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MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY NGUYEN SY LANH RESEARCH ON THE HISTOPATHOLOGICAL CLASSIFICATION OF DIFFUSE GLIOMAS OF THE BRAIN USING THE CLASSIFICATION[.]
MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY NGUYEN SY LANH RESEARCH ON THE HISTOPATHOLOGICAL CLASSIFICATION OF DIFFUSE GLIOMAS OF THE BRAIN USING THE CLASSIFICATION OF THE WORLD HEALTH ORGANIZATION (WHO) IN 2007 Specialized: Anatomical Pathology and Forensic Medecine Major: Biomedical Science Code: 9720101 SUMMARY OF DOCTORAL THESIS HA NOI - 2022 THE SCIENTIFIC WORK WAS COMPLETED AT HANOI MEDICAL UNIVERSITY Academic supervisors: Assoc Prof Dr Nguyen Phuc Cuong Prof Dr Nguyen Thuy Huong Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended in front of the school-level doctoral thesis examination committee held at Hanoi Medical University Time of organization: ……hours….date……month… 2022 The thesis can be found at: Vietnam National Library Hanoi Medical University Library PUBLISHED RESEARCH WORKS RELATED TO THE THESIS Nguyen Sy Lanh, Nguyen Phuc Cuong, Nguyen Thuy Huong Applying the classification of the World Health Organization (WHO) in 2007 in the diagnosis of diffuse gliomas of the cerebral hemisphere at Viet Duc Hospital from 6/2014 to 10/2017 Journal of Vietnamese Medicine Episode 461 December - special issue - 2017: 335-344 Nguyen Sy Lanh, Nguyen Phuc Cuong, Hoang Xuan Su, Nguyen Thuy Huong Application of immunohistochemistry in evaluating the expression characteristics of IDH1 and IDH2 genes in diffuse gliomas of brain Vietnamese Journal of Internal Medicine Issue 18/2020: 15-23 Nguyen Sy Lanh, Nguyen Phuc Cuong, Nguyen Thuy Huong Study on histopathological classification and some immunohistochemical markers of diffuse gliomas of the brain using the World Health Organization classification in 2007 Vietnam Medical Journal Episode 513 - April - Issue - 2022: 249-253 INTRODUCTION Brain tumor is the conventional term for tumors in the skull, in the central nervous system According to the definition of the International Agency for Recording of Cancer (IARC), the annual incidence of brain tumors ranges from to per 100,000 population and this number is increasing The frequency of the disease is mainly seen in age groups from to 12 and 40 to 70 years old Gliomas occur in any location in the brain such as the frontal lobes, temporal lobes, parietal lobes, occipital lobes, pons, brainstem, and cerebellum Gliomas includes low-grade gliomas and high-grade gliomas Diffuse gliomas or highgrade gliomas that typically grows, diffuse, and increase in malignancy over time or from the time of presentation presents as a highly malignant glioma such as glioblastomas In order to effectively apply the treatment methods, we must make the correct histopathological diagnosis and histopathological types using the new classification of the World Health Organization Then, we have to evaluate each specific case whether there are favorable factors with the treatment methods such as: IDH1 mutant; 1p19q co-deleted; p53 mutant or p53 gene overexpression on immunohistochemical staining From that fact, we conducted a study on the topic " Research on histopathological classification of diffuse gliomas of the brain using the classification of the World Health Organization (WHO) in 2007" with the following purposes: Description of histopathological and immunohistochemical characteristics of diffuse gliomas of the brain using the classification of the World Health Organization in 2007 Analysis of the relationship between the expression of immunohistochemical markers with histopathological types and histopthological grade in the group of patients studied NEW CONTRIBUTIONS OF THE THESIS - The thesis has new contributions that are given the rate of histopathological types and histopathological grade of diffuse gliomas of the brain by using WHO classification in 2007 - The first study in Vietnam using many immunohistochemical markers (7 markers) for diffuse gliomas, in which there are new and highly significant markers for clinical practice such as GFAP, OLIG2, IDH1, INA, P53, Ki67 and ATRX Giving the positive rate of these markers in the research results helps pathologists have a look and apply in diagnostic practice - The study