KR101807392B1 - Detection of glioma on the ventricule wall which is not enhanced on magnetic resonance image by photosensitizer induced-fluorescence - Google Patents

Abstract

The present invention relates to the detection of gliomas by photosensitizer-induced fluorescence in a magnetic resonance image. Using the method according to the invention, 5-ALA was used as a photosensitizer in the ventricular wall where no enhancement on the magnetic resonance image was observed and no invasive tumor was observed under the microscope, If it is not observed under blue light, it may be judged that there is no brain tumor and unnecessary resection of brain tissue may not be performed. In addition, if brain wall fluorescence is observed under blue light by 5-ALA, brain tumor may occur. Therefore, it is possible to detect the onset of brain tumor through close examination, thereby providing accurate information on the occurrence of brain tumor during surgery, Abstinence can be avoided.

Description

{Detection of glioma on the ventricule wall by intracavitary photosensitizer induced fluorescence in the ventricular wall without contrast enhancement in magnetic resonance imaging {

The present invention relates to the detection of glioma cells by photosensitizer-induced fluorescence in the ventricular wall during surgery.

Malignant glioma is the most common malignant type of brain tumor in children and adults. The incidence of malignant glioma is very low compared to other tumors, but the mortality rate is remarkably high. In particular, GBM (Glioblastoma Multiforme) is one of the most lethal cancers in humans. It is a malignant brain tumor with local invasion and is notoriously well penetrated into the parenchyma. It causes the worst prognosis after usual treatment including chemotherapy. Despite the development of anticancer drugs, most of the patients with gliomas are dead due to local carcinogenesis. Current best practice for patients is to maintain patient survival for 14 months using surgical resection, chemoradiotherapy with Temozolomide, and external beam radiation therapy.

There are many studies on the surgical resection range as a method to evaluate the prognosis of patients with gliosis. Various techniques including intraoperative neurosurgery, intraoperative magnetic resonance imaging, intraoperative ultrasonography, and electrical stimulation during surgery are helpful in setting optimal resection range in patients with gliomas. Recently, studies on the prognosis of glioma subtypes in brain tissue using 5 - aminolevulinic acid (ALA) - guided fluorescence as a photosensitizer have been actively conducted as a tumor treatment using a photosensitizer. However, there is little research on how to diagnose gliomatosis accurately and easily during surgery.

Therefore, the present inventors have tried to develop a method for accurately diagnosing glioma during surgery. As a result, it has been found that there is no enhancement on magnetic resonance imaging and macroscopic infiltration of tumor cells during gliosis surgery guided by 5-ALA I found fluorescence in the wall of the absent brain. Although 5-ALA is widely used to improve the operation of malignant gliomas, detection of fluorescence in the ventricular wall induced by 5-ALA is not uncommon when the ventricles are open during surgery. The present invention has been accomplished by detecting glioma cells using the relationship between fluorescence and anatomical examination results on the ventricular wall without improvement on magnetic resonance imaging.

Friesen SA et al., 2002, Int J Oncol, 21 (3): 577. Regula J et al., 1995, Gut, 36 (1): 67. Ohgari Y et al., 2005, Biochem Pharmacol, 71 (1-2): 42. Teng L et al., 2011, Br J Cancer, 104 (5): 798. Valdes PA et al., 2012, J Neuropathol Exp Neurol, 71 (9): 806. Ennis SR et al., 2003, Brain Res, 959 (2): 226. Stummer W et al., 1998, Acta Neurochir (Wien), 140 (10): 995. Stummer W et al., 1998, Neurosurgery, 42 (3): 518. Chaichana KL et al., 2014, World Neurosurg, 82 (1-2): e257. Lacroix M et al., 2001, J Neurosurg, 95 (2): 190. McGirt MJ et al., 2009, J Neurosurg, 110 (1): 156. Pichlmeieri et al., 2008, Neuro Oncol, 10 (6): 1025. Sanai N et al., 2011, J Neurosurg, 115 (1): 3. Stummer W et al., 2008, Neurosurgery, 62 (3): 564.

DISCLOSURE OF THE INVENTION The object of the present invention is to provide a 5-ALA guided fluorescence in the wall of the ventricle, which is observed during surgery, to detect a magnetic resonance image or an invasive glioma The method comprising the steps of:

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a method for detecting glioma by measuring fluorescence in a sample of a ventricle wall in which there is no improvement in a magnetic resonance image during 5-ALA treatment or a tumor is not observed on a microscope.

