The Study of Glioblastoma Differentiation Possibility

Along with a regular course of a tumor process — gradual transformation in low-grade and undifferentiated tumors — the transformation of malignant tumors into ones with lower anaplasia degree is also possible — reverse transformation (differentiation, reversion). Experimental studies demonstrate the possibility of differentiation induction of glial tumor cells into mature glial cells and proliferation inhibition [1–4]. The authors describe various factors causing differentiation; however, the mechanism of the phenomenon is unclear [5].

There are a few reports in literature describing the differentiation of malignant brain tumors confirmed morphologically in surgical reinterventions [6, 7]. Glioblastoma is the most common malignant brain tumor. According to different authors, total survival rate of patients with glioblastomas is 10–12 months, and patients with anaplastic astrocytic tumors — 20–24 months [8, 9].

A progression-free period, which can last from 1 to 12 months, is a significant criterion of efficiency assessment. A true cause of early or late continuous tumor growth is unknown. Total glioblastoma excision is considered to extend patients’ life [10, 11].

Glial tumors are characterized by invasive growth extending in the surrounding marrow far beyond the main tumor node, and in 47% cases tumor cells are found in the opposite sphere [11, 12]. In general oncology and neuro-oncology infrared (IR) tissue spectroscopy is just the technique to detect tumor cells in the surrounding brain matter and determine tumor morphological character to high precision. The studies [13–17] show infrared spectra of tumor and non-tumor cells, grey and white matter, astrocytoma and glioblastoma tissues to differ from each other.

A tumor is pathological change of structural and functional condition of not only newly formed tissue, its microenvironment but also the whole body. All processes proceeding in the body, some systems and cell groups are known to affect blood serum. Glial tumor cells are known to produce certain substances causing the blood-brain barrier breakdown. As a result, under-oxidized products and waste products of tumor cells enter bloodstream [11, 18].

Our previous studies of blood serum using IR spectroscopy in patients with brain tumors revealed significant dependence between IR spectroscopy findings, morphological nature of tumors and tumor grade [19–21]. Based on these findings we examined the patients with glioblastomas preoperatively and postoperatively to determine if it is possible to reduce anaplasia degree of glioblastomas from Grade IV preoperatively to Grade III consistent with postoperative anaplastic astrocytomas, i.e. if there is differentiation of glioblastomas.

The aim of the investigation was to assess the possibility of glioblastoma differentiation using infrared spectroscopy, proton magnetic resonance spectroscopy and immunohistochemistry.

Materials and Methods. We carried out a single blind prospective cohort study of 43 patients with malignant brain tumors undergoing medical treatment in Nizhny Novgorod Interregional Neurosurgical Centre and Nizhny Novgorod Research Institute of Traumatology and Orthopedics, Ministry of Health of the Russian Federation, over the period 2009–2013. Glioblastomas (Grade IV) were diagnosed in 22 patients (51.16%), among them primary tumors (de novo) were detected in 21 patients, and one patient had secondary glioblastoma resulted from malignant transformation of excised anaplastic astrocytoma. Anaplastic astrocytomas (Grade III) were diagnosed in 21 patients (48.84%). Control group consisted of 28 healthy volunteers. All patients underwent blood serum IR-spectroscopy. Preoperative and postoperative IR-spectroscopy findings of 16 patients with glioblastomas were compared.

The study complies with the declaration of Helsinki (adopted in June, 1964 (Helsinki, Finland) and revised in October, 2000 (Edinburg, Scotland)), and was performed following approval by the ethic committee of Nizhny Novgorod Research Institute of Traumatology and Orthopedics. Written informed consent was obtained from all patients.

The research technique was as follows. 5.0 ml blood from cubital vein was centrifuged (1000 rpm) within 15 min. Separated serum (1.0 ml) was dried in a plate in a dry-air sterilizer at 25°С for 24 h. Dry residue was crushed and suspensized in liquid paraffin. The research was carried out on spectrophotometer Specord 75 IR (Carl Zeiss, Germany) in spectral range 1200–1000 cm^–1. It enables to reveal full spectrum of substances in blood serum containing Р–О and С–О bonds, ordinary bonds and ester groups С–ОО, Р–ОО, all lipids, phosphorylated proteins, carbohydrates, creatine phosphokinase and other compounds referring to them. At first we determined peak height of absorption band with maxima 1170, 1165, 1160, 1150, 1140, 1130, 1125, 1100, 1070, 1050 and 1025 cm^–1. To exclude errors in the assessment of IR-spectroscopy findings we studied not absolute values of peak height of absorption bands but calculated the ratio of peak height of absorption bands (cm^–1/cm^–1): 1 — 1165/1160, 2 — 1165/1070, 3 — 1165/1150, 4 — 1165/1140, 5 — 1040/1070, 6  — 1165/1130, 7  — 1070/1025, 8 — 1165/1050, 9  — 1165/1025, 10  — 1100/1050, 11 — 1170/1150, 12 — 1170/1160, 13  — 1125/1165. Fasting blood was withdrawn in the morning, before taking medicines, on the eve of the surgery (days 1–3), as well as postoperatively (days 3–10).

