1B_General pathology_oncology and molecular pathology.docx

**THEME 1B: GENERAL PATHOLOGY: ONCOLOGY AND MOLECULAR PATHOLOGY...................................................................** **INTRODUCTION** Determining the correct diagnosis of a disease is dependent on clear classification which is the core of pathology. A correct classification is cruc...

**THEME 1B: GENERAL PATHOLOGY: ONCOLOGY AND MOLECULAR PATHOLOGY...................................................................** **INTRODUCTION** Determining the correct diagnosis of a disease is dependent on clear classification which is the core of pathology. A correct classification is crucial to determine therapy and prognosis. Classical pathology deals with morphological changes of histology, specific stainings and expression of proteins as detected by immunohistochemistry. But since the progress in human genomics much information has become available on the role of genetic alterations in pathological processes and DNA/ RNA testing is becoming a crucial tool to identify both somatic and germline aberrations in patient material. Nowadays the analysis of nucleic acids is integrated in regular pathology diagnostics and is used to confirm diagnosis but also to determine treatment options. Also prognostic genetic alterations are described, although these are less commonly applied in patient care. Molecular analysis is of particular importance in the diagnosis and treatment of cancer. Classification of tumors is performed using histological examination and location in the body, but in the last two decades the identifications of specific DNA mutations have become more important for tumor pathology. In this theme 'Oncology and Molecular Pathology' you will be introduced to tumor classification and to the methods that are used to identify genetic alterations in patient material. There are special issues with this type of analyses, comparable with those in forensic investigations. Especially the quality of the tissue is not always optimal, because it may be formalin fixed, partly necrotic and contaminated with non-diseased tissue. You will be acquainted with the concept of personalized medicine, which is based on specific genetic aberrations in the diseased tissue, especially in tumors. Much of the information presented in this theme will be handled in more depth in the year 3 course: 'Molecular Biology and Oncology'. The technologies that will be presented have mostly been dealt with in year 1 in the courses: 'Biomoleculen' and 'Molecular Biology'. Molecular biology and cancer will come back in theme 4 of this course "Hereditary tumors'. There is a lot of development in Molecular Pathology and it is not yet routine technology in all pathology departments. The analyses require specially trained molecular biologists. Since a few years a new function has been formulated, the clinical molecular scientist in pathology (KMBP) who is in charge of translating the results of nucleic based assays into a report that can help in defining the right diagnosis and/or therapy. Results of molecular analyses are discussed in multidisciplinary consultations, more specifically for cancer: Molecular Tumor Boards. In this theme emphasis will be on histological tumor classification and mutation analysis. In the theme mesenchymal differentiation (theme 3) there will be a presentation on molecular alterations that are specific for mesenchymal tumors. Theme 4 will elaborate on hereditary genetic alterations and cancer. **LEARNING OBJECTIVES** - Know the basics of tumor classification (benign vs. malignant, carcinoma vs sarcoma vs hematological tumors) (1) - Explain the technologies used in molecular pathology (repetition of the course 'Molecular Biology') (5) - Know which issues can hamper molecular pathology analyses (5) - Recognize how molecular aberrations can contribute to disease (2) - Understand how molecular