Recently, photodynamic therapy (PDT) has been developed in addition to surgery, chemotherapy, and radiation therapy as a cancer treatment method. Photodynamic therapy is a treatment using a rich oxygen in the body, an externally supplied laser, and a photosensitizer that reacts sensitively to laser light. The principle of photodynamic therapy is that the photosensitizer is selectively administered only to cancer tissues after a certain period of time, and when a laser of a specific wavelength is irradiated, It is a cancer treatment that removes it by enemy. For photodynamic therapy, photosensitizers that are activated at specific wavelengths of light are essential, and currently developed photosensitizers are phorphyrin, cholorin, bacteriochlorin, porphycene, Photogem and aminolevulinic acid compounds have been used.

On the other hand, 5-aminolevulinic acid (5-ALA) is a natural metabolic precursor in the hematin biosynthetic pathway, and recently, as a photosensitizer exhibiting strong fluorescence in cancer tissues than in normal tissues, ≪ / RTI > A large amount of 5-ALA orally administered leads to synthesis and selective accumulation of protoporphyrin IX (Protoporphyrin IX, PpIX), which overloads the hematin pathway and fluoresces in tumor cells and epithelial tissues (Non-Patent Documents 1 and 2). Increased tubular permeability and decreased levels of ferrochelatase activity in tumor cells due to the collapse of tumor BBB (Blood-Brain-Barrier) have been observed in 5-ALA-mediated protoporphyrin Synthesis of IX (protoporphyrin IX) and selective accumulation (Non-Patent Documents 3 to 6). PpIX in glioma tumors, which are present at a higher rate than normal brain tissue, emits red-violet fluorescence under blue light, allowing objective measurement of tumor invasion during surgery, while visually dividing the tumor boundary 7 and 8). Evidence of high sensitivity and specificity for 5-ALA-induced PpIX fluorescence in gliomas confirms the accuracy of 5-ALA in diagnosis. As a result, 5-ALA fluorescence guidance can be widely used to improve the extent of tumor resection.

Fluorescence-guided surgery with 5-ALA has been commonly used to excise malignant gliomas because 5-ALA-induced fluorescence is a predictor of malignant glioma tissue and helps in the detection of invasive tumors. During 5-ALA-guided surgery, we found fluorescence of the ventricular wall that lacked enhancement of magnetic resonance imaging and no tumor on the microscope. Fluorescence in the ventricular wall is a known phenomenon in previous studies, but the study of this was limited by the few cases and the loss of negative control samples. Thus, the present inventors have verified the relationship between the 5-ALA fluorescence during surgery and the histopathological characteristics of the ventricular wall whose enhancement has been deleted on the magnetic resonance image through a number of cases. We used a non-fluorescent specimen of the ventricular wall where improvement was lost on magnetic resonance images as a parietal control. Further, to maintain the uniformity of the patient group in the present invention, only the patients newly diagnosed with glioma subtype (GBM) were included in the patient group. In order to eliminate the possibility of false-positive 5-ALA-induced fluorescence in the ventricular wall samples, patients who were diagnosed with recurrent glial leukemia were excluded.

There are two types of brain tumors: primary brain tumors in the brain and metastatic brain tumors arising from cancer in other organs. There are two types of primary brain tumors, benign and malignant. Even benign tumors often cause difficulties in treatment, such as malignant tumors, because they cause neurological dysfunction by pressing the normal brain. Each year, 190,000 people in the United States and 10,000 in Canada are diagnosed with primary or metastatic brain tumors. Brain tumors have the highest mortality among solid tumors in children under 20 years of age. Brain tumors, even benign, have a lower survival rate than breast cancer patients. The incidence of metastatic brain tumors is 10-15%, but the incidence of metastatic brain tumors is increasing as the survival rate of tumor patients increases. In the United States, there are more than 29.5 cases per 100,000 population, and glioma accounts for 50% of all brain tumors.

