Những kết luận mới của luận án: - Đây là nghiên cứu đầu tiên tại Việt Nam áp dụng mô bệnh học, hóa mô miễn dịch trong chẩn đoán sarcôm mô mềm (SMM) ở các vị trí ngoại vi và trung tâm theo phân loại của tổ chức y tế thế giới năm 2013. - Nghiên cứu đã phân loại được típ mô bệnh học trên 363 trường hợp SMM, có 11/12 nhóm nguồn gốc, trong đó nhiều nhất là nhóm sarcôm nguyên bào xơ cơ (29,2%), tiếp đến là sarcôm mỡ (26,7%); sarcôm có nguồn gốc không chắc chắn 14,9%;…; không gặp trường hợp nào thuộc nhóm quanh mạch. U mô đệm dạ dày ruột ngoài tiêu hóa chiếm 2,2% (8/363) trong SMM nói chung và chiếm 5,9% (8/136) các SMM ở sau phúc mạc – trong ổ bụng. Hóa mô miễn dịch không những có vài trò quan trọng trong phân loại típ mô bệnh học của SMM mà một số dấu ấn còn có giá trị xác định bản chất phân tử của khối u như MDM2, CDK4, INI1, MUC4, TFE3, WT1, H3k27me3. - Độ mô học theo hệ thống phân độ SMM của Pháp và độ mô học theo Ki-67 càng cao càng làm tăng tỷ lệ diện cắt phẫu thuật dương tính (R1-R2) với p < 0,05. - Phân tầng nguy cơ di căn của u xơ đơn độc ác tính (≥ 4 nhân chia/10 vi trường) trên50 trường hợp: nguy cơ thấp 56%, nguy cơ trung bình 34%, nguy cơ cao 10%.
Trang 1HANOI MEDICAL UNIVERSITY
HO DUC THUONG
INVESTIGATE HISTOPATHOLOGY, IMMUNOHISTOCHEMISTRY AND SOME PROGNOSTIC FACTORS OF COMMON SOFT TISSUE SARCOMAS
Trang 2HANOI MEDICAL UNIVERSITY
The thesis will be defended at Board of Examiners of Hanoi Medical
University At: 14:00 Date: 10/ 09 / 20
The thesis can be found at:
1 National library of Vietnam
2 Library of Hanoi Medical University
Trang 3TO THE CONTENT OF THE THESIS
1 Ho Duc Thuong, Nguyen Thi Khuyen (2018), “Histopathological characteristics and TFE3 expression of niche soft tissue sarcoma: Four case studies at Viet Duc Hospital and reviewing medical literature”,
Vietnam Cancer Journal, Issue No 5 - 2018, pp 256-261
2 Ho Duc Thuong, Le Dinh Roanh (2020), “Histological type classification, expression of MDM2/CDK4 markers and some
prognostic factors of fatty sarcoma at Viet Duc Hospital”, Medical Practice Journal, Issue No.1139, July 2020, pp 74-77
3 Ho Duc Thuong, Nguyen Thi Luan, Nguyen Sy Lanh, Le Dinh Roanh (2020), “Histological characteristics, expression of STAT6 and CD34
markers in 62 isolated fibroid cases at Viet Duc Hospital” , Medical Practice Journal, Issue No 1139, July 2020, pp 112-116
4 Ho Duc Thuong, Nguyen Thi Quynh, Dao Thi Luan, Nguyen Sy Lanh, Nguyen Duc Thang (2021), “Low-grade mucinous fibrosarcoma: A case study at Viet Duc Hospital and a review of the medical literature”,
Vietnam Journal of Oncology, Issue No.1 - 2021, pp 367-372
Trang 4In recent times, with the appearance of many new molecularly significant antibodies, IHC has shown an increasingly important role in diagnosing and determining the histopathological type of STS
So far, there are many classifications of STS, in which the Fourth World Health Organization (WHO) classification of STS in 2013 has highlighted the role of IHC and molecular biology in stool histopathological type of STS In early 2020, the WHO's fifth classification of soft tissue and bone tumors was born, in addition to adding some new histopathological types and molecular biology, the classification also updated a new element has new prognostic significance of some common tumor types, dedifferentiated liposarcomas, solitary fibrous tumors
In terms of treatment, surgery is the first indication for STS, however,
it is difficult to remove the entire tumor, because the tumor often has extensive local invasion, the tumor size is often large and varies with anatomical position This is also one of the important factors in the prognosis of STS For the above reasons, the project titled “ Investigate histopathology, immunohistochemistry and some prognostic factors of common soft tissue sarcomas” has been carried out for the following
purposes:
1 Study on histopathology and immunohistochemistry of soft tissue sarcomas according to the classification of the WHO 2013
2 Analysis of some macroscopic and microscopic factors with
prognostic significance of some common soft tissue sarcomas
NEW CONTRIBUTIONS OF THE THESIS
1 This is the first study in Vietnam to apply histopathology, IHC in the diagnosis of STS in peripheral and central locations according to the classification of the WHO 2013
