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[.]
Trang 1MINISTRY 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
Trang 2THE SCIENTIFIC WORK WAS COMPLETED
AT HANOI MEDICAL UNIVERSITY
The thesis can be found at:
Vietnam National Library
Hanoi Medical University Library
Trang 3PUBLISHED RESEARCH WORKS RELATED
TO THE THESIS
1 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
2 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
3 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 1 - 2022: 249-253
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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 3
to 5 per 100,000 population and this number is increasing The frequency of the disease is mainly seen in 2 age groups from 3 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 high-grade 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:
1 Description of histopathological and immunohistochemical characteristics of diffuse gliomas of the brain using the classification of the World Health Organization in 2007
2 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
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STRUCTURE OF THE THESIS
The thesis consists of 124 pages: Introduction 2 pages; Overview document 37 pages; Research subjects and methods 15 pages; Research results 32 pages; Discussion 35 pages; Conclusion 2 pages and Recommendations 1 page The thesis has 48 results tables, 4 charts and 42 images, 157 references in Vietnamese, English and French
Chapter 1 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
Grade Astrocytic tumors
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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 1 are tumors that do not meet any of the criteria; Grade 2 are tumors that have one criterion, usually “Nuclear atypia” criterion; Grade 3 are tumors with two criteria, usually: “Atypical nuclear” and
“Mitose”; Grade 4 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
2 gliomas, respectively When IDH1 mutation, more than 93% at the R132H position The product of the mutation has been used to produce selective anti-R132H 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
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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 2 and 3 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 X-linked 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
Trang 85 + 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 2 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 2 to grade 4 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
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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
Trang 107 + Evaluation of tumor necrosis index: Find the presence of tumor necrosis, including 2 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
Trang 118 yellow Coloration of less than 1% of tumor cells was considered negative Color staining 1-10% of tumor cells were considered weakly positive (+) Color staining of > 11% of tumor cells was considered strongly positive (+++)
- The ATRX marker is evaluated with the following criteria: Coloring in
the nucleus of tumor cells Expression is positive when the tumor cell nucleus is not brown yellow with sufficient intensity to be discerned under the optical microscope The result is negative when the tumor cell nucleus becomes brown yellow Loss of staining in less than 10% of tumor cells was considered negative Loss of staining expression 11-90% of tumor cells were considered weakly positive (+) Loss of staining expression > 90% of tumor cells was considered strongly positive (+++)
- For Ki-67: Ki67 was positive when any nuclei of tumor cells are brown
yellow with sufficient intensity to be seen under the optical microscope The Ki67 proliferation index (Ki67-LI) was recorded as the percentage of tumor-positive cells per tumor cell count after counting at least 1000 cells in a high power- field (x 400) In the region of increased mitotic activity, positive standard color of the staining method, have internal control, then calculate the percentage (%)
+ The formula to calculate the proliferation index Ki67:
( )
( )
- Take microscopic photos of typical illustrative specimens
Positive test and negative test
Positive control: GFAP: Reactive astrocytes in the normal brain parenchyma around the tumor; OLIG2: oligodendrocytes; IDH1: Macrophages, compared with PCR results and sent to a control test in the French Republic; Ki67: The epithelium of the tonsils is benign; INA: Neuron; P53: Serous papillary carcinoma of the ovary; ATRX: astrocytoma
Negative control: Do not coat the first antibody on the slide for all cases of negative control slide staining
2.4 Data processing using the statistical software SPSS 16.0
Data were collected and processed using the statistical software SPSS 16.0
in difficult cases Histochemical staining results are positive and negative Data
is carefully checked before entering and processing
2.6 Ethics in research
- Research is carried out on tissue samples or parafin blocks of research patients, only for the purpose of improving the quality of diagnosis, treatment, assessment of disease prognosis and quality of life for patients The research objective was approved by the Proposal Evaluation Committee and approved by the Ethics Committee in Biomedical Research, Hanoi Medical University by
Trang 129 'Acceptance from the Ethics Council in Biomedical Research' NUMBER: 187/HDĐHYHN and consensus of Viet Duc Friendship Hospital All information extracted from patients and medical records are kept confidential, only for research purposes Research results are not used for commercial purposes, absolutely do not disclose information about patients in the study There was no distinction between study patients and non-study patients
Chapter 3 STUDY RESULTS 3.1 Characteristics of age, gender and some common clinical and laboratory signs of patients with diffuse gliomas of the brain
3.1.1 Characteristics of distribution according to age group
- The mean age in the study was 47.42±13.53 years old In which, the most common age group is from 31 to 40 years old, accounting for 25.46% Second is the age group from 41 to 50 years old (23.15%) The age group ≤ 20 accounts for the lowest percentage, accounting for 0.5%, the rest is 21.30% in the age group > 60, 18,98% in the 51 - 60 group and 10.65% in the 21 - 30 age group
3.1.2 Characteristics of distribution according to sex
- In total 216 patients Men accounted for a higher proportion of 53.70% and women accounted for 46.30% Male/Female ratio = 1.16/1
3.2 Histopathological images of diffuse glioma of the brain
3.2.1 Distribution of histopathological types using the World Health Organization Classification 2007
Table 3.6: Distribution of histopathological types using the WHO
Comment: Glioblastoma was the most common, accounting for 37.96%
Anaplastic astrocytoma accounted for the second with 14.35% In addition, some other common types such as: anaplastic oligodendroglioma accounted for 13.43%, oligodendroglioma accounted for 9.26%, oligoastrocytoma accounted for 8.80%, diffuse astrocytoma is 8.33% The least common histopathological type is anaplastic oligoastrocytoma accounting for 7.87%
3.2.2 Histological distribution characteristics of diffuse gliomas
- The most common grade 4 glioma was 37,96%, the second most common grade 3 accounted for 35.65% and grade 2 accounted for at least 26.39%
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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)
Oligoastrocytoma 19 1,42±0.61 1 3 Oligodendroglioma 20 1,5±0,76 1 3 Diffuse astrocytoma 18 1,11±0,32 1 2 Anaplastic oligoastrocytoma 17 7,06±3,29 1 18 Anaplastic oligodendroglioma 29 8,31±3,75 5 22 Anaplastic astrocytoma 31 8,13±3,93 4 23
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 4 gliomas have the highest mitotic rate of 19.7±14.44, the rest are grade
3 gliomas of 8.0±3.72 and grade 2 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 2 and grade 3 diffuse gliomas
Comment: Area under the curve is 0.992, with P<0.001 The cut off value for
the mitotic ratio between the grade 2 gliomas and grade 3 gliomas is 3.5 mitotic/10 HPF with J=0.987 (the range of the mitotic ratio with sensitivity and specificity >0.8 is from 2, 5 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 3 gliomas and grade 4 gliomas