Neurons are the cells between neurons and nerve cells, between neurons and blood vessels, and serve to supply nutrition or oxygen to nerve cells. Gliomas are tumors of the glial cells that nourish nerve cells. Gliomas are generally malignant, and are classified by their type or nature. Glioma is the most common type of primary brain tumor and is usually associated with a serious prognosis. High-grade astrocytoma, including glioblastomas (GBM) and degenerated astrocytoma (AA), is the most common endogenous brain tumor in adults. Although the degree of understanding of molecular genetics of high grade astrocytoma has increased, the origin cell type (s) is still unclear, and molecular determinants of disease aggression are not well understood.

These gliomas are histologically classified according to nuclear atypia, cell necrosis, vascular endothelial cell proliferation, mitotic figures, etc., and classified as the most malignant grade 4, which is diagnosed as glioblastoma multiforme (GBM) (Non-Patent Document 5). Glioblastoma multiforme (GM) is the most common and most aggressive primary malignant tumor of human origin. It is the most malignant tumor of various types of brain tumors. It is associated with glial cells and accounts for the largest portion of all intracranial tumors . The annual incidence of GM occurs in 2 to 3 out of 100,000 people in Europe and North America. Polymorphic glioma subtype (GM) is recognized as the most common and fatal form of central nervous system cancer. The prognosis is poor, with an average survival rate of roughly one year, and only about 3% survive for more than three years, so the disease is almost invariably fatal. The median survival after diagnosis is 3 months without treatment, and it is about 1 year as a complete therapy currently in use. The greater the age (> 60 years), the greater the risk of a prognosis. The cause of death is usually an increase in cerebral edema and / or intracranial pressure, and additionally a mass effect that damages blood circulation and causes brain prolapse.

Recently, extensive gene expression studies on glioblastoma have revealed that there are four subtypes of subglottic subtype subtypes. The four types of glioma subtype are proneural subtype, mesenchymal subtype, classical subtype, and neural subtype, respectively, , The mean age of patients, and the response to existing treatments (Non-Patent Document 6). There is a large difference in the overexpression and mutation patterns of important genes in the glial subtype subtype. Epidermal growth factor receptor (EGFR) is abnormally overexpressed in classical subtypes, and unlike the other three subtypes, no mutation occurs in the tumor suppressor gene 'p53'. Patients with classical subtypes are known to have superior outcomes over the other subtypes. In contrast to the classical subtype, the proportion of the p53 gene mutation is high in the anterior nervous subtype and the mutation rate of IDH1 (Isocitrate dehydrogenase) gene and PDGFRA (Platelet-derived growth factor receptor-α) gene is also high. Mutations in these genes are crucial for the proliferation of cancer cells, and mutations in the PDGFRA gene are present only in all nerve subtypes. Patients with anterior nerve subtypes are younger than the other subtypes, but the survival time does not increase with current therapies. Mutations in the p53 gene, as well as mutations in the NF1 and PTEN tumor suppressor genes, are also high in the hepatic subtype. Neurogenic subtypes have mutations in the genes found in other subtypes but do not have characteristic high or low mutation ratios.

Because many studies have demonstrated that Extent of Resection (EOR) is associated with an improved prognosis for patients with gliomas, neurosurgeon exclusions are limited by the difficulty of resection resulting from the invasive nature of glioma subtype (Non-patent documents 9 to 14). A large, randomized, controlled, multi-centered Phase III surgical procedure, guided by 5-ALA fluorescence for malignant glioma, induces a significantly higher ablation rate, compared to conventional microsurgery guided by white light The survival time was longer. In addition, a variety of techniques have been introduced to enable optimal resection, including neuronavigation during surgery, magnetic resonance imaging during surgery, ultrasound during surgery, and electrical stimulation during surgery so that safe resection ranges And improved the survival rate of patients with gliomatosis. The increased range of resection of glioma subspecies to gain the advantage of this survival improves the chances of entering the ventricular system during the course of cell loss. Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