Trang 52 The results from the study show that:
The study classified the histopathological type on 363 cases of STS with 11/12 groups of origin, of which the most common group was fibroblastic /myofibroblastic sarcomas (29.2%), followed by adipocytic sarcomas (26,20%); sarcomas of uncertain differentiation 14.9%;…; There were no cases of perivascular tumour group Extra-digestive GIST accounts for 2.2% (8/363) of STSs in general and 5.9% (8/136) of retroperitoneal-intra-abdominal STSs IHC not only plays an important role in the histopathological classification of STSs, but some markers are also valuable
in determining the molecular nature of the tumor such as MDM2, CDK4, INI1, MUC4, TFE3 , WT1, H3k27me3
- The higher histological grading of STS according to FNCLCC and Ki-67 grade is, the higher the rate of positive surgical resection (R1-R2) with p < 0.05 is increased
- The stratification of the risk of metastasis of malignant solitary fibrous tumours (≥ 4 mitoses/10 HPF) in more than 50 cases: low risk 56%, medium risk 34%, high risk 10%
THE STRUCTURE OF THE THESIS
The thesis consists of 147 pages, including the following parts: Introduction (2 pages), Chapter 1: Literature Overview (38 pages), Chapter 2: Research Subjects and Methods (21 pages); Chapter 3: Research results (35 pages); Chapter 4: Discussion (48 pages); Conclusion (2 pages); Recommendations (1 page) In the thesis, there are 36 tables, 6 charts and 2 figure References have 222 documents (8 documents in Vietnamese and
214 documents in English) The appendix includes a list of patients, illustrations, and sample research records
CHAPTER 1 LITERATURE OVERVIEW
1.1 Research situation on soft tissue sarcoma in the world and in Vietnam
Recent studies on molecular biology and IHC in the field of STS have helped to diagnose the genetic nature of many histological types, discovering many new histologic types from undifferentiated/unclassified STSs, leading to the introduction of the new classification of STSs of the WHO 2013 and more recently the WHO 2020 Studies on biochemical markers IHC with molecular value is realized and increasingly applied in diagnostic practice
Trang 61.2 Histopathological classification of soft tissue sarcomas
The first globally accepted histopathological classification for STS was the WHO classification (1969) Advances in IHC and molecular biology have led to the creation of new classifications that better serve the diagnosis, treatment, and prognosis Up to now, there have been 05 classifications of STS of the WHO, the latest 02 classifications were born in
Some new points of the 2013 classification compared to the previous ones:
- Adipocytic tumours: The most notable change in this tumor
category is the removal of the term round cell liposarcoma, and the classification of this tumor in the group of high-grade myxoid liposarcomas Mixed type liposarcomas have also been excluded from this classification and are classified as dedifferentiated liposarcomas
- Fibroblastic/myofibroblastic tumours: Dermatofibrosarcoma
Protuberans (DFSP) is closely related to giant cell fibroblastoma and is included in the WHO 2013, both tumors had rearrangements of chromosomes 17 and 22, which resulted in the formation of the mosaic gene PDGFB-COL1A DFSP is classified as a tumor that rarely metastasizes (intermediate), although it has the potential to metastasize in the presence of a fibrosarcomatous component (Dermatofibrosarcoma Protuberans- variant Fibrosarcomatous - DFSP-FS) The subtypes of hemangiopericytomas were eliminated and classified as solitary fibrous tumours due to the discovery of the same NAB2-STAT6 gene The WHO
2013 classification recognizes the close relationship between low-grade fibromyxoid sarcoma and a subtype of sclerosing epithelioid fibrosarcoma
- So-called fibrohistiocytic tumours: The term “malignant fibrous
histiocytomas” was dropped from the 2013 WHO classification The term is
Trang 7obsolete, it includes many types of tumors that now exist can be precisely classified as specific types of sarcomas Unclassified/undifferentiated
sarcomas now have their own classification
- Rhabdomyosarcoma: The classification of “sclerosing/spindle cell
rhabdomyosarcoma” was separated from embryonal rhabdomyosarcoma due to the detection of genetic differences
