performed as well as applicable international norms and standards but these must not be allowed to reduce or eliminate any of the protections for research subjects set forth in this Declaration. The committee must have the right to monitor ongoing studies. The researcher must provide monitoring information to the committee, especially information about any serious side effects. No change to the protocol may be made without consideration and approval by the committee.
16. Medical research involving human subjects must be conducted only by individuals with the appropriate scientific training and qualifications. Research on patients or healthy volunteers requires the supervision of a competent and appropriately qualified physician or other health care professional. The responsibility for the protection of research subjects must always rest with the physician or other health care professional and never the research subjects, even though they have given consent.
17. Medical research involving a disadvantaged or vulnerable population or community is only justified if the research is responsive to the health needs and priorities of this population or community and if there is a reasonable likelihood that this population or community stands to benefit from the results of the research.
18. Every medical research study involving human subjects must be preceded by careful assessment of predictable risks and burdens to the individuals and communities involved in the research in comparison with foreseeable benefits to them and to other individuals or communities affected by the condition under investigation.
19. Every clinical trial must be registered in a publicly accessible database before recruitment of the first subject.
20. Physicians may not participate in a research study involving human subjects unless they are confident that the risks involved have been adequately assessed and can be satisfactorily managed. Physicians must immediately stop a study when the risks are found to outweigh the potential benefits or when there is conclusive proof of positive and beneficial results.
21. Medical research involving human subjects may only be conducted if the importance of the objective outweighs the inherent risks and burdens to the research subjects.
22. Participation by competent individuals as subjects in medical research must be voluntary. Although it may be appropriate to consult family members or community leaders, no competent individual may be enrolled in a research study unless he or she freely agrees.
23. Every precaution must be taken to protect the privacy of research subjects and the confidentiality of their personal information and to minimize the impact of the study on their physical, mental and social integrity.
24. In medical research involving competent human subjects, each potential subject must be adequately informed of the aims, methods, sources of funding, any possible conflicts of interest, institutional affiliations of the researcher, the anticipated benefits and potential risks of the study and the discomfort it may entail, and any other relevant aspects of the study. The potential subject must be informed of the right to refuse to participate in the study or to withdraw consent to participate at any time without reprisal. Special attention should be given to the specific information needs of individual potential subjects as well as to the methods used to deliver the information. After ensuring that the potential subject has understood the information, the physician or another appropriately qualified individual must then seek the potential subject's freely-given Informed Consent, preferably in writing. If the consent cannot be expressed in writing, the non-written consent must be formally documented and witnessed.
25. For medical research using identifiable human material or data, physicians must normally seek consent for the collection, analysis, storage and/or reuse. There may be situations where consent would be impossible or impractical to obtain for such research or would pose a threat to the validity of the research. In such situations the research may be done only after consideration and approval of a research ethics committee.
26. When seeking Informed Consent for participation in a research study the physician should be particularly cautious if the potential subject is in a dependent relationship with the physician or may consent under duress. In such situations the Informed Consent should be sought by an appropriately qualified individual who is completely independent of this relationship.
27. For a potential research subject who is incompetent, the physician must seek Informed Consent from the legally authorized representative. These individuals must not be included in a research study that has no likelihood of benefit for them unless it is intended to promote the health of the population represented by the potential subject, the research cannot instead be performed with competent persons, and the research entails only minimal risk and minimal burden.
28. When a potential research subject who is deemed incompetent is able to give assent to decisions about participation in research, the physician must seek that assent in addition to the consent of the legally authorized representative. The potential subject's dissent should be respected.
29. Research involving subjects who are physically or mentally incapable of giving consent, for example, unconscious patients, may be done only if the physical or mental condition that prevents giving Informed Consent is a necessary characteristic of the research population. In such circumstances the physician should seek Informed Consent from the legally authorized representative. If no such representative is available and if the research cannot be delayed, the study may proceed without Informed Consent provided that the specific reasons for involving subjects with a condition that renders them unable to give Informed Consent have been stated in the research protocol and the study has been approved by a research ethics committee. Consent to remain in the research should be obtained as soon as possible from the subject or a legally authorized representative.
30. Authors, editors and publishers all have ethical obligations with regard to the publication of the results of research. Authors have a duty to make publicly available the results of their research on human subjects and are accountable for the completeness and accuracy of their reports. They should adhere to accepted guidelines for ethical reporting. Negative and inconclusive as well as positive results should be published or otherwise made publicly available. Sources of funding, institutional affiliations and conflicts of interest should be declared in the publication. Reports of research not in accordance with the principles of this Declaration should not be accepted for publication.
C.  ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH MEDICAL CARE
31. The physician may combine medical research with medical care only to the extent that the research is justified by its potential preventive, diagnostic or therapeutic value and if the physician has good reason to believe that participation in the research study will not adversely affect the health of the patients who serve as research subjects.
32. The benefits, risks, burdens and effectiveness of a new intervention must be tested against those of the best current proven intervention, except in the following circumstances:
•    The use of placebo, or no treatment, is acceptable in studies where no current proven intervention exists; or
•    Where for compelling and scientifically sound methodological reasons the use of placebo is necessary to determine the efficacy or safety of an intervention and the patients who receive placebo or no treatment will not be subject to any risk of serious or irreversible harm. Extreme care must be taken to avoid abuse of this option.
33. At the conclusion of the study, patients entered into the study are entitled to be informed about the outcome of the study and to share any benefits that result from it, for example, access to interventions identified as beneficial in the study or to other appropriate care or benefits.
34. The physician must fully inform the patient which aspects of the care are related to the research. The refusal of a patient to participate in a study or the patient's decision to withdraw from the study must never interfere with the patient-physician relationship.
35. In the treatment of a patient, where proven interventions do not exist or have been ineffective, the physician, after seeking expert advice, with Informed Consent from the patient or a legally authorized representative, may use an unproven intervention if in the physician's judgment it offers hope of saving life, re-establishing health or alleviating suffering. Where possible, this intervention should be made the object of research, designed to evaluate its safety and efficacy. In all cases, new information should be recorded and, where appropriate, made publicly available.