In one embodiment according to the present invention, a. Obtaining a magnetic resonance image of a ventricular wall in a brain tissue of a patient; b. Administering a photosensitizer to the patient; c. Obtaining a magnetic resonance image for the ventricular wall; d. Examining the tumor in the wall of the ventricle under a microscope; e. Detecting fluorescence from the wall of the ventricle and detecting fluorescence from the wall of the brain, thereby providing information for diagnosis of a brain tumor in the patient. In this embodiment, there is provided a method of detecting brain stem wall fluorescence having a probability of not being a brain tumor of less than 60%, preferably less than 55%, even if fluorescence derived from the ventricular wall is detected, thereby providing information for diagnosis of brain tumor in a patient. In this embodiment, the photosensitizer is selected from the group consisting of phorphyrin, cholorin, bacteriochlorin, porphycene, photogem, and aminolevulinic acid. , Wherein the photosensitizer is 5-aminolevulinic acid administered in an amount of 2 to 25 mg per 1 kg body weight of the patient, and the detection is performed under blue light, To provide information about the onset of the disease. In the above embodiment, a method of detecting brain wall-derived fluorescence in which brain tumor is not generated in the brain tissue of the patient when fluorescence is not detected in the step f, and thereby providing information about the onset of brain tumor in the patient Respectively. In the above embodiment, when there is no enhancement of the magnetic resonance image at the c-th stage compared to the magnetic resonance image of the a-th stage, no tumor is observed at the d-th stage, and fluorescence is detected at the e-th stage, , And if the presence of a brain tumor in the patient is confirmed by an additional test, the tumor may be an invasive tumor and may detect a glioma-derived glioma-derived fluorescence from the ventricular wall, which is any of WHO class II, III or IV To provide information on the onset of brain tumor in a patient. In another embodiment according to the present invention, there is provided a method of treating a brain tumor patient comprising administering a photosensitizer to a brain tumor patient; And detecting fluorescence against the wall of the ventricle. The present invention provides a method for providing information for diagnosis of a brain tumor in a patient. In this embodiment, there is provided a method of detecting brain stem wall fluorescence having a probability of not being a brain tumor of less than 60%, preferably less than 55%, even if fluorescence derived from the ventricular wall is detected, thereby providing information for diagnosis of brain tumor in a patient. In this embodiment, the photosensitizer provides 5-aminolevulinic acid as a therapeutic agent for detecting brain stem-derived fluorescence from 2 to 25 mg / kg body weight to provide information for diagnosis of brain tumor in a patient Respectively. In this embodiment, the brain tumor has provided a method of detecting brain stem wall fluorescence, which is any one of the WHO class II, III or IV, to provide information on the onset of brain tumor in a patient.

Unless defined otherwise herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs.

In the present invention, aminolevulinic acid (ALA) is a photosensitizer used in the treatment of photodynamic kinetics and is a precursor of heme, which is biosynthesized by two biosynthetic systems (C4 and C5 pathway) The C4 system found in animals, fungi and aerobic bacteria is produced by the condensation of glycine and succinyl-CoA, which is a pyridoxal phosphate dependent enzyme It is catalyzed by ALA synthase. Another pathway, the C5 system, has been found in plants, birds, and Escherichia coli. This pathway has been linked to glutamate-glutamyl-tRNA synthetase, glutamic-tRNA hydrodeinase dehydrogenase and glutamate-1-semialdehyde (GSA) aminotransferase (transaminase).

In the present invention, " glioma " is the most common type of primary brain tumor and is usually associated with a severe prognosis. Gliomas are generally malignant and are classified by their type or nature.

The term " glioblastoma (GBM) " in the present invention means a tumor originating from glial cells, which are normally abundantly present in brain tissue. Specifically, the glioma subtype is the most common single tumor occurring in the brain, accounting for 12 to 15% of all brain tumors and accounting for 50 to 60% of brain gliomas.

Hereinafter, the present invention will be described in detail.

Using the method according to the present invention, 5-ALA induced 5-ALA-induced ventricular wall fluorescence in the ventricular wall where there was no improvement in magnetic resonance imaging and no microscopic invasive tumor was observed with the 5-ALA as photosensitizer If it is not observed under blue light, it may be judged that there is no brain tumor and unnecessary resection of brain tissue may not be performed. In addition, if brain wall fluorescence is observed under blue light by 5-ALA, brain tumor may occur. Therefore, it is possible to detect the onset of brain tumor through close examination, thereby providing accurate information on the occurrence of brain tumor during surgery, Abstinence can be avoided.