pathology can contribute to diagnosis (3) - Understand how molecular pathology can contribute to selection of the best therapy (4) **LECTURES** In this theme there will be a lecture on general nomenclature in oncology and the classification of different tumor types. In a second lecture the application of molecular analyses in pathology is presented and what is needed to obtain a robust and reliable conclusion despite the often suboptimal tissue from which DNA or RNA must be examined. Examples of particular analyses and how this is incorporated in the final diagnosis and treatment of the patients will be shown. **ACTIVITIES** - Study the pages indicated in the reading list in advance. - Perform the SSA-test (Brightpace) before attending the work group - Keep your course book (Robbins and Cotran Pathologic Basis of Disease) available the work group - The workgroups is in on campus with a tutor **READING LIST AND STUDY GUIDELINES** Read the pages from Robbins and Cotran Pathologic Basis of Disease, Kumar, Abbas & Fausto, Elsevier Saunders, 10th edition, 2020, that are indicated. - Chapter 5, page 180-187, molecular genetic diagnosis... end - Chapter 7, page 267-338, chapter 7: neoplasia **MOLECULAR PATHOLOGY: R-ASSIGNMENT** SH: 2 hrs You will get an assignment to develop an application in R for the analysis of next generation sequencing data **GENERAL PATHOLOGY: INTRODUCING PATHOLOGY (25-03-2024 10:15-11:00).....................................................................** **Pathology** = study of disease (study of functional and structural changes in cells, tissues and organs that cause disease) **Histology** = tissue **Cytology** = cells **Etiology**: cause **Pathogenesis**: mechanism of its development **Morphologic and molecular changes**: structural alterations induced in cells and organs of the body **Clinical significance**: relation to the clinical picture **Detection methods:** - Standard histology (such as HE staining - Special stains (such as histochemical stains and immunohistochemical stains - Molecular assays - Electron microscopy **GENERAL PATHOLOGY: ONCOLOGY (25-03-2024 11:15-12:00)................................................................................................** ***Unifying nomenclature of neoplasia*** **LEARNING OBJECTIVES:** - Being able to use the oncologic terminology regarding benign and malignant tumors and their pre-malignant stages. - Can distinguish (pathologically; clinically) between benign and malignant tumours, and name the differences. - Can specify the properties of tumours that can serve as guidance for therapeutic purposes. **SOME DEFINITIONS** - Neoplasm literally means "new growth" - Neoplasm = Tumour - Tumour = swelling so 'strictly' - Neoplasm or inflammation! - So: Tumour is not per se Neoplasm - Tumour in case of neoplasm: benign or malignant - Cancer: malignant tumour **PREFIXES IN TERMINOLOGY** - Ana-, meaning absence - Dys-, meaning disordered - Hyper-, meaning an excess over normal (e.g. Hyperthyroidism) - Hypo-, meaning a deficiency below normal - Meta-, meaning a change from one state to another (e.g. Metaplasia) - Neo-, meaning new **SUFFIXES IN TERMINOLOGY** -itis, meaning an inflammatory process -oid, meaning resembling (e.g. Epithelioid) -penia, meaning lack of -ectasis, meaning dilatation (e.g. Bronchiectasis) -opathy, meaning an abnormal state lacking specific characteristics (e.g. Lymphadenopathy) **EPONYMOUS DISEASES** Named after a person or a place associated with it: - Hodgkin disease: a neoplasm of lymph nodes characterized by the presence of Reed-Sternberg cells - Paget disease: (of bone (hyperostosis) or nipple (breast cancer) extending from the ducts into the nipple) **Definition of neoplasia:** "A neoplasm is an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues and persists in the same excessive manner after cessation of the stimuli which evoked the change" **CLASSIFICATIONS