also showed the relationship between pairs of histochemical markers contributing to the grouping of gliomas for treatment and prognosis of patients PRACTICAL VALUE OF THESIS - This study helps to make an accurate diagnosis of histopathological type, grade and grouping prognosis of patients based on histopathological-molecular subtypes of each patient This helps the treatment and prognosis of patients to be reasonable for each specific patient - The study also opens up a new direction for individualized analysis of each patient, helping to apply appropriate, safe and economical treatment methods for patients, avoiding under- or over-treatment STRUCTURE OF THE THESIS The thesis consists of 124 pages: Introduction pages; Overview document 37 pages; Research subjects and methods 15 pages; Research results 32 pages; Discussion 35 pages; Conclusion pages and Recommendations page The thesis has 48 results tables, charts and 42 images, 157 references in Vietnamese, English and French Chapter OVERVIEW DOCUMENT 1.1 Epidemiology With an annual incidence of 6-7 cases per 100,000 population, malignant gliomas are the most common CNS primary tumour, accounting for more than half of adults and the one of the top 10 causes of cancer death Every year in France, 3,000 patients with malignant glioma are seen In the US, the incidence of brain tumors is 4.5/100,000 population, the mortality rate ranks fifth after other liver cancer, lung cancer, stomach cancer and esophageal cancer In Vietnam, according to statistics in 2000, the rate of brain tumors accounted for 1.3/100,000 people and recorded a number of cases Data from Globocan Vietnam in 2020, brain tumor occupies the 15th position 1.2 Histopathological classification 1.2.1 Classification of World Health Organization The WHO classification is based on two theories that define the histopathological types of tumors depending on the predominant tumor cell type and the histopathological grade of the tumor based on the dedifferentiation properties Each tumor has an official WHO name, ICD-O code Table 1.1: World Health Organization Classification 2007 Type Astrocytic tumors Piloytic astrocytoma +Pilomyxoid astrocytoma Subependymal giant cell astrocytoma Pleomorphic xanthoastrocytoma Diffuse astrocytoma + Fibrillary astrocytoma + Protoplasmic astrocytoma + Gemistocytic astrocytoma Anaplastic astrocytoma Glioblastoma + Giant cell glioblastoma + Gliosarcoma Gliomatosis cerebri Oligodendroglial tumors Oliogodendroglioma Anaplastic oligoastrocytoma Oligoastrocytic tumors Oligoastrocytoma Anaplastic oligoastrocytoma ICD code Histological Grade 9421/1 9425/3* 9384/1 9424/3 9400/3 9420/3 9410/3 9411/3 9401/3 9440/3 9441/3 9442/3 9381/3 I II I II II II II II III IV IV IV IV 9450/3 9451/3 II III 9382/3 9382/3 II III 1.2.3 Histopathological classification of gliomas - The first edition of the International Classification of Diseases (ICD) in 1993 We are currently using the ICD-10, which entered use in the United States in 2015, and is revised annually Histopathological grading according to World Health Organization - The WHO histological grading system is also based on the same histopathological criteria as the St Anne-Mayo as follows: nuclear abnormalities (atypial nuclear); Multiplication; Proliferation of endothelial cells, not angiogenesis; Tumor necrosis Staging system for malignancy according to St Anne-Mayo has four grades of tumor: Grade are tumors that not meet any of the criteria; Grade are tumors that have one criterion, usually “Nuclear atypia” criterion; Grade are tumors with two criteria, usually: “Atypical nuclear” and “Mitose”; Grade are tumors with three or four criteria The WHO classification also defines the histopathological types of the tumor according to the predominant tumor cell type and the histopathological grade based on the signs of tumoral cell dedifferentiation This classification was modified in 1993, 2000, 2007 and 2016 to incorporate biological and molecular genetic factors 1.4 Immunohistochemical markers in diffuse gliomas of the brain 1.4.1 Glial Fibrillary Acidic Protein (GFAP) - GFAP is the main glial fiber, specific for astrocyte differentiation, is an immunohistochemical marker that has a useful role in identifying tumor cells of glial origin GFAP is a reliable marker in