Figure 1 summarizes the histopathological measurement procedure of a ventricular wall sample according to the present invention.
FIG. 2 is a histopathological analysis result of the patient 13 in the present invention. FIG. 2A is a pre-operative magnetic resonance image showing contrast enhanced tumor features of the right temporal lobe extending to the frontal lobe and islands, FIG. 2C relates to an intraoperative microscopic image under blue light representing the wall of the ventricle with varying intensity of fluorescence and fluorescence, FIG. 2D relates to a high-grade nerve The present invention relates to histopathological analysis of fluorescent tissues in which contrast enhancement is lost in a magnetic resonance image as a result of demonstrating the presence of a tumor corresponding to glioma (WHO grade III), and Fig. 2E demonstrates the presence of a tumor corresponding to GBM As a result of histopathologic analysis of the fluorescent tissue showing enhancement in magnetic resonance images 2F is a histopathological analysis of non-fluorescent tissue deficient in contrast enhancement in a magnetic resonance image as a result of demonstrating the absence of tumor and FIG. Lt; RTI ID = 0.0 > T1-weighted < / RTI >
Figure 3 illustrates the histopathological analysis results for patient 15 in the present invention, wherein Figure 3A relates to preoperative, axial, T1-weighted, and post-contrast magnetic resonance images indicating that the tumor in the lateral ventricle is involved, 3B relates to an intraoperative microscopic image under blue light representing the exposed lateral ventricle, FIG. 3C relates to an intraoperative microscopic image under blue light indicative of a ventricular wall with strong fluorescence, and FIG. 3D shows the presence of a tumor corresponding to GBM FIG. 3E relates to axial, T1-weighted, and post-contrast magnetic resonance imaging showing tumor resection, and FIG. 3F shows the histopathological analysis of fluorescence Is a pre-operative axial T1-weighted post-contrast magnetic resonance image showing the horn of the temporal lobe of the lateral ventricle where the tumor is unrelated, Figure 3G relates to an intraoperative microscopic image under white light illumination representing the horn of the exposed temporal lobe of the lateral ventricle, Figure 3H relates to an intraoperative microscopic image under blue light indicative of a fluorescent ventricular wall and an intact ventricular wall, Fig. 3J is a histopathological analysis of fluorescent tissue deficient in contrast enhancement in a magnetic resonance image as a result of demonstrating the absence of a tumor, 3K depicts an axial T1-weighted post-magnetic resonance imaging showing tumor resection involving the ventricular wall as part of the planned boundary for resection.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Patient Information

From December 2012 to May 2015, 19 patients with newly diagnosed GBM and undergoing fluorescein angiography at Shinchon Severance Hospital were included in the study. The approval was obtained from the Institutional Audit Committee of Yonsei University Severance Hospital. Subject consent was provided under the Helsinki Declaration. Of the 19 patients, 12 were male and 7 were female and the ages ranged from 45 to 74 years (mean, 58.5 years). All patients were newly diagnosed with GBM and had no experience of surgery, chemotherapy or radiotherapy. In order to eliminate the possibility of false-positive 5-ALA induced fluorescence in the ventricular wall samples, patients with recurrence of GBM who received prior therapy including surgery and radiation therapy were excluded from the analysis. All tumors showed a typical pattern of enhancement of GBM on magnetic resonance images after contrast agent administration. In all patients, the ventricles were open during tumor resection, and the ablation site was located near the lateral ventricle. The characteristics of 19 patients are summarized in Table 1 below.

Clinical and molecular characteristics of 19 GBM patients patient
No age,
gender tumor
location Ablation range In the lateral ventricle
About
Tumor contact IDH1
transition EGFR P53
transition Ki-67
LI MGMT
Promoter
Methylation 1 pLOH /
19qLOH One 61, M Rt, FT Subtotal Yes No 2+ 40% 60% none No / Yes 2 66, F Rt, TP Total Yes No 3+ 3% 50% none Yes / Yes 3 61, M Rt.
Temporal Total Yes No 3+ 5% 20% none Yes / No 4 70, F Rt,
TPO Subtotal Yes No 3+ none 30% has exist No / No 5 62, F Rt,
Frontal Supratotal No No 3+ none 5% has exist No / No 6 53, M Lt.
Temporal Total Yes No 3+ 2% 60% none No / No 7 45, M Rt.
Frontal Total Yes No 3+ none 50% has exist Yes / No 8 45, M Rt, TPO Subtotal No No 3+ 30% 40% none No / No 9 58, F Lt.TP Total Yes No 2+ 60% 30% has exist Yes / Yes 10 56, M Rt.
Temporal Supratotal Yes No 1+ 2% 5% has exist No / No 11 51, M Lt.FT Subtotal Yes No 2+ 40% 15% none No / No 12 51, M Lt.
Frontal Total Yes No 0 25% 7% has exist Yes / Yes 13 62, F Rt.
Temporal Subtotal Yes No 1+ 5% 20% none No / No 14 58, F Lt.TP Total Yes No 3+ none 3% none No / No 15 67, M Rt.TPO Subtotal Yes No 3+ 90% 40% has exist Yes / Yes 16 74, M Lt.
Temporal Subtotal Yes No 1+ 70% 30% none No / No 17 65, F Rt.
Temporal Total No No 0 One% 3% none No / No 18 46, M Rt.
Temporal Total No No 3+ 5% 20% none No / No 19 60, M Rt.PO Supratotal Yes No 3+ 20% 25% none No / No

