Utility of Molecular Diagnostics in Cancer Management

Question 1

Outline tumour pathology.

Answer 1

• Tumours evolve slowly as a consequence of gene mutation.
• Tumours grow locally and then metastasise to distant sites.
• Tumours can secrete a variety of proteins and tumour cells can be shed (e.g. sputum, urine, blood).
• Tests may involve evaluation of secreted products, evaluation of presence of tumour cells, evaluation of tumour nucleic acids, evaluation of tumour tissue.

Question 2

What may tests for tumour pathology involve?

Answer 2

Tests may involve evaluation of secreted products, evaluation of presence of tumour cells, evaluation of tumour nucleic acids, evaluation of tumour tissue.

Question 3

What is the purpose of tumour staging?

Answer 3

• Tumour staging is about mapping out how far a tumour has spread.
• Tumours grow locally at first - measured by the T-stage. Eventually tumours will metastasise to distant sites and lymph nodes (M- Stage).
• The greater the stage, the poorer the prognosis is going to be.

Question 4

What is CEA?

Answer 4

• CEA is carcinoembryonic antigen.
• CEA is expressed at low levels in the normal colon but is expressed at high levels and secreted by colorectal cancers.
• It is a prognostic factor and it can be monitored to test for recurrence.

Question 5

What antigen can be monitored to test for the recurrence of colorectal cancer?

Answer 5

• CEA is carcinoembryonic antigen.
• CEA is expressed at low levels in the normal colon but is expressed at high levels and secreted by colorectal cancers.
• It is a prognostic factor and it can be monitored to test for recurrence.

Question 6

What are serological markers of tumours?

Answer 6

Many tumours aberrantly produce proteins which can be secreted (e.g. CEA in CRC). May be new proteins or proteins that are found normally but are produced at a much higher level by tumours.

These proteins can be used for diagnosis, prognosis, monitoring, and possibly screening.

Examples include:

• CEA produced by CRC
• CA125 produced by ovarian cancers
• AFP produced by hepatocellular cancers
• CA19.9 produced by pancreatic cancers
• PSA produced by prostate cancers
• Markers may have different values in different settings.

Question 7

List some serological tumour markers.

Answer 7

• CEA produced by CRC
• CA125 produced by ovarian cancers
• AFP produced by hepatocellular cancers
• CA19.9 produced by pancreatic cancers
• PSA produced by prostate cancers

Question 8

What type of cancer is CEA a marker for?

Answer 8

CEA is a marker for CRC.

Question 9

What type of cancer is CA125 a marker for?

Answer 9

CA125 is a marker for ovarian cancers.

Question 10

What type of cancer is AFP a marker for?

Answer 10

AFP is a marker for hepatocellular cancers.

Question 11

What type of cancer is CA19.9 a marker for?

Answer 11

CA19.9 is a marker for pancreatic cancers.

Question 12

What type of cancer is PSA a marker for?

Answer 12

PSA is a marker for prostate cancers.

Question 13

How can tumour cells be detected/collected?

Answer 13

• Tumour cells can be shed from external surfaces or into the blood.
• Tumour cells can also be aspirated from masses.
• Abnormal fluid collections (such as pleural effusions) can be tested for the presence of tumour cells.
• Circulating tumour cells (CTC) can be detected in the blood.
• Externally shed cells, blood samples or aspirated cells can be directly visualised for in the presence of tumour cells (i.e. cytological examination).
• Supplementary tests can be performed on the cells.
• This methodology can be used for screening, diagnosis, surveillance, and possible prognosis.

Question 14

What tests can be done on collected tumour cells? Outline some examples of cytological examination.

Answer 14

• Cytological examination - just looking at the actual cells rather than histological specimens preserved in wax etc.
• Sometimes may be easier just to take smear of cells.
• Variety of different stains used. For histology specimens use haematoxin and eosin as general rule. For cytology use predominantly PAP smear or Giemsa. Diagnosis usually depends on assessing the size and morphology of the nuclei.
• The cervical cancer screening program is dependent on cytological examination of cells.
• Surveillance for bladder cancer can be done by cytological examination of urine.
• Generally prefer to use biopsy but cytological examination of masses can give rapid diagnosis or can be used when biopsy may be difficult (e.g. lung, biliary tract).
• Abnormal fluid collections (such as pleural effusions) can be tested for the presence of tumour cells.

Question 15

What are the main 2 stains used in the examination of cytology specimens?

Answer 15

For cytology use predominantly PAP smear or Giemsa.

Question 16

Give some examples of where cytology is used as a cancer screening tool.

Answer 16

• The cervical cancer screening program is dependent on cytological examination of cells.
• Surveillance for bladder cancer can be done by cytological examination of urine.

Question 17

When would we generally use cytological examination of masses rather than biopsy followed by histological examination?

Answer 17

• Generally prefer to use biopsy but cytological examination of masses can give rapid diagnosis or can be used when biopsy may be difficult (e.g. lung, biliary tract).

Question 18

How can we detect circulating tumour cells in the blood? What can they tell us?

Answer 18

• Circulating tumour cells (CTC) can be detected in the blood.
• Blood samples can be purified for epithelial cells or epithelial markers may be used - blood should only contain lymphocytes, polymorphs, leukocytes etc. and should not contain epithelial cells.
• Can stain cytokeratin fluorescently tagged antibody, DAPI to stain nuclei, fluorescently tagged andibody to stain CD45 - all epithelial markers.
• High CTC number may have poor prognosis (possible that CTC numbers are a reflection of tumour load).
• May be used for screening in the future.