diagnostic practice for gliomas, which is significant in determining astrocyte differentiation Tumors were defined as astrocytomas if the tumor cells had abnormal nuclear characteristics and the tumor cell cytoplasm reacted to the GFAP marker to varying degrees Reactive astrocytes also express the GFAP marker with multiple cytoplasmic and elongated cytoplasmic branches 1.4.2 Oligodendrocyte transcription factor (OLIG2) - OLIG2 is a recently identified transcription factor that plays a role in the differentiation of oligodendrocytes Studies in human brain tumors indicate significantly higher OLIG2 mRNA expression in oligodendrogliomas compared with other types of gliomas OLIG2 expression on immunohistochemical staining in oligodendrogliomas confirmed this Studies have also shown that using the OLIG2 antibody on immunohistochemical staining can help differentiate the histopathologic types of brain gliomas, although the distinction between oligodendrogliomas and astrocytomas of the brain may not be really obvious 1.4.3 Isocitrate Dehydrogenase (IDH) - The genes for IDH1 and IDH2 are located on chromosomes 2q33.3 and 15q26.1 Determination of IDH mutation status is a valuable factor in the diagnosis as well as in the prognosis for patients with glioma Mutations in the IDH1 gene are much more common than the IDH2 gene in 85% and 3% of grade gliomas, respectively When IDH1 mutation, more than 93% at the R132H position The product of the mutation has been used to produce selective antiR132H antibodies and is used in routine diagnostic practice with formalin-fixed and paraffin-embedded tissue samples IDH2 mutation at codon position 172 (R172K) Other rare IDH mutations can only be identified by gene sequencing 1.4.4 Alpha internexin (INA) - The alpha-internexin (INA) is a type of intermediate fiber present in the cells of the central nervous system whose dominant gene is located on the long arm of chromosome 10 (10q24.33) INAs are expressed to varying degrees in oligodendrogliomas that have 1p19q co-deleted but retain the long arm of chromosome 10 All gliomas with 1p19q co-deleted have mutations in the IDH gene Expression of the INA marker is a good prognostic factor, increasing susceptibility to chemotherapy 1.4.5 P53 - The gene p53, located on the short arm of chromosome 17, encodes for a nuclear protein that is responsible for cell cycle regulation but also for proliferation control Using immunohistochemical staining with monoclonal or polyclonal antibodies on parafin-embedded specimens, not only known mutant Protein53 is detected as well as wild-type Protein53 Protein53 overexpression, including mutant and wild type, plays an important role and was very common in grade and astrocytomas or secondary glioblastomas and unstable to the oligoastrocytomas 1.4.6 Ki67 - Ki67 is a nuclear antigen, appearing between the G1, S, G2 phases and the entire M phase of the cell cycle, which is characteristically detected on immunohistochemical staining The gene encoding the Ki-67 protein was discovered by Scholzen and Gerdes in 2000 The expression of the Ki-67 protein is associated with the proliferative activity of tumor cell populations in malignancies Ki 67 is also a marker for tumor invasion The role of the Ki-67 indexes in determining prognosis has been studied quite a bit before, so it is considered as a potential marker for tumors of the central nervous system 1.4.7 Alpha thalassemia X-linked mental retardation (ATRX) - The ATRX regulatory gene is located on chromosome Xq21.1 and encodes a nuclear protein weighing 280 kDa, which is involved in many cellular functions, including DNA recombination, repair, and transcriptional regulation The human ATRX mutation leads to the development of α-thalassemia, an Xlinked mental retardation syndrome, and other genetic conditions ATRX mutations are present in at least 15 types of human tumors, including neuroblastoma, osteosarcoma, and neuroendocrine tumors of the pancreas The ATRX mutation is strongly associated with the IDH mutation and strongly associated with KD of the CpG island methylation phenotype 1.5 Histopathology-immunohistochemistry subtypes and pathogenesis of diffuse gliomas - In clinical practice, we will encounter combinations of three markers IDH1, INA and P53 according to D Figarella-Branger et al