All histological diagnoses in a postoperative period were confirmed morphologically, and in 25 complicated cases (39.7%) — by immunohistochemistry (10 cases of glioblastomas, and 15 cases of anaplastic astrocytomas). We assessed the results of morphological studies using WHO classification [22]. The presence of two of four main malignant tumor criteria: nuclear polymorphism, mitoses, endothelial proliferation, necroses — is consistent with Grade III; the presence of at least three criteria corresponds to Grade IV [23].

Seven patients with glioblastomas underwent proton magnetic resonance spectroscopy (PMRS) of tumors and identical areas of perifocal zones preoperatively and postoperatively. MRI scanner Magnetom-Symphony 1.5 Т (Siemens, Germany) with head coil was used. At first conventional brain examinations were performed with Т2-, Т1-weighed MR images in axial, sagittal and coronal planes, Т2 Flair — in axial planes. Based on the obtained images we determined a region of interest for 2D-multi-voxel spectroscopy. After preset adjustment of MRI-scanner: shimmimg and suppression of water molecule proton signal we initiated protocol csi_se 30 using Spectroscopy Evaluation. Further, after data post-processing we obtained graphic presentations of spectra parametric maps of metabolites and their ratios.

The findings were processed by univariate statistic techniques using Statistica 6.1. The results were presented as М±σ, where М — arithmetic mean, σ — standard deviation. Paired within-group and intergroup comparisons were performed using Student t-test, Wilcoxon and Mann–Whitney tests depending on the fulfillment of applied conditions. The samplings were considered to belong to different general populations if p≤0.05.

Results. To handle the problem of determining the diagnostic capability of different morphological types of tumors with different grades using IR-spectroscopy, we first studied the ratio values of absorption band peaks depending on histological type of brain tumors in preoperative period (Table 1).

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Table 1. The values of absorption IR spectrum band peak ratio of blood serum in patients with glioblastomas (Grade IV) and anaplastic astrocytomas (Grade III) in a preoperative period

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In the survey we revealed significant correlations between histological diagnoses confirmed by morphological (n=43) or immunohistochemical (n=25) studies, and ratio values of 13 absorption band peaks obtained by IR-spectroscopy of blood serum of the same patients.

In accordance with the findings, to have differential diagnostic profiles, we plotted diagrams consisting of 13 radial beams coming from the centre, with 30° angle between them. Each of the beams corresponds to a certain ratio of absorption bands. The obtained ratio values were plotted on these beams, and the endpoints of the segments connected yielded plane polygons representing differential and diagnostic profiles of brain tumors (Fig. 1).

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Fig. 1. Diagnostic profile based on averaged data: а — glioblastomas (Grade IV); b — astrocytomas (Grade III)

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As a result of the examination of 22 patients with morphologically verified glioblastoma using IR-spectroscopy, we obtained certain repeated values of ratios common only to brain tumors — Grade IV glioblastomas (See Table 1, Fig. 1, а). The examination of 21 patients with postoperatively morphologically verified anaplastic astrocytoma gave certain repeated values of ratios typical of brain tumor — Grade III anaplastic astrocytoma (See Table 1, Fig. 1, b).

To identify the capabilities of glioblastoma differentiation in a postoperative period using blood serum IR-spectroscopy we examined 16 patients with Grade IV glioblastoma and compared the findings with those of the same patients before the operation (Table 2).

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Table 2. Blood serum IR-spectroscopy findings of 16 patients with Grade IV glioblastomas based on the volume of incised tumor in a postoperative period

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It was found that in 12 cases of total tumor resection, in the postoperative period 13 values of absorption peak ratios and their diagrams changed and by their characteristics were consistent with the diagnosis “anaplastic astrocytoma” (Grade III). In four cases of subtotal tumor resection, postoperative IR-spectroscopy findings underwent no changes compared to preoperative findings and the diagnosis “glioblastoma” (Grade IV) remained. The volume of incised tumor was assessed by postoperative brain contrast CT and MRI. The absence of contrast accumulation in the operative intervention area was considered to be total tumor resection.