Question 19

Other than cytological analysis via visualisation studies, what other tests can we perform on tumour cells?

Answer 19

• Once tumour cells have been obtained we can do other tests using these cells such as PCR, FISH etc. to look for mutations / cytogenetic abnormalities.

Question 20

What else may we be able to do to test for tumour cells in a fluid etc. when no actual cells can be detected?

Answer 20

• Where cells have become degraded they can release their DNA into the bloodstream. You can then purify the DNA from whatever fluids we are examining and perform PCR to look for the presence of mutations.
• Can also detect tumour cells metastisising to difference sites such as in lymph nodes via detection of tumour nucleic acids.
• Can use the evaluation of nucleic acid to look for specific mutations / epigenetic changes (e.g. circulating tumour DNA / RNA).
• Where you have a small number of cells in an organ that has large numbers of normal cells in there, e.g. looking for tumour cells in a lymph node, if you are just doing PCR then you will amplify the lymphocytes in preference to the tumour cells DNA - by knowing specific mutations or epigenetic changes you can enrich for the presence of your mutant alleles and detect whether they are there or not. For example, if you know that there is a specific KRAS mutation in your primary tumour and you are looking for circulating cells/lymph node metastases then you can do an ARMS PCR for specific KRAS mutations that will tell you if they are there or not.
• Can use one-step nucleic acid amplification to directly detect lymph node metastases while patients are on the operating table rather than trying to use frozen sections etc. Can interrogate the whole of the lymph node for epithelial cytokeratin markers that shouldn’t be there. If we find these markers then we know that there is a very high chance of metastasis being present - can then change operation based on PCR results.

Question 21

How can we use analysis of free tumour DNA to guide operations to remove cancers?

Answer 21

• Can use one-step nucleic acid amplification to directly detect lymph node metastases while patients are on the operating table rather than trying to use frozen sections etc. Can interrogate the whole of the lymph node for epithelial cytokeratin markers that shouldn’t be there. If we find these markers then we know that there is a very high chance of metastasis being present - can then change operation based on PCR results.

Question 22

Where might we find tumour cell DNA in the case of CRCs specifically?

Answer 22

• In stool samples.
• High detection rates of colorectal neoplasia by stool DNA testing has been demonstrated using a novel digital melt curve assay (2009).
• Better detection rate than faecal occult blood tests.

Question 23

What epigenomics biomarker has recently been shown successful in the early detection of CRC?

Answer 23

• Epigenomics biomarker SEPT9 for the early detection of cancer in a simple blood sample has demonstrated continuously highest performance in multiple clinical studies with in total more than 3,000 individuals tested.
• Based on PCR on DNA extracted from peripheral blood.

Question 24

Outline the general journey of a cancer specimen.

Answer 24

1) . Specimen resected - if left ischaemic for too long before being fixed can affect the integrity of the DNA and the proteins.
2) . Fixed in formalin - very rarely will be examined fresh. Formalin has an effect on tissues because of the protein and DNA cross linking that it causes. The length of time that the specimen spends in formalin may affect the preservation of the tissue for other tests such as IHC. Thus excessive fixation can affect both IHC an PCR tests.
3) . Specimen cut up, processed, and H&E stained.
4) . Goes to pathologist and they make the diagnosis - may request adjunctive tests (special stains or IHC) if diagnosis can’t be made.

Question 25

What are the main points of specimen compromise on the journey from a specimen being taken?

Answer 25

1) . If left ischaemic for too long before being fixed can affect the integrity of the DNA and the proteins. 2) . Fixed in formalin - very rarely will be examined fresh. Formalin has an effect on tissues because of the protein and DNA cross linking that it causes. The length of time that the specimen spends in formalin may affect the preservation of the tissue for other tests such as IHC. Thus excessive fixation can affect both IHC an PCR tests.

Question 26

What can adjunctive/further histological tests be used for?

Answer 26

1). To aid diagnosis - e.g. to pin down where neoplastic cells have originated from such as using IHC for CK7+ cells to exclude primary colonic cancer as a posibility when neoplastic cells identified in the colon - identify metastasised breast cancer cells using ER+ and Progesterone receptor staining. 2). As complimentary tests for other investigations - e.g. to try and pin down which MMR protein is missing in Lynch syndrome.

Question 27

All the information we can gather about a patient's cancer should be integrated......

Answer 27

- Once a cancer is suspected, it needs to be appropriately investigated - History and examination - Blood tests - Pathology - Radiology - Specific tumour markers - Molecular tests if relevant - Discussion of patient (overall assessment of the data) is undertaken at the Multi-Disciplinary Team Meeting (MDT) and management decisions made.

Question 28

Outline the general patient pathway in cancer.

Answer 28

1) . Patient presentation to clinical services. 2) . If a mass is identified or cancer is suspected then a biopsy is taken 3) . On results of biopsy a management decision is made at MDT. If malignant then management decision will either be straight to surgery, or neo-adjuvent chemotherapy. 3) . Once surgical resection is done then it comes back to the pathologist for evaluation and staging. 4) . Goes back to MDT and management decision is made as to what kind of follow up to do and whether to give any further treatment or not.

Question 29

Summary:

Answer 29

- Specific features of tumour cells can be the basis of investigative tests. - Tests can interrogate the nucleic acids or the proteins