as follows: + Groups of IDH1(-)/INA(-)/P53(+) include mainly: glioblastomas and a few anaplastic astrocytomas + Groups of IDH1(+)/INA(-)/P53(+) include mainly: anaplastic astrocytomas, a number of glioblastomas, and a number of anplastic oligodendrogliomas + Groups of IDH1(+)/INA(+)/P53(+) include: anaplastic oligodendrogliomas, a few cases of glioblastomas and a few cases of astrocytomas + Groups of IDH1(+)/INA(+)/P53(-) include: olgiodendrogliomas and a small number of anaplastic oligodendrogliomas + Groups IDH1(+)/INA(-)/P53(-) include mainly: astrocytomas and a few oligodendrogliomas + Groups IDH1(-)/INA(-)/P53(-) include: a small number of astrocytomas and oligodendrogliomas + Groups of IDH1(-)/INA(+)/P53(+) include: a very small number of anaplastic oligodendrogliomas and anaplastic astrocytomas + Groups of IDH1(-)/INA(+)/P53(-) include a very few cases of olgidendrogliomas Chapter OBJECT AND METHOD OF RESEARCH 2.1 OBJECT - Including 216 patients with diffuse gliomas who underwent surgical resection at Viet Duc Friendship Hospital, with the pathological diagnosis of diffuse gliomas from grade to grade according to WHO classification in 2007, in the period from June 2014 to January 2020 2.1.1 Standard patient The patient must meet all of the following criteria: - Had brain tumor surgery at Viet Duc Friendship Hospital - Histopathological result was diffuse gliomas - The medical record has full information on clinical symptoms, with MRI and/or CT scan - There are full slides and parafin blocks - The tissue sample is still large enough in quantity, ensuring enough for immunohistochemical staining - Tissue samples are still antigenic when immunohistochemical staining is based on positive and negative control staining 2.1.2 Patient elimination standard - The patient underwent surgery to remove the brain tumor but there were not enough specimens for immunohistochemical staining - There is not enough information on medical records, MRI or CT scans, specimens and archival parafin blocks - Tissue samples are not in sufficient quantity for immunohistochemical staining and no longer show antigenicity based on positive and negative control staining 2.1.3 Study size and method of the study - Study size: 216 patients who met the selection criteria for inclusion in the study - Method of the study: non-probability, purposeful, ensuring full selection criteria and not getting caught in exclusion criteria The patients had a complete medical history, underwent brain tumor resection, were confirmed as diffuse glioma of the brain, and had enough samples in terms of quantity and quality to perform immunohistochemistry staining 2.1.4 Time and place of study - Period: From June 2014 to January 2020 - Location: Department of Pathology, Viet Duc Friendship Hospital 2.2 Research Methods 2.2.1 Research design Cross-sectional, retrospective and prospective descriptive studies 2.2.2 Indicators and variables used in the study - Age group: - Gender: number and percentage of men and women - Some clinical symptoms in patients with diffuse glioma of the brain: - Location of diffuse glioma of the brain: - Tumor size: based on the size on preoperative MRI or CT scan, measured in millimeters - Histopathological type and histological grade: determining the histopathological type and histological grade of diffuse gliomas of the brain according to the WHO classification in 2007 - Mitotic ratio: multiplier divided by /10 high power field (HPF), counting at the most active areas of the tumor - Tumor necrosis: to evaluate the presence or not of necrotic tissue on the specimen, including: tumoral necrosis and hypotrophic necrosis - Expression rate of immunohistochemical markers of diffuse gliomas (n=130) - Expression rate of immunohistochemical phenotypes applied to diffuse gliomas (n=130) - The relationship between immunohistochemical markers with histopathological type and histopathological grade - The relationship between immunohistochemical phenotypes of IDH1, INA and P53 markers with histopathological type and histopathological grade - General characteristics of the research subjects: Collect data on age and gender; Some common clinical and paraclinical signs of patients with diffuse gliomas of the brain through treatment records, CT or CT scans and detailed design of