Procedures and Instructions
Safety procedures
 Serious Side Effects Report
Any serious side effects must be reported, including serious deviations in laboratory tests developed in the period after signing the Informed Consent and till six weeks after AHSC with apoptosis inducing NK transplantation. The period after transplantation can be expanded if the investigator supposes association with transplantation. All serious side effects must be recorded in the period when the study protocol is joined to standard therapy. Repeating episodes, complications and progress of initial SSE must be recorded as following observation after initial episodes within 24 hours after the investigator is informed on observation. SSE that happened in other time is considered either unassociated with previous one or must be registered as a new side effect.
Information on all SSE must be gathered and registered in the Serious Side Effects Report. The investigator must evaluate association with investigated transplant, fill the report on SSE and send filled and signed report to the Main Investigators during 24 hours after information on SSE is received. Contact phone and fax numbers are provided. Original of Report and faxed confirmation must be stored together with case history files at the trial site.
Information on the observation should be forwarded to the same Investigator who received SSE Report with the note saying this is the observation following previously registered SSE and its date. Every relapse, complication or progression of initial event must be registered as follow-up observation of the event independent from the time of onset. The record on the observation must inform whether the event was coped with or it persists, what treatment the patient receives for it, if the patient is still in the trial or is withdrawn from the trial.
Specific questions involving the patient and serious side effect must be forwarded to the Main Investigator. Issues on SSE report transfer are forwarded to the Main Investigator.
                          Pregnancy report
Any pregnancy or fatherhood happened 6 months post transplantation of AHSC with apoptosis inductor NK must be registered in the Patient Report.
To guarantee the patient’s safety every pregnancy of trial participant must be reported to the Main Investigator within 24 hours upon reporting. Pregnancy must be registered in PR and the investigators must be aware of it. Pregnancy must be followed till outcome, including spontaneous or voluntary interruption, birth details, birth defects, hereditary disorders, complications in mother and/or infant. Pregnancy follow-up must be registered in PR and contain evaluation of possible outcome association with investigated transplant. Any SSE registered during pregnancy must be registered in SSE Report.
In case the father received transplantation of the investigated material, the Informed Consent to provide information on pregnancy outcome must be obtained from the mother.
Serious Side Effect Reporting