Abbreviations given in Table 1 are shown in Table 2 below.

Abbreviation Abbreviation Contents Abbreviation Contents FT (frontotemporal)  Frontal temporal TPO (temporoparietooccipital) Temporal parietal larynx PO (parietooccipital)  Parietal larynx Loss of Heterozygosity (LOH) Loss of heterozygosity L.I. (Labeling Index)  Cover index M (Male) male Subtotal  Bottom part F (Female) female Supratotal  Top

Surgical procedure

5-ALA (Gliolan; Photonamic GmbH & Co. KG, Wedel, Germany) was orally administered at a dose of 20 mg / kg of body weight of the patient 3 hours before the patient's anesthesia. In order to avoid skin phototoxicity, patients were protected from direct exposure to light sources for 20 hours after 5-ALA inhalation. Preoperative, high-resolution, contrast-enhanced, T1-weighted axial magnetic resonance images were obtained from each patient on the day of surgery. All tumor resection was performed under the guidance of a neural navigation system using a magnetic resonance image (Stealth Station Treon [Medtronic, Minneapolis, MN, USA] or Stryker [Stryker Instruments, Kalamazoo, MI, USA]). Additional functional magnetic resonance images and diffusion tensor images were used appropriately according to tumor location. The Zeiss OPMI Pentero microscope (Carl Zeiss Surgical GmbH, Oberkochen, Germany) was used for all patients as a device capable of switching from standard standard xenon light used for fluorescence visualization to Namolla excitation light filtered. In all procedures, the tumor was resected to the extent possible, and all of the above resections were performed on some patients with tumors in nonfunctional areas. After opening the lateral ventricle, the fluorescence of the ventricular wall was measured using a microscope by switching between white light and Namolan excitation light. Neurological surgeons who underwent surgery labeled areas with positive or negative for 5-ALA-induced fluorescence, and samples from these areas were collected and analyzed for anatomy. Before collecting the samples, areas of the ventricular wall were visually checked for tumor relevance and verified with the help of a neurological navigation system. Tumor involvement was determined as enhancement in the presence of macroscopic invasion of the tumor or in a T1-weighted magnetic resonance image used for neurological navigation enhanced by preoperative contrast agents.

Histopathology analysis

Samples derived from GBM patients were immediately received from the operating room. Samples of the ventricular wall for histopathologic analysis were classified according to the presence or absence of 5-ALA-induced fluorescence by the neurosurgeon performing the surgery and delivered to the neuropathology part. Histopathological analysis was performed on tissues derived from hematocylin and eosin (H & E) -staining, formalin-fixed, paraffin-embedded major tumor masses and ventricular wall. Immunohistochemical staining for neurofibrillary acidic protein (GFAP), proliferative marker Ki-67, epithelial growth factor receptor (EGFR), and p53 was performed to establish a complete diagnosis. The methylation position of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter and the isocitrate dehydrogenase 1 mutation were analyzed by PCR and the heterozygosity loss in chromosomal 1p and 19q (LOH, Loss of Heterozygosity) was measured by fluorescence homo-hybridization. Experienced neuropathologists diagnosed the shape and grade of each sample based on the World Health Organization (WHO) 2007 Rating System. To reduce bias, the neuropathologist did not see the 5-ALA fluorescence state. Samples with no identified tumor cells and no detectable increased levels of proliferation were considered tumor free.