the research record - Histopathological study: * Macroscopic research: + Measure the size of the patient in dimensions with the smallest unit being millimeters + Comment on the characteristics: color, hardness, cystization or necrosis and border with the normal area * Histopathological research + The slides were read on an optical microscope with a magnification of 50 - 400 times + Evaluation of mitotic index: Select the area with the most mitotic activity area on each specimen + Formula for calculating the mitotic index of 10 HPF according to WHO: ( ) + Evaluation of tumor necrosis index: Find the presence of tumor necrosis, including types of neoplastic necrosis, map and hypotrophic necrosis + Formula to calculate tumor necrosis index: ( ) ( ) + Determine the histopathological type of diffuse gliomas according to the classification criteria of the World Health Organization classification in 2007 + Determination of histologic grade of diffuse glioma according to the 2007 World Health Organization classification - Immunohistochemical research: All immunohistochemical staining were performed at the Department of Pathology, Viet Duc Friendship Hospital + Antibodies and antibody concentrations: Antibodies used in the study by BioSP, Sigma and Dako Antibody dilutions according to the manufacturer's instructions + Evaluation of results: - Conditions for evaluate results: There are negative control samples, external positive controls and internal positive controls Compare with Hematoxylin eosin stain to know clearly where antigens need to be determined in the nucleus, cytoplasm or cell membrane such as: The markers GFAP, INA and IDH1 were positive stain in the cytoplasm; The markers Oligo2, P53, Ki-67 and ATRX were positive stain at the cell nucleus - Evaluate the result: Negative: only green and Positive: brown yellow + How to evaluate the results of immunohistochemical staining - The markers GFAP, IDH1, ATRX, Oligo2, INA, Ki67, P53 were evaluated for intensity, positive charecteristics and quantified - GFAP and IDH1 markers were evaluated with the following criteria: Coloring in tumor cells cytoplasm Positive expression is when the tumor cell cytoplasm has a yellow-brown color with sufficient intensity to be seen under the optical microscope The result is negative when the cytoplasm of tumor cells is not brown yellow Coloration of less than 1% of tumor cells was considered negative Color staining 1-10% of tumor cells are considered weakly positive (+) The staining of 11 - 50% of tumor cells is considered to be moderately positive (++) Color staining > 51% of tumor cells was considered strongly positive (+++) - Oligo2 and P53 markers were evaluated with the following criteria: Coloring in tumor cell nucleus Expression is positive when the tumor cell nucleus was brown yellow with sufficient intensity to be seen under the optical microscope The result was negative when the tumor cells nucleus was not brown yellow Coloration of less than 1% of tumor cells was considered negative Color staining 1-10% of tumor cells are considered weakly positive (+) The staining of 11 - 50% of tumor cells is considered to be moderately positive (++) Color staining > 51% of tumor cells was considered strongly positive (+++) - INA markes was evaluated with the following criteria: Coloring in tumor cells cytoplasm Positive expression was when the tumor cell cytoplasm has a yellow-brown color with sufficient intensity to be seen under the optical microscope The result was negative when the cytoplasm does not stain brown 10 3.2.3 Characteristics of the mitotic index of histopathological types and histopathological grade of diffuse glioma of the brain Table 3.8: Mitotic index of histopathological types of diffuse gliomas of the brain using the WHO Classification 2007 (n=216) Mitotic ratio /10 Type Number Min Max HPF Oligoastrocytoma 19 1,42±0.61 Oligodendroglioma 20 1,5±0,76 Diffuse astrocytoma 18 1,11±0,32 Anaplastic oligoastrocytoma 17 7,06±3,29 18 Anaplastic oligodendroglioma 29 8,31±3,75 22 Anaplastic astrocytoma 31 8,13±3,93 23 Glioblastoma 82 19,65±14,44 82 Total 216 10,70±11,83 82 Comment: Glioblastomas have the highest mitotic index of 9.65±14,44 Anaplastic gliomas have the second highest mitotic index, including: anaplastic astrocytomas are 8.13±3,93; anaplastic