Every serious side effect must be registered in a Side Effect Report. Every side effect must be described according to:
1.    Duration (date of onset and terminal)
2.    Severity (degrees 1-4)
3.    Association with investigated transplant (supposed/not supposed)
4.    Measures taken
Detection of side effect severity provides for quality of evaluation of side effect intensity according to evaluation of investigator or patient’s report. Severity does not show clinical meaning of the event, only intensity or degree (e.g. strong nausea, minor cramps) and does not reflect association with the investigate transplant.
Severity Grades for Events not included ОКТ NCI/NIH
1= 1 grade    I- light
2=2 grade    II-moderate severity
3=3 grade    III-severe
4=4 grade    IV-life threatening

Association of the investigated implant and side effect is described through one of the categories as supposed or not supposed (absent) by the investigator.
Association of the investigated to immunotherapy.
0=not supposed    Temporal associations of the clinical effect and investigated material with immunotherapy make cause and effect relations unlikely, or other medications, interventions or hidden states present sufficient explanations of the observed event.
1=supposed    Temporal associations of clinical event and investigated material make cause and effect relations possible other medications, interventions or hidden states do not present sufficient explanations of the observed event.
Actions taken to cope with the side effect are described through numerical scale from 1 to 6 points including different options. One or more options can be chosen.
Actions to cope with the side effect
0=No actions taken
1=additional medication
2=Drug-free therapy
3=Hospitalization/extended hospitalization
                                  Management
Changes in the Program Protocol
Any changes or additions to the protocol require written amendment approved by ZAO NeuroVita Clinic and the Main Investigator before applying. Amendments that considerably influence safety, trial size or scientific quality demand additional approval of Ethical Committee, or if necessary, with other organizations. The copy of written approval of Ethical Committee becomes part of the protocol and must be given to the Main Investigator. Examples of amendments that require approval include:  
1.    Changes in transplant treatment
2.    Considerable changes in the trial design (e.g. adding or deleting control group)
3.    Increase of the number of interventions
4.    Addition or elimination of testing procedures for safety monitoring.
These demands to the approval must not prevent from immediate measures taken by the investigator or ZAO NeuroVita Clinic for the safety of the patients. In case the Main Investigator considers amendments urgent and initiates them for safety reasons, ZAO NeuroVita Clinic must be informed on them, and Ethical Committee in the course of 10 days.
Amendments influencing only administrative part of the trial do not require changes in the protocol or Ethical Committee approval. Examples of administrative amendments not requiring formal changes in the protocol or Ethical Committee approval include:
1.    Changes in the staff who monitor trial (E.g. staff of the ZAO NeuroVita Clinic)  
2.    Minor changes in the package or labeling of the investigated product.

                                                      Monitoring procedures
During the trial a supervisor from ZAO NeuroVita Clinic will visit the trial site regularly, examine reports and how they are filled, protocol adherence, inclusion into the trial, as well as control storage, recording and distribution of the investigated transplant according to specifications. The investigators and key personnel must be provided for the visit.
The supervisor must be provided the access to the records to confirm their conformity to the PR. All information will be kept confidential.  
The copy of PR is stored at the Main Investigator who guarantees its storage with other documents, such as protocol, the Investigator brochure and any amendments to the protocol in a safe place.
                                             Data registration/Documents storage  