PMRS is known to enable to specify morphological tumor type, estimate anaplasia degree by the content and ratio values of metabolites in the tumor and peritumoral area [24–27].

Seven patients with glioblastomas underwent PMRS; we studied the tumor and peritumoral area condition preoperatively and postoperatively. Preoperative tumors of all patients were found to have significant increase of choline (Cho) peak height, significant decrease of NAA peak height and high double-peak of lactate (Lac). Choline/creatine (Cho/Cr) ratio was high (up to 20.1). There was also observed the significant increase of Lac/Cr ratio (up to 39). All the above mentioned data suggest high tumor grade — Grade IV. In the peritumoral area 1.0 cm away from a visible tumor edge there were observed moderate increase Cho peak height, insignificant decrease of NAA peak height and insignificant growth of Lac peak height. We found marked decrease of myoinositol/creatinine ratio (Ins/Cr) — 0.15±0.12, the increase of glutamine–glutamate/creatinine ratio (Glx/Cr) — 0.36±0.04. Сho/Cr ratio values in different voxels were 1.36±0.70. The obtained results were also consistent with Grade IV anaplasia. In all cases the diagnosis “glioblastoma” was confirmed by histological studies of different tumor parts. No heterogeneous tumors with different histological structure in one tumor node were found in our survey.

The same 7 patients after surgical resection of glioblastomas underwent PMRS of identical peritumoral areas. The comparative analysis of metabolic changes before and after tumor resection showed that postoperatively in the peritumoral area there was the reduction of Cho peak height, insignificant increase of Ins/Cr ratio values (0.22±0.29), the decrease of Glx/Cr ratio values (0.21±0.08), and the decreased Cho/Cr ratio values up to 0.69±0.23 indicating the reduced anaplasia degree up to Grade III–II.

The analysis of a proliferative activity marker Ki-67 in a solid part of the tumor and tumor cells of perifocal area in 10 patients with glioblastomas after surgery showed proliferation index Ki-67 in the tumor to be on average 15–25% that is consistent with Grade IV glioblastoma (Fig. 2, а). In all cases we studied different tumor areas. There were revealed no mixed histological tumor types. The study of peritumoral areas of totally removed tumors, where tumor cells were found, Ki-67 averaged to 5–10% corresponding to Grade III–II tumors (Fig. 2, b) [22].

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Fig. 2. Ki-67 expression in nuclei of tumor cells: а — the main node of glioblastoma: 25%; ×200; b — peritumoral area: 10%; ×100

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Below a clinical example is given.

Patient S. was operated on for continuous tumor growth in the right temporal lobe 23.09.2013. Pathology report dated 01.10 — glioblastoma.

The patient underwent preoperative MRI (Fig. 3) and PMRS (Fig. 4, а), and in a postoperative period: a follow-up MRI (Fig. 5) and PMRS (Fig. 4, b).

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Fig. 3. Contrast MRI of patient S. dated 01.08.2013. Continuous growth of cystic tumor of the right temporal lobe

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Fig. 4. Patient S., 2D-multi-voxel PMRS. Color parametric maps. Cho/Cr ratio: а — preoperative MRS dated 17.09.2013; b — postoperative MRS dated 29.09.2013

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Fig. 5. Patient S., contrast MRI dated 29.09.2013. The condition after the right temporal lobe tumor resection. The postoperative cyst edema. No contrast accumulation

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In a postoperative period 20 of 21 compared voxels (95.2%) have decreased value of Cho/Cr ratio (p<0.01, Mann–Whitney criterion).

In four cases of subtotal tumor resection, preoperative and postoperative blood serum IR-spectroscopy findings underwent no changes and corresponded to “glioblastoma” diagnosis. The comparison of PMRS findings of identical parts of the peritomoral area in preoperative and postoperative periods revealed that in some parts of the peritumoral area after subtotal tumor resection there was an insignificant decrease of Cho/Cr, Cho/NAA, Glx/Cr ratio values, and insignificant increase of NAA/Cr and Ins/Cr ratio values that indicates the reduced tumor grade, however, to a lesser degree compared to the total tumor resection. In adjacent peritumoral zones there were metabolic changes consistent with Grade IV that were not found in total tumor resection.

All four cases of subtotal tumor resection, morphological and immunohistochemical studies in peritumoral area revealed tumor cells of glioblastoma with high Ki-67 consistent with Grade IV.

In all four cases of subtotal tumor resection there was observed an early (up to a month) continuous tumor growth.