Ventricular wall  Fluorescence and sample collection

In each patient, the tumor was clearly visible during surgery and showed 5-ALA-induced fluorescence under blue light. 5-ALA-induced fluorescence in the ventricular wall was confirmed in 11 patients (57.9%) after opening the lateral ventricles. The fluorescence domain of the ventricular wall varied from patient to patient. All 25 samples of the ventricular wall were classified into five areas (front horn, trunk, labial, laryngeal, and temporal horns) along the vertical axis and 19 GBM patients (5 from the front horn, And 12 from temporal horns) were collected during surgery. Of the 25 samples, 11 clearly showed 5-ALA-induced fluorescence during surgery, whereas 14 samples were not shown (Table 3). In all patients, the samples for the ventricular wall area did not show macroscopic evidence of tumor involvement and showed no enhancement on magnetic resonance imaging.

5-ALA Fluorescence Characterization and Pathologic Symptoms in 19 GBM Patients Patient No
Ventricular wall sample
Acquired site In tumor
5-ALA Fluorescence Ventricular wall tissue Tumor cell
(WHO grade) sample water 5-ALA Fluorescence One Horn of temporal horn
(Temporal horn) Yes One Yes Yes (II) 2 Horn of temporal horn Yes One Yes No 3
Horn of temporal horn
Yes
2
Yes Yes (II) No No 4
Horn of temporal horn
Yes
2
Yes Yes (III) No No 5 Frontal horn
(Anterior horn) Yes One No No 6
Horn of temporal horn
Yes
2
Yes No No No 7 Frontal horn Yes One No No 8 Horn of the larynx
(Occipital horn) Yes One Yes No 9 Horn of temporal horn Yes One No No 10
Horn of temporal horn
Yes
2
Yes No No No 11 Frontal horn Yes One No No 12 Frontal horn Yes One No No 13
Horn of temporal horn
Yes
2
Yes Yes (III) No No 14 Atrium Yes One Yes Yes (IV) 15
Horn of temporal horn
Yes
2
Yes No No No 16 Horn of temporal horn Yes One No No 17 Horn of temporal horn Yes One No No 18 Horn of temporal horn Yes One No No 19 Horn of the larynx Yes One Yes No

Histopathological analysis of ventricular wall samples

Histopathologic measurements of major tumor tissues demonstrated WHO grade IV GBM in all patients (Figure 1). Eleven ventricular wall samples with 5-ALA-induced fluorescence during identifiable surgery were analyzed; Five samples (45.5%) were positive for the presence of tumor cells, while the remaining six samples (54.5%) were not tumor cells. Of the five ventricular wall samples representing the presence of tumor cells, two samples (40%) corresponded to high-grade (WHO grade III) gliomas and one sample (20%) corresponded to WHO grade IV GBM . Fourteen ventricle wall samples without 5-ALA-induced fluorescence during identifiable surgery were analyzed; No tumor cells were identified in 14 samples (Table 3). 5-ALA showed 100% sensitivity and 70% specificity (95% confidence interval (CI): 45.72% -88.11%) in tumor detection of the ventricular wall. The predictive value of positive and negative predictive value were 45.5% (95% CI: 16.75% -76.62%) and 100%. The overall accuracy of the method is 76%. Typical results for 5-ALA fluorescence, intraoperative microscopic image, magnetic resonance imaging, and histopathology analysis during surgery are shown in FIG. 2 and FIG.

Concrete example  One

1.1 Patient 1

A 62-year-old woman (patient 13) has a history of headache for 1 month and left paraplegia is underway. The pre-operative magnetic resonance image shows contrast enhancement of the right temporal lobe extending to the frontal lobe and the islands (Fig. 2A). The right frontal temporal lobe was performed using 5-ALA-fluorescence guidance and the tumor was partially removed using temporal lobectomy. Histopathologic analysis of the tumor was diagnosed as glioma subtype. During tumor resection, it entered the temporal horn of the left lateral ventricle (Fig. 2B) and detected distinct fluorescence in a portion of the ventricular wall (Fig. 2C). The ventricular wall samples obtained as part of the planned temporal lobectomy were analyzed histopathologically. The pathological diagnosis of fluorescent tissue with enhancement of contrast on magnetic resonance imaging is gliosis (Figure 2D). The presence of tumors corresponding to high grade glioma (WHO grade III) was demonstrated in samples of fluorescent tissue without contrast enhancement in magnetic resonance images (FIG. 2E). No tumor cells were identified in samples from non-fluorescent tissues deficient in contrast enhancement in magnetic resonance images (FIG. 2F). Postoperative MRI showed that the tumor was resected to the greatest possible extent and additional right temporal lobectomy was performed (FIG. 2G).

