Week 13 - Molecular Pathology

Question 1

Goals of Molecular Testing

Answer 1

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

Question 2

Molecular Anatomical Lab Main Diagnostic Functions

Answer 2

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

Question 3

Technical Aspects Prior to Molecular Testing

Answer 3

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%

Question 4

What is Lymphoma

Answer 4

Lymphomas are malignant proliferations of lymphocytes or lymphoblasts

Question 5

Why are Diagnostic Tests Used to Diagnose Lymphoma

Answer 5

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

Question 6

Molecular Changes Detected in Lymphoma

Answer 6

Antigen receptor gene rearrangements
Chromosomal translocation: BCL2, BCL1 and NPM-ALK
Epstein-Barr virus encoded RNA (EBV is associated with some types of lymphoma)

Question 7

PCR for Lymphoma Analysis - Method

Answer 7

Amplify DNA target using specific primers, analyse PCR products, agarose, acrylamide gels, capillary electrophoresis

Question 8

PCR for Lymphoma Analysis - Uses

Answer 8

Antigen receptor gene combinations: IgH; IgK, TCRB, TCRG
Chromosomal translocations: BCL1, BCL2, NPM-ALK, fusion mRNA detected using reverse transcriptase PCR

Question 9

Lymphoma PCR Analysis - Narrow and Broud Band

Answer 9

Narrow product band of expected size
- AgR gene monoclonal
- translocation detected
Broad product back – AgR gene polyclonal

Question 10

Lymphoma PCR Interpretation - False Negatives

Answer 10

Somatic hypermutation in (Follicular lymphoma)
Major, minor chromosomal breakpoint clusters/in particular when primers do not anneal correctly

Question 11

Lymphoma PCR Interpretation - False Positives

Answer 11

Contamination
Insufficient polyclonal target DNA

Question 12

Advantages of PCR Analysis of Lymphoma

Answer 12

Very sensitive test for monoclonal proliferation and chromosomal translocation
Small amounts of target nucleic acid needed
Partly degraded DNA may be suitable
Rapid
Inexpensive

Question 13

Disadvantages of PCR Analysis of Lymphoma

Answer 13

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

Question 14

Importance of Molecular Pathology in Medicine

Answer 14

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

Question 15

Types of Specimens Required

Answer 15

FFPE
Cytology samples
- FNA
- CB
- cell scrapes
Liquid biopsy

Question 16

Molecular Dx in Breast Ca - Intrinsic Molecular Types

Answer 16

Luminal A
Luminal B
Her2 enriched
Basal like

Question 17

Luminal A Breast Ca

Answer 17

Can be further subdivided into 4 groups on copy number and usually have a worse prognosis

Question 18

Luminal B Breast Ca

Answer 18

Display a higher level of genes related to proliferation

Question 19

Her2 Breast Ca

Answer 19

ER negative encompasses the HER2 subgroup characterised by high level expression of genes of the HER2 amplicon (17q11)

Question 20

Triple Negative Breast Cancer

Answer 20

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

Question 21

Oncotype Dx in Breast Ca

Answer 21

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

Question 22

HER2 (c-erb-B2) Gene

Answer 22

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

Question 23

HER2 and it’s Association with Breast Cancer

Answer 23

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

Question 24

Herceptin

Answer 24

Recombinant, humanised monoclonal antibody against HER2 protein that is beneficial for cancer patients with HER2 gene amplification

Question 25

What does FISH, CISH and SISH Stand For?

Answer 25

FISH - Fluorescent in situ hybridization
CISH - Chromogenic in situ hybridization
SISH - Silver in situ hybridization

Question 26

Evaluation of HER2 in CISH Staining

Answer 26

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

Question 27

Gene Abnormalities and the Associated Outcomes

Answer 27

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