Discussion. The obtained results of postoperative differentiation of glioblastoma (Grade IV) in anaplastic astrocytoma (Grade III) can be explained theoretically from the point of view of tissue oncogenesis theory, according to which a self-regulating system (homeostasis) controls the constancy of qualitative and quantitative composition of cells, which have the potency to independent growth. In accordance with this theory, tissue control abnormality results in uncontrolled division of clonogenic tumor stem cells having activated oncogenes [28]. This theory is sustained by the experiment of human glioblastoma stem cell transplanted in caudal nucleus of genetically modified nude thymic mouse brain. Histological type of tumors detected 10–19 days later in both cerebral hemispheres, and anaplasia degree depended on tumor site (microenvironment). So, in caudal nucleus or in adjacent parenchyma, tumor structure resembled human glioblastoma multiforme. Its subependumal layer looked like ependymoastrocytoma, in choroid plexus the tumor had histological signs of choroid plexus carcinoma [29]. The effect of microenvironment on morphological differentiation of cells was confirmed by other researchers as well [30–32]. In oncodifferentiation processes the critical part is assigned to cell molecular and metabolic microenvironment of tumor cells including the relationship between tumor and non-tumor cells, stromal component factors, neuronal stem cells, tumor stem cells, as well as oxygen tension, acidity, lactate and glucose content, energy deficiency, interstitial fluid flow, interstitial hypertension, different factors produced by microenvironment cells, the factors having an effect on proliferation, cell division and differentiation [28, 33–35].

In total resection of a tumor node, at a single step there were removed the bulk of actively proliferating tumor cells, the majority of chemo- and radioresistent tumor cells, tumor stem cells located in tumor stroma, necrotic foci, most tumor neovasculature, growth factors produced by tumor cells, cytokines, inflammatory mediators, antibodies, proteases and other enzyme types, different metabolites, and other indices. Postoperative blood flow, vascular permeability, acidity, osmotic and oncotic pressure, oxygen partial tension, etc. change significantly in peritumoral area. All the above mentioned significantly changes homeostasis of surrounding tissues. The residual tumor cells in the postoperative peritumoral zone are in the changed microenvironmental conditions. Microenvironmental homeostatic changes after the resection of the main tumor node if there is no mass effect of the tumor theoretically can contribute to the differentiation of glioblastoma (Grade IV) into less malignant brain tumor — anaplastic astrocytoma (Grade III).

The collected empirical and experimental data show genetic abnormalities to not always result in malignant transformation and active tumor growth. The results we obtained demonstrate that in case of total glioblastoma resection the postoperative diagnosis “glioblastoma” is not consistent with reality relying on the changes in the perifocal area that is supported by the findings of three different research techniques: IR-spectroscopy, PMRS, immunohistochemistry. Preoperative spectral ratio studied by IR-spectroscopy of blood serum correspond to Grade IV tumors, postoperative — Grade III tumors. According to PMRS findings compared with the identical peritumoral areas before and after surgery there is a reduced level of tumor metabolites and their ratios that indicates the decrease of anaplasia degree to Grade III in the postoperative period. Immunohistochemical analysis of proliferative activity of tumor cells by Ki-67 expression in a tumor node determines anaplasia degree: Grade IV that is consistent with gliblastoma diagnosis. In peritumoral zone the index decreases up to Grade III–II compared to the main part of the tumor that corresponds to the diagnosis of anaplastic astrocytoma.

Conclusion. A complex of techniques applied (IR-spectroscopy, proton and magnetic resonance spectroscopy and immunohistochemistry) enables to assess how effective and total the surgery was, and if it contributed to postoperative glioblastoma differentiation, and estimate how the tumor will develop in an early postoperative period: as glioblastoma — with early continuous growth or as anaplastic astrocytoma — with the longer recurrence-free period.

Total resection of glial brain tumors is impossible due to their infiltrative growth character. However, surgery is still the principal component of malignant brain tumor treatment. Our studies show that total tumor resection is required to prevent early continuous tumor growth. Differentiation of Grade IV glioblastomas into Grade III anaplastic astrocytomas is accompanied by an increased progression-free period.

The revealed regularity enables to understand the mechanisms of malignant transformation and brain tumor differentiation, and offers the prospects of possible development of a target effect of the process.

Study Finding. The research was supported using extra-budgetary funds of Nizhny Novgorod State Medical Academy and Nizhny Novgorod Research Institute of Traumatology and Orthopedics.

Conflict of Interests. The authors have no conflict of interests to declare.