OF TUMOURS** \> Behavioral classification: benign or malignant \> Histogenetic classification: cell of origin (recapitulates the morphology of a certain differentiated cell) \> Precise classification of individual tumours is important for planning treatment **BENIGN VERSUS MALIGNANT** [Benign]: small (usually); slowly growing; non-invasive (expansive); well-differentiated; localized [Malignant]: large/small; fast growing; invasive growth; poorly differentiated; metastasize **BENIGN (EPITHELIAL) NEOPLASMS** *In general --oma means benign, but there are exceptions:* - -oma = benign neoplasm (NOT carcin-, sarc-, lymph-, or melan- !!!) - Epithelial neoplasms (ecto- or endo- derived) - Aden-oma: tumour forming glands - Papill-oma: tumour with finger-like projections - Papillary cystaden-oma: papillary and cystic tumour forming glands - Polyp: a "tumour", NOT a true neoplasm, that projects above a mucosal surface **MALIGNANT (EPITHELIAL) NEOPLASMS** - Carcinoma: malignant epithelial neoplasm - Adenocarcinoma - Squamous cell carcinoma keratin producing tumor Intercellular bridges with desmosomes p40/p63 protein expression - Neuroendocrine carcinoma - Transitional type carcinoma - Undifferentiated carcinoma **BENIGN (MESENCHYMAL) NEOPLASMS** - -oma = benign neoplasm (NOT carcin-, sarc-, lymph-, or melan-) - Mesenchymal neoplasms (mesodermal derived) lip-oma: fat neoplasm Chondr-oma: cartilaginous neoplasm Leiomy-oma : smooth muscle neoplasm Rhabdomy-oma: striated muscle neoplasm **MALIGNANT (MESENCHYMAL) NEOPLASMS** - -sarcoma = malignant mesenchymal neoplasm (NOT , lymph-, or melan-) lipo-sarcoma: fat neoplasm chondro-sarcoma: cartilaginous neoplasm Leiomyo-sarcoma : smooth muscle neoplasm Rhabdomyo-sarcoma: striated muscle neoplasm Osteo-sarcoma: osteoid producing/bone tumour **NOMENCLATURE: THINGS THAT DON'T FIT** - Tumours comprised of cells from more than one germ layer Arise from pluripotent cells (usually gonads) Can be benign (ovary) or malignant (testis) - - Melanoma: malignant skin neoplasm of melanocytic origin Seminoma: malignant testicular neoplasm Lymphoma: malignant neoplasm of lymphocytes **EXAMPLES** Afbeelding met tekst, schermopname, Lettertype Automatisch gegenereerde beschrijving **MICROSCOPIC APPEARANCE OF CANCER CELLS**  **MORPHOLOGICAL/CYTOLOGICAL FEATURES** **Benign** Look like normal cells Low proliferation rate \- low mitotic index \- Low Ki-67-fraction (marker for cells being in the cell cycle) No necrosis **Malignant** Cells look "very" abnormal Polymorphic cells High N/C ratio (nucleotide to cytoplasmic ratio) High proliferation rate \- High mitotic index \- abnormal mitotic figures \- High Ki-67 (marker for cells being in the cell cycle) Often necrosis **SUMMARY OF TYPING OF TUMOURS** Typing tumours: **Morphology (microscope):** Epithelial? Mesenchymal? melanocytic? Other? **Immunohistochemistry:** keratin (epithelial), vimentin (mesenchymal), desmin (muscle), MelanA (melanocytic) **Molecular analysis**: Chromosomal translocations, gene-alterations **WHY GRADING TUMOURS?** *Grading means how malignant a tumor is* - For treatment and prognosis (risk-assessment) - Low grade → better prognosis (outcome of disease) - High grade → worse prognosis - Important in treatment of → Breast cancer Prostate cancer Lymphomas Astrocytomas (brain tumors) Ovarian and endometrial cancer etc. **HOW TO GRADE TUMOURS ?** - - - growth pattern (glandular/solid) - extent of differentiation - nuclear aspects - cell cycle information If you do a IHC for Ki-67, you'll stain for proliferating cells. Note that when grading tumors, that there is a 30% interobserver variation **GENERAL PRINCIPLES OF GRADING** ✓ Microscopic appearance of cancer cells ✓ 2-4 degrees of severity (tumor dependent!!) ✓ the more degrees the more interobserver variability **Grade:** GX Grade cannot be assessed undetermined grade G1 Well-differentiated (Low grade) G2 Moderately differentiated (Intermediate grade) (G4 Undifferentiated (High grade)) METHOD OF GRADING OF BREAST CANCER Example: Afbeelding met tekst, schermopname Automatisch gegenereerde beschrijving **OTHER GRADING SYSTEMS** Interobserver variability, better more objective systems? - - - **TP53-MUTATIONS IN CANCER** *DNA damage, cell cycle abnormalities and hypoxia induce p53. P53 either induces apoptosis or it induces cell cycle arrest and DNA repair.