- Vascular tumours: A newly recognized term "pseudomyogenic
haemangioendothelioma" (and also “epithelioid sarcoma-like haemangioendothelioma”) is included in this category
- Gastrointestinal stromal tumour: For the first time, GIST was
included in the soft tissue tumour classification of the WHO (2013), previously GIST belonged to the classification of digestive system tumours
It is worth noting that in the GIST classification there is recognition of SDH deficiency GIST
- Tumours of uncertainly differentiation: There are 2 new groups
added to the 2013 classification: "Haemosidrotic fibrolipomatous tumour" and “Phosphaturic mesenchymal tumor” The term "primitive neuroectodermal neoplasm" (PNET) (synonymous with Ewing's sarcoma) has been dropped from this classification This is to reduce complex histological and genomic differences, similarity of names, PNETs of the central nervous system, and female genital tract
- Undifferentiated/unclassified sarcomas: This group of tumors is
new to the WHO 2013 classification, and is referred to as a tumor that cannot
be classified into any other class because it cannot be proven otherwise histological, IHC, or molecular biology
1.2.2 Histological classification of soft tissue sarcomas of the 5th World Health Organization (2020)
The fifth edition of the WHO's Classification of Bone and Soft Tissue Tumors was published in early 2020, seven years after the fourth edition New updates on classification are based on new genetic databases, prompting the introduction of several new histopathological subtypes and regrouping of some tumor types The chapter on soft tissue tumors in particular includes the addition of recently described tumor types; In some cases, more and more molecular biology research results are used to inform the reclassification of soft tissue tumors or to change the nomenclature and management of the disease Despite the increasing contribution of molecular biology, new classifications continue to emphasize the central diagnostic role of morphology In addition to introducing “new” soft tissue tumor types, this classification also provides prognostic updates for familiar
Trang 8tumors such as dedifferentiated liposarcomas and solitary fibrous tumours The classification also includes discussions focusing on a range of genetic alterations recently described in soft tissue tumours The past decade has been full of new discoveries of gene fusions in rare soft tissue tumors These include genetic alterations that are relevant to diagnostic practice An example is the chapter on undifferentiated small round cell sarcomas; The discovery of new gene fusions has led to the subtype of formerly "Ewing-like" sarcomas into new types with distinct clinical, morphological and immunohistochemical manifestations
1.3 The role of immunohistochemistry in the diagnosis of soft tissue sarcoma
IHC is a special staining technique that uses specific antibodies to determine the presence of antigens on a slice of tissue or on different types
of cells present in the tissue The basic principle is that when applying a tissue-specific antibody, if the antigen is present in the tissue, an antigen-antibody combination reaction will occur In the diagnosis of STS, IHC not only detects the differentiation of tumor cells, helps to accurately classify each histopathological type and distinguishes it from other cancers, but also detects abnormal proteins generated from combined genes due to tumor-specific translocations or abnormal proteins produced by mutations in some genes involved in tumorigenesis Therefore, although it is an immunological technique, it reflects the lesion characteristics at the molecular level Nowadays, the introduction of automated IHC staining machines has improved the technical quality Many new studies on IHC and many new markers are significant in the diagnosis of STS In STS diagnostic practice, IHC can be used with 3 main roles:
- Distinguish with benign lesions (pseudo-STS)
- Distinguish with non-sarcoma undifferentiated malignancies
- Classification of sarcomas
1.4 Prognosis of soft tissue sarcomas
1.4.1 Significant factors in the prognosis of soft tissue sarcoma
