Week 13 - Molecular Pathology Flashcards
Goals of Molecular Testing
Nail a diagnosis based on a molecular target or abnormality
Strengthen a diagnosis of malignancy or sometimes to reassure the diagnosis obtained from a Histopathology or Cytology sample
Optimise treatment or to find a specific treatment target (“personalised medicine”)
To determine prognosis
Monitoring of disease process (residue/resistance mutations)
To improve our understanding of the disease and to satisfy professional curiosity
Molecular Anatomical Lab Main Diagnostic Functions
Lymphoma clonality detection - diagnose/classify lymphoma
Mutation detection - assist oncologist to select a molecular targeted therapy e.g.: vemurafenib (Zelboraf) for malignant melanoma
- cytology - KRAS mutation in pancreatic cyst fluid
HER2 gene amplification in breast and gastric cancer to evaluate for Herceptin (trastuzumab) therapy
Microsatellite instability detection - to investigate patients for HNPCC (Lynch Syndrome)
FISH/CISH/SISH
Technical Aspects Prior to Molecular Testing
Avoid decalcification and mercury based fixatives
Tumour cellularity is more important than sample size
Validation on cytology material in the first instance
CB and cyto smears are suitable for PCR based techniques, CB preferred for FISH
Be aware of the limit of detection of the different molecular techniques:
- sanger sequencing 15-20%
- RT-PCR 0.5-5%
What is Lymphoma
Lymphomas are malignant proliferations of lymphocytes or lymphoblasts
Why are Diagnostic Tests Used to Diagnose Lymphoma
Morphology, immunophenotyping may be inconclusive
Small proportion of malignant cells in a benign background, insufficient sample (PCR highly sensitive)
To confirm other clinical or lab findings Eg: flow cytometry
To detect presence or recurrence of a clone (implies malignancy)
To detect specific chromosomal translocations
To aid prognosis and treatment
Molecular Changes Detected in Lymphoma
Antigen receptor gene rearrangements
Chromosomal translocation: BCL2, BCL1 and NPM-ALK
Epstein-Barr virus encoded RNA (EBV is associated with some types of lymphoma)
PCR for Lymphoma Analysis - Method
Amplify DNA target using specific primers, analyse PCR products, agarose, acrylamide gels, capillary electrophoresis
PCR for Lymphoma Analysis - Uses
Antigen receptor gene combinations: IgH; IgK, TCRB, TCRG
Chromosomal translocations: BCL1, BCL2, NPM-ALK, fusion mRNA detected using reverse transcriptase PCR
Lymphoma PCR Analysis - Narrow and Broud Band
Narrow product band of expected size
- AgR gene monoclonal
- translocation detected
Broad product back – AgR gene polyclonal
Lymphoma PCR Interpretation - False Negatives
Somatic hypermutation in (Follicular lymphoma)
Major, minor chromosomal breakpoint clusters/in particular when primers do not anneal correctly
Lymphoma PCR Interpretation - False Positives
Contamination
Insufficient polyclonal target DNA
Advantages of PCR Analysis of Lymphoma
Very sensitive test for monoclonal proliferation and chromosomal translocation
Small amounts of target nucleic acid needed
Partly degraded DNA may be suitable
Rapid
Inexpensive
Disadvantages of PCR Analysis of Lymphoma
Potential for contamination
Difficult to interpret significance of faint monoclonal bands in a polyclonal bg
Only useful for translocation detection if breakpoints cluster in defined region
Importance of Molecular Pathology in Medicine
Targeted drug therapy and check point mutations are transforming current treatment strategies in oncology
- therapeutic antibodies
- kinase inhibitors
- poly (ADP-ribose)
- polymerase (PARP) inhibitors
They all require prior diagnosis to identify molecular alterations that may serve as possible targets
Types of Specimens Required
FFPE
Cytology samples
- FNA
- CB
- cell scrapes
Liquid biopsy
Molecular Dx in Breast Ca - Intrinsic Molecular Types
Luminal A
Luminal B
Her2 enriched
Basal like
Luminal A Breast Ca
Can be further subdivided into 4 groups on copy number and usually have a worse prognosis
Luminal B Breast Ca
Display a higher level of genes related to proliferation
Her2 Breast Ca
ER negative encompasses the HER2 subgroup characterised by high level expression of genes of the HER2 amplicon (17q11)
Triple Negative Breast Cancer
Vast heterogeneity with 7 molecular subtypes:
- Basal like 1 (BCL1)
- Basal like 2 (BCL2)
- Mesenchymal (M)
- Mesenchymal stem like (MSL)*
- - Immunomodulatory (IM)
- Luminal androgen receptor (LAR)
- Unstable group
Oncotype Dx in Breast Ca
Oncotype Dx is a reverse transcriptase (RT-PCR) which measures the relative expression of 21 genes including;
- 16 cancer related genes and five reference genes
- produces a recurrent score (RS) from 0 to 100
- assigning patients into different risk categories
- LR (RS <18), IR (RS 18 – 30), HR (RS ≥ 31)
- determines the risk of distant recurrence at 10yrs and the benefit of additional chemotherapy in ER +, HER2-, node –ve breast cancer patients
HER2 (c-erb-B2) Gene
Human epidermal growth factor receptor 2 gene
Located on chromosome 17q12-21
HER2 encodes a cell surface transmembrane receptor protein (p185kDa)
- has tyrosine kinase (phosphorylating) activity
Ligands are epidermal growth factor and heregulin;
HER2 forms homo or heterodimers following ligand binding at its extracellular domain, and activates intracellular signaling pathways by autophosphorylation of tyrosines at its intracellular domain
HER2 and it’s Association with Breast Cancer
Multiple copies of HER2 gene in breast cancer cells
Gene amplifications causes increased HER2 protein expression in breast cancer and is implicated in cancer progression
Herceptin
Recombinant, humanised monoclonal antibody against HER2 protein that is beneficial for cancer patients with HER2 gene amplification
What does FISH, CISH and SISH Stand For?
FISH - Fluorescent in situ hybridization
CISH - Chromogenic in situ hybridization
SISH - Silver in situ hybridization
Evaluation of HER2 in CISH Staining
Not amplified: 1 to 5 dots in >50% of carcinoma cells
Clearly amplified: >10 dots in >50% (the majority) of carcinoma cells; or large clusters; or mixtures of multiple dots with large or small clusters in >50% carcinoma cells
Borderline: count dots in 30 carcinoma cells and calculate the average dots per carcinoma cell; >5 dots/cell is amplified, 1 to 5 dots/cell is not amplified
Gene Abnormalities and the Associated Outcomes
MYB/NF1B translocation - adenoic cystic ca
ETV6-NTRK3 - secretory carcinoma
HER2 amplification - predication of response to anti-HER2 agents and chemotherapy in adjuvant, neoadjuvant and metastatic settings
FGFR amplification - predictive factor for response to FGFR1 tyrosine kinase inhibitors