* - Mutated in \>50% of all human tumors ('guardian of the genome') - p53 protective pathways affected in \>90% of all tumors - Loss of p53 =\> loss of G1-S checkpoint/reduced apoptosis =\> proliferation of cells with DNA damage =\> mutations, chromosomal aberrations =\> genomic instability - p53 mutations lead to loss of protein function and accumulation of dysfunctional protein in tumor cells - p53 mutation often related to worse prognosis **STAGING OF CANCER** - **Stage** refers to the extent of cancer, such as how large the tumor is and if it has spread - Understand how serious cancer is and chances of survival - Plan the best treatment - Identify clinical trials that may be treatment options for you - There are many staging systems. Some, such as the **TNM staging system**: - The T refers to the size and extent of the main tumor. The main tumor is usually called the primary tumor. - The N refers to the number of nearby lymph nodes that have cancer - The M refers to whether the cancer has metastasized. This means that the cancer has spread from the primary tumor to other parts of the body **TNM SYSTEM OF STAGING** - **Primary tumor (T)** - TX: main tumor cannot be measured - T0L main tumor cannot be found - T1, T2, T3, T4: Refers to the size and/or extent of the main tumor. The higher the number after the T, the larger the tumor of the more it has grown into nearby tissues. T's may be further divided to provide more detail, such as T3a and T3b. - **Regional lymph nodes (N)** - NX: Cancer in nearby lymph nodes cannot be measured. - N0: There is no cancer in nearby lymph nodes. - N1, N2, N3: Refers to the number and location of lymph nodes that contain cancer. The higher the number after the N, the more lymph nodes that contain cancer. - **Distant metastasis (M)** - MX: Metastasis cannot be measured. - M0: Cancer has not spread to other parts of the body. - M1: Cancer has spread to other parts of the body.  **GENERAL PATHOLOGY: MOLECULAR PATHOLOGY 26-03-2024 (09:15-10:00)......................................................................** **MOLECULAR PROFILE TUMOR DNA** **Copy number variation \> genomic instability** \> Gain or loss of chromosomes \- Aneuploidy: Presence of an abnormal number of chromosomes in a cell \- Nuclear hyperchromasia: increased DNA content \> Gain or loss of parts of chromosomes/focal **Nucleotide variation** 1\) Non (unlikely) pathogenic variants: Single Nucleotide Polymorphism (SNP), silent variation 2\) Variants with unknown significance (VUS) 3\) Pathogenic variants: \- Driver mutations in oncogene \- Frameshift/ non-sense mutations in tumor-suppressor genes \- Passenger mutations (caused by loss of function caretakers) **GENOMIC ALTERATIONS** **Nucleotide variations** Single nucleotide polymorphism, frameshift variants, or in-frame deletions of insertions - Pathogenic variants: activating or inactivating - Variant with unknown significance (VUS) **Copy number variations** - Amplification: activating - Deletion: inactivating **Gene rearrangements** - Fusion genes: activating - Loss of inhibitory domain: activating **ONCOGENES: THERAPEUTIC TARGETS** **Activation of proto-oncogene -\> abnormal activation -\> cancer** Proto-oncogenes are dominant on cellular level, abnormal activation is achieved with activation of 1 allele. **Examples of proto-oncogenes**: - - - - - **Types of mutations \> gain-of-function** - point-mutations (regulatory elements) - missense mutations \> change amino acid - in-frame deletions/insertions - splice site \> exon skipping **TARGET RNA-SEQ DETECTION OF FUSION GENES** *This is an example of a fusion gene. Two genes are being merged together. In both genes a break occurs. When the break is faultily repaired, it results in a chimeric gene. If gene B is a oncogene, it will be under de control of gene A, this can result in cancer.