oligodendrogliomas are 8.31±3.75 and anaplastic olioastrocytomas are 7.06±3.29 Groups with lower mitotic index include: oligodendrogliomas are 1.5±0.76; oligoastrocytomas are 1.42±0.61 The lowest mitotic index in the diffuse astrocytomas was 1.11±0.32 Histopathologically mitotic index of diffuse gliomas (n=216) - Grade gliomas have the highest mitotic rate of 19.7±14.44, the rest are grade gliomas of 8.0±3.72 and grade gliomas are 1.4±0.61 Figure 3.1: The ROC curve finds the cut off point of the mitotic index to distinguish between grade and grade diffuse gliomas Comment: Area under the curve is 0.992, with P0.8 is from 2, mitotic/10HPF to 5.5 mitotic/10 HPF) Figure 3.2: The ROC curve finds the cut off point of the mitotic ratio to distinguish grade gliomas and grade gliomas 11 Comment: The area under the curve is 0.895, with P 0.6 is from 7, mitotic/10 HPF to 12.5 mitotic/10 HPF) 3.3 Immunohistochemical characterization of diffuse gliomas of the brain 3.3.1 Characterization of the expression of GFAP marker in diffuse gliomas - The average value of the expression level of the GFAP marker is 77.76±20.11% (5-98) In which, GFAP has a strong expression level, accounting for 87.50% and 0.93% of GFAP cases are positive with mild or negative level Moderate disclosure accounted for 11.57% 3.3.2 Characterization of the expression of OLIG2 marker in diffuse gliomas - The average value of the expression level of the OLIG2 marker is 83.53±16,41% (5-98) The strong positive rate of the OLIG2 marker accounted for 93.06% and the mild and moderate expression level accounted for 0.46% and 6.48% 3.3.3 Characterization of the expression of IDH1 marker in diffuse gliomas - In 216 cases, there were 126 cases with IDH1-marker showing for 58.33% and 90 cases not revealing with IDH1-marking, accounting for 41.67% 3.3.4 Characterization of the expression of INA markers in diffuse gliomas - In total 216 cases of the study, 130 cases were immunohistochemically stained with INA marker, the results showed that 45 cases were positive, accounting for 34.62% and 85 cases were negative, accounting for 65.38% 3.3.5 Characterization of the expression of ATRX markers in diffuse gliomas - In 216 studied patients, 41 cases were stained with ATRX marker, in which the rate of loss of ATRX marker accounted for 36.59% and 63.41% of cases did not lose expression with ATRX 3.3.6 Characterization of the expression of P53 markers in diffuse gliomas - In 216 case study patients, the number of revealed cases with P53 was 156, accounting for 72.22%, 2.6 times higher than the number of negative cases, accounting for 27.78% 3.3.7 Characterization of the expression of Ki67 markers in diffuse gliomas - In 216 studied patients, the number of cases with Ki67 expression was 216, accounting for 100.0% at different levels, proving that the samples for immunohistochemistry all showed guaranteed antigenicity 3.3.8 Characterization of the expression of a combination of immunohistochemical markers IDH1, INA and P53 of diffuse gliomas - In 216 cases, there were 130 cases where the full set of markers IDH1, INA and P53 were completed The group with IDH1 (-), INA (-), p53 (+) accounted for the highest rate of 28.46%; ranked second is the group with IDH1 (+), INA (-), p53(+) accounting for 20.00% The group with IDH1 (-), INA (+), p53 (-) accounted for the lowest rate of 0.77% 12 3.4 The relationship between the expression of immunohistochemical markers of diffuse glioma with histopathological type and histological grade 3.4.1 The relationship between IDH1 marker expression with histopathological type and histopathological grade The relationship between IDH1 marker expression and histopathological type (n=216) - The group of oligoastrocytoma, oliodendroglioma, astrocytoma and anplastic oligodendroglioma have a higher positive rate of IDH1 than other groups with statistical significance with p < 0.05 The IDH1-positive glioblastomas have a lower positive rate for IDH1 than other groups with statistical significance with p0.05 The relationship between IDH1 marker expression and histopathological grade (n=216) - Grade gliomas, the probability of being positive for IDH1 marker was higher than other groups with OR of 9.52 in the confidence interval from 3.87 to 23.45, statistically significant with p