General instructions on filling PR are given in the PR.
The patients’ data in PR will be documented anonymously and the patient will be named by identification number, birth date, and, if necessary, by initials. In case identification of the patient is necessary for the safety or approval procedures, ZAO NeuroVita Clinic and the investigators are obliged to keep the information confidential.
All information required by the protocol must be provided and any omissions must be explained. All PRs must be filled and provided for analysis no later than 7 days after the visit of the patient, so that the supervisor is able to control accurateness, exactness and readability.
All data must be entered accurately, in a blue pen to provide readability. No corrections in a correcting fluid should be made.
         The investigator must fill all basic reports for the trial. All information of RP should be traceable in the basic documents attached to the case history. Basic documents must contain all demographic and medical information including laboratory data, electrocardiograms and the copy of Informed Consent with the number and name of the trial.
            The investigator must store important documents as long as required according to the state and international standards (usually 10 years after termination of the trial).  
Significant documents include:
1. EC approvals of the protocol and all amendments
2. All basic documents and laboratory records
3. PR copies
4. Informed Consent
5. Any other trial concerning documents
EXAMPLE
Informed Consent
For participation in clinical trial
        I, hereby, (name of the patient) ______________________________________________________________________________________, born «___» __________ sign the present Informed Consent  for participation in limited clinical trial organized by ZAO “NeuroVita” Clinic, FGBU N. N. Blokhin Russian Cancer Research Center of RAMN, FGBU “Federal Research Center for Specialized Types of Medical Care and Medical Technologies” of FMBA of Russia according to protocol №__________________________________________________________________________________________________________________________________________________________________voluntarily and in my own hand.
I (or my authorized representative) received clear and detailed explanation on the nature of the trial, its potential risk and benefit and possible discomfort. I (or my authorized representative) are informed that participation in the trial is volitional and I (they) can withdraw from the trial at any time and withdrawal of the consent will not influence further therapy and/or attitude of attending doctor.
I (or my authorized representative) am informed that I am (they are) offered a novel therapy involving immunotherapy with the cell preparation from the hematopoietic progenitor cells (HPCs) or neural precursors (NPs) of my (their) close relative (sister, brother, mother or father) or a histocompatible donor. The goal of the therapy is regulation and restoration of certain functional anticancer properties of the cells of my (their) immune system. I (they) understand that the goal of the proposed trial is evaluation of safety and efficiency of a new cell preparation and a whole complex of given anticancer immunotherapy.
I (they) received an explanation on the essence of the proposed immunotherapy that consists in various transplantations (subcutaneous, intramuscular, intraventricular, intrathecal, intravenous or intraarterial) of immune cell preparation. I was (they were) explained that the components of immune cell preparations migrate to the site of pathology or peri-tumoral edema following concentration of inflammatory chemokines and then evenly distribute through the site of pathology. The cells of applied transplant and patient’s tumor cells trigger acute immune reaction “graft-versus-host” initiating regulatory mechanisms of differentiation or apoptosis.
I (they) were informed that cell transplant was prepared by mobilization of hematopoiesis precursor cells to peripheral blood of histocompatible donor using granulocyte colony stimulating factor, further harvest of the cells, their separation and immunohistochemical isolation of specialized cells according to specific cell surface markers.  The preparation passes HLA-typing (in the group of patients with donor allogeneic preparation) to test for tissue compatibility and the preparation is used for immunotherapy only if tissue compatibility is confirmed. To obtain preparation of neural cell precursors, the cells are isolated from olfactory sheath of a nose and then cultured.   
     I am (they are) warned that the cell preparation obtained in the above mentioned way expands the range of therapies for tumors, diseases and injuries of central and peripheral nervous systems but cannot guarantee complete recovery, because its clinical effects are still evaluated. I have no claims against the personnel of the Clinic in case of complications.
The Informed Consent is signed by me before one witness and assisting doctor. In case written consent is impossible, an oral consent is acceptable provided that two witnesses not involved into trial are present and the reason preventing the patient from signing is stated.      
Patient _____________________________________________________________________________________________________________________________________________________
Signature _______________________________________ Date