* **TUMOR SUPPRESSOR GENES** *Tumor suppressor genes are often not direct targets for therapies, but they cause processes which can be targeted.* **Tumor supressor genes:** - - - - - - - **Types of mutations \> loss-of-function** - - - - - **Loss-of-function \> bi-allelic** Mutations in allele 1 and second hit: 1. Mutation in allele 2, or 2. Loss of wild-type allele (LOH) **LOSS-OF-FUNCTION, TUMOR-SUPPRESSOR GENES**  - - - - **CDKN2A AND RB1 REGULATE CELL CYCLE CONTROL** \> Loss of CDKN2A function and RB1-wt can be treated with CDK4/6 inhibitor. If p16 is lost, then the cell cycle progression is not inhibited, this is often seen in cancer. **FROM BIOPSY TO DNA ISOLATION** Biopsy -\> formaline -\> IHC to selects cells of interest -\> then you annotate the cells of interest and you do a microdissection (MD). This is a way for enrichment of neoplastic cells. **DNA EXTRACTED FROM FORMALING-FIXED TISSUES CONTAINS VARIOUS TYPES OF DAMAGE** *Formalin fixation has a result on the DNA.*  1. 2. 3. 4. 5. 6. 7. 8. This are similar changes that occur in cells. Afbeelding met tekst, diagram, Perceel, Lettertype Automatisch gegenereerde beschrijving **INFORMATION FROM R ASSIGNMENT LECTURE:** FFPE DNA often contain artifacts from formalin fixation that result in enhanced cytosine deamination resulting in a high percentage of C\>T or G\>A substitution. You need to compensate for these paraffin artifacts, these are not true mutations that happened in the cell.  **MOLECULAR BIOLOGY TECHNIQUES** **Next generation sequencing** - DNA: mutations, copy-number variations - RNA: mutations, translocations, expression **Methylation-specific PCR** - Epigenetic changes (MLH1 promoter methylation) **Fragment length analysis** - B-/T cell clonality, STR genotyping, Microsatellite instability **Multiplex ligation-dependent probe amplification (MLPA)** - Large chromosomal deletions **Fluorescent in-situ hybridization (FISH)** - Translocations, amplification **Silver-stained ISH (SISH)** - Detection Epstein-Barr virus encoded RNA, amplification (HER2/NEU) **Reverse hybridization assay** - Detection human papilloma virus **NGS WORKFLOW (ION TORRENT PLATFORM)** Afbeelding met tekst, schermopname, scherm, ontwerp Automatisch gegenereerde beschrijving **ACTIONABLE TARGETS IN LUNG CANCER** **EGFR mutations in lung cancer** Luncg cancers often have mutations in the EGFR gene in the tyrosine kinase domain - Without growth factors, the EGFR is inactive - With growth factors, the EGFR is active and cell growth and survival - EGFR mutations, EGFR is active without growth factors, and there is growth and survival  **EGFR tyrosine kinase inhibitors** EFGR TKI binds at the same positions as ATP. But there is resistance against this TKI, this is cause by a second mutations. This mutations prevents the TKI to bind to the binding position, this allows ATP to bind and activated. There is a 3^rd^ generation EGFR TKI, this binds at the same position but in a different way. Afbeelding met tekst, diagram, schermopname, tekenfilm Automatisch gegenereerde beschrijving **BIOMARKERS IN COLORECTAL CARCINOMA** \> Treatment of colorectal carcinoma depends on the location of the tumor and on the gene mutation RAS is downstream of EGFR, so if you have a RAS mutation it will not help if you target the EGFR  **ACTIONABLE TARGETS IN BREAST CANCER** Breast cancer subtypes: Afbeelding met tekst, schermopname Automatisch gegenereerde beschrijving Chapter 5, page 180-187, molecular genetic diagnosis... end Chapter 7, page 267-338, chapter 7: neoplasia