1.2 Patient 2

A 67-year-old man (patient 15) presented with a two-week history of disruption, left hemianopsia, and left dyspepsia. The preoperative magnetic resonance image demonstrated a contrast enhanced tumor of the right temple apical portion, including a posterior portion of the temporal horn of the right lateral ventricle (Figs. 3A, 3B). The tumor was excised under 5-ALA fluorescence guidance and showed strong red fluorescence under blue light. Histopathologic diagnosis of major tumor masses is glioma subtype. After opening the ventricle (Fig. 3C, Fig. 3D), fluorescence in the wall of the ventricle without contrast enhancement was confirmed in magnetic resonance images (Fig. 3E, Fig. 3F). The ventricular wall samples obtained from the planned lesion resection and temporal lobectomy were analyzed histopathologically. The pathologic diagnosis of a fluorescent enhancement that is enhanced in a magnetic resonance image is gliosis (Fig. 3G). No tumor cells were identified in samples from fluorescent or non-fluorescent tissue deficient in enhancement in magnetic resonance images (FIG. 3H, FIG. 3I). Postoperative MRI showed that the tumor was resected to the extent possible and additional right temporal lobectomy was performed (Fig. 3J, Fig. 3K).

All references, articles, publications and patents and patent applications cited herein are hereby incorporated by reference in their entirety. Accordingly, the spirit and scope of the following claims should not be limited to the description of the preferred embodiments described above.

Claims (18)

a. Obtaining a magnetic resonance image of a ventricular wall in a brain tissue of a patient other than a human;
b. Administering a photosensitizer to a patient other than a human;
c. Obtaining a magnetic resonance image for the ventricular wall;
d. Examining the tumor in the wall of the ventricle under a microscope;
e. Detecting fluorescence against the ventricular wall; And
f. Detecting fluorescence from the ventricular wall wall including the step of determining that no brain tumor has occurred in the brain tissue of the patient when the fluorescence is not detected in the step e, and providing information for diagnosis of a brain tumor in a patient other than a human Way.
The method according to claim 1,
A method of detecting brain stem wall fluorescence having a probability of not being a brain tumor even if fluorescence derived from the wall of the brain is detected less than 60%, thereby providing information for diagnosis of a brain tumor in a patient other than a human.
3. The method of claim 2,
A method for detecting brain stem-derived fluorescence having a probability of less than 55% and providing information for diagnosis of a brain tumor in a patient other than a human.
The method according to claim 1,
The photosensitizer is any one selected from the group consisting of phorphyrin, cholorin, bacteriochlorin, porphycene, photogem, and aminolevulinic acid. A method for detecting brain stem wall fluorescence and providing information for diagnosis of a brain tumor in a patient other than a human.
The method according to claim 1,
Wherein the photosensitizer detects 5-aminolevulinic acid-derived fluorescence from the wall of the brain to provide information for diagnosis of a brain tumor in a patient other than a human.
6. The method of claim 5,
Wherein the 5-aminolevulinic acid is administered in an amount of 2 to 25 mg per 1 kg of body weight of a patient except for a human, thereby detecting information on the brain tumor in a patient other than a human.
The method according to claim 1,
Wherein said detection is performed under blue light to detect fluorescence originating from the ventricular wall to provide information for diagnosis of a brain tumor in a patient other than a human.
delete The method according to claim 1,
When there is no enhancement of the magnetic resonance image at the c-th stage compared to the magnetic resonance image at the a-th stage, no tumor is observed at the d-th stage, and fluorescence is detected at the e-th stage, A method for detecting brain stem-derived fluorescence, which is an additional test, to provide information for diagnosis of a brain tumor in a patient other than a human.
10. The method of claim 9,
A method for diagnosing brain tumors in a patient other than a human by detecting fluorescence from the ventricular wall, wherein the tumor is an invasive tumor when the presence of a brain tumor in the patient is confirmed by the additional test. 11. The method of claim 10,
Wherein the invasive tumor detects fluorescence from the ventricular wall, which is glioma, to provide information for diagnosis of a brain tumor in a patient other than a human.
12. The method of claim 11,
Wherein said glioma detects fluorescence derived from the ventricular wall, which is any of WHO class II, III or IV, and provides information for diagnosis of a brain tumor in a patient other than a human.








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