Witness
______________________________________________________________________________________________________________________________________________________

Signature _____________________________________ Date
Doctor__________________________________________________________________________________________________________________________________________________

Signature ___________________________________   Date
Literature
 
1.    Брюховецкий А.С. Трансплантация нервных клеток и тканевая инженерия мозга при нервных болезнях- М.:ЗАО Клиника
«Нейровита», 2003.-400с.
2.    Брюховецкий А.С., В.П. Чехонин, Г.Л.Менткевич А.Ю. Стволовые клетки в нейроонкологии: здоровые и раковыестволовые клетки , их возможная роль и место в канцерогенезе и современных высокотехнологичных сценариях лечения опухолей мозга //                         Материалы  науч-практ-конференции «Высокие технологии в терапии и реабилитации  заболеваний нервной системы».-М.,2008.- С.43-44
3.    Введение в молекулярную медицину /  под ред. А.А.Пальцева/ .- М.,Медицина.-2006 .- 370с.
4.     Гродинз Ф. Теория регулирования и биологические системы .-М.,1966.- 254 с.
5.    Фокин С.В., Беркинблит М.Б. Математические проблемы в биологии.-М.,1973.-199 с.
6.    Неймарк Ю.И., Коган Р.Я., Савельев В.П. Динамические модели теории управления М.: Наука ,1985
7.    Цымбалюк В.И..Медведев В.В. Нейрогенные стволовые клетки : Монография-К., Изд-во «Коваль», 2005.- 596 с.
8.    Чехонин В.П. Дмитриева Т.Б. Гурина О.И.
9.    Коbayashi N., Navarro-Alvares N.,Soto-Guetierrez A., Kavamoto Hironobu. et al. Cancer  Stem Cells Research: Currently
10.    Situation and Problems//Cell Transplantation.-2008, Vol.17 .- P.19-25
11.    Marshall C.T., Guo Z., Lu C. et al. // Brain Res. – 2005 – Vol. 1045 – P. 45-56.
12.    Marshall C.T., Lu C., Winstead W. et al. // Histol. Histopatol. – 2006 – Vol. 21 – P. 633-643.
13.    Murrell W., Feron F., Wetzig A. et al. // Dev. Dyn. – 2005 – Vol.233 – P. 343-350
14.    Neimark Ju.I Mathematical models in Natural Science and Engineering,2003
15.    Roisen F.J., Klueber K.M., Lu C.L. et al. // Brain Res. – 2001 – Vol. 890 – P. 11-22.
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17.     Zhang X., Cai J., Klueber K.M. et al. // Stem Cells – 2005 – Vol. 23 – P. 442-53.
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20.    ZigovaT.,E.Snyder, Sanberg P.  Neural Stem Cells for Brain and Spinal Cord Repair,2003.-350 р.
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for malignant gliomas//  Neuro Oncol Advance Publication, published on August 1, 2008 as DOI: 10/1215/15228517-2008-056

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Thank you for visiting our webpage!

Today cancer cannot be cured completely, but it can be transferred into a chronic and non-lethal disease, the survival rates can be significantly increased and the life quality can be improved. Alternatively, we can help provide a dignified departure from life, free from pain and humiliation.

Sincerely yours,
Professor,
PhD,
MD

Andrey S. Bryukhovetskiy

NeuroVita

Address

23 Kashirskoye shosse
Moscow
115478
Russia

Contact us

Telephone:
007 (499) 324-9339
007 (499) 324-9389
Fax:
007 (499) 324-9350
Email: neurovita@mail.ru

Opening time

Administration:
Monday-Friday from 9 am to 8 pm
Saturday and Sunday from 9 am to 4 pm
Patients are admitted 24/7