Prognostic factors include 3 main groups: patient-related factors, tumor-related factors, and treatment-related factors Some factors are significant in the prognosis of STS such as: age, tumor location, tumor size, surgical resection margin (R), histopathological type, histological grade, TNM
1.4.2 Ki-67 marker in the prognosis of soft tissue sarcoma
Several histological grading systems based on IHC have been proposed, of which Ki-67 - a protein involved in cell proliferation - is the
Trang 9most widely studied marker Furthermore, this method shows high validity and reproducibility in histological classification of STS by semi-quantitative method In addition, several studies have demonstrated that Ki-
67 expression level is an independent predictor of survival in STS
1.4.3 Update on prognostic factors of some soft tissue sarcomas according to WHO 2020
Table 1.3 WHO 2020 Classification: New concepts in nomenclature,
grade and risk factor stratification
Malignant melanotic
nerve sheath tumour
Change in nomenclature (formerly melanoma Schwann cell tumor) to indicate clinical malignancy
Dedifferentiated
liposarcoma
Adverse effects of high histologic grade according
to FNCLCC, as well as with muscle differentiation (especially rhabdomyolysis) were noted
Solitary fibrous tumor Prognostic model predicts risk of metastasis,
using patient age, mitotic rate, tumor size, and necrosis
CHAPTER 2 RESEARCH SUBJECTS AND METHODS 2.1 Research subjects
2.1.1 Research subjects
The study was carried out on STS cases according to the classification
of the WHO 2013, at the Department of Pathology - Viet Duc Hospital, from January 1, 2016 to December 31, 2020
2.1.2 Selection criteria
The patient must meet all of the following criteria:
- Primary STSs of head and neck, extremities, trunk, retroperitoneum - intra-abdominal, pleura - mediastinum
- Type of specimen: Surgical specimen
- There are templates and paraffin blocks for storage
- Tissue samples are sufficiently exploited, still have antigenicity (based on staining of negative and positive controls), and tumor tissue samples have enough IHC staining
Trang 10- No more paraffin blocks or poor quality paraffin blocks
2.3 Research contents
2.3.1 Research variables and indicators
2.3.1.1 Some clinical features
- Age: Percentage of age groups (<20; 20-39; 40-59; ≥ 60)
- Gender: Percentage of men and women in STS
- Location: Percentage of groups of STS sites
2.3.1.2 Histopathological and immunohistochemical features
- Based on the characteristics of the HE specimen, IHC and some genetic analysis, to determine the histopathological type of STSs according
to the WHO’s Histological Classification of Soft Tissue Tumors in 2013 Updated according to WHO 2020
+ Type Histopathological WHO 2013 classification of STSs
+ Percentage of histopathological types according to the origin of STS + Percentage of histopathological types according to groups of origin + Describe histopathological characteristics of some common and rare types Evaluation of histopathological characteristics on tumor cell morphology, stromal, tumor structure, multiplication rate, activity, and cell differentiation
+ Expression rate of IHC markers by type or group of subtypes
+ Intensity reveals some imprints
+ The relationship of some IHC markers with histopathological type + Identify genetic characteristics of some cases
1.3.1.3 Some macroscopic and microscopic factors have prognostic significance
- Size: Divide tumor size into 04 groups according to AJCC: ≤ 5 cm;
>5 to ≤10 cm; >10 to ≤15 cm; >15 cm Determine the percentage
- Tumor depth: Determine the percentage of tumors in the superficial
or deep (in large muscle mass, intra-abdominal, mediastinum, meninges )
- Surgical resection margin: Determine the percentage of R0, R1, R2 according to AJCC
Trang 11R0: Grossly and microscopically negative (tumor 1 mm from the cutting area)
R1: Microscopically positive (tumor less than 1 mm from the cutting area)
R2: Grossly positive
Negative cross-section is R0, positive cross-section is R1 and R2
- Histological grading: The histological grading of STSs according to FNCLCC is based on necrosis, multiplication index and the grade of differentiation of the tumor
- Assessing Ki67:
+ The Ki-67 marker was determined by the proliferation index of the nucleus, determined based on the percentage of tumor cell nuclei that caught color, divided into 3 levels according to Hasegawa et al.: grade 1: little positive ( <10%), grade 2: moderately positive (10-29%) and grade 3: strongly positive (≥30%) Determine percentages by groups
+ Histological classification based on Ki-67: applied on the basis of the FNCLCC system, replacing the multiplier index with the Ki-67 index
- Prognostic stratification of the risk of metastasis of solitary fibrous tumours based on 4 factors (age, size, mitosis, and necrosis) divided into 3 groups of levels: low risk, medium risk, high risk Calculate the percentage
of groups
- EGIST prognosis: EGIST risk subgroup was applied according to the National Institute of Health (NIH) classification and modified NIH criteria
It changes based on two characteristics: tumor size and mitotic index
2.3.2 Techniques and tools for collecting information
- Collect information about age, gender, tumor location, tumor size, disease spread based on test request form (surgeon's), medical records, diagnostic imaging
- Collect specimens and/or paraffin blocks in the archives
- Handling macroscopic specimens, performing histopathological (HE) and IHC testing techniques
- For difficult and rare cases, send samples for further consultation at K hospital or in the US and do other IHC markers or molecular biology
Table 2.2 Some general information about diagnostic techniques
Number of consultations in the US 126/363 (34.7%) Number of cases of molecular biology (FISH,
done in the US)
4/363 (1.1%) Number of IHC staining cases 292/363 (80.4%)
Trang 12Number of cases without IHC staining (HE
alone)
71/363 (19.6%) Total number of antibodies used 86
Minimum number of antibodies used/a case 1 (2 cases)
Maximum number of antibodies used/a case 19 (1 case)
Average number of antibodies used/case 5.19
Set of most used antibodies/a case 7 (47 cases)
2.4 Data processing
The obtained data and results were processed by computer, using the statistical software SPSS 16.0 Analyze and present data according to the topic's objectives
- Qualitative variables: Calculated as a percentage (%)
- Normally distributed continuous variables: Analyze the relationship between variables using χ2 test (in case the expected sample is less than 5, the exact test method will be used (Exact Probability Test: Fisher and Phi and Cramer's ) The difference is statistically significant when p < 0.05
2.5 Errors and how to fix them
- Sampling error: only select cases that meet the required research criteria to be included in the study
- Error due to data mining: only taking the medical records with enough information in the research form to study
- Test error: Comply with the testing process of the Ministry of Health, Viet Duc Hospital and the manufacturer's recommendations There is always a negative control and a positive control in each test run 2 independent readers (student and instructor) In case of disagreement, the results will be consulted with domestic and foreign doctors (Brigham and Women's Hospital of Harvard University, USA)
Trang 132.6 Ethics in research
The study was conducted on patient tumor specimens, from archival specimens and paraffin blocks, without performing procedures on patients The study was approved by the ethics committee in biomedical research – Hanoi Medical University, certificate No 219/HĐĐĐHYHN, December 30, 2016
CHAPTER 3 RESEARCH RESULTS 3.1 Some general results on age, gender, tumour position
3.2 Histopathological and immunohistochemical features
3.2.1 Some common features
Table 3.3 Subgroups of soft tissue sarcomas by origin
Group of Soft tissue sarcoma origin Quantity (n) Ratio (%)
Comments: Fibroblastic and myofibroblastic sarcomas accounts for the
highest proportion with 29.2%, followed by liposarcoma with 26.7%, sarcoma of uncertain differentiation group with 14.9% The group of so-called fibrohistiocytic tumour and chondro-osseous tumour had 1 case (accounting for 0.3%) There are no cases in the perivascular group
3.2.2 Histopathological characteristics, immunohistochemistry of some histopathological subtypes
● Liposarcoma:
- Histopathological type: Among the total 97 cases of liposarcoma, the
dedifferentiated type had the highest rate (50.5%), followed by the differentiated type (37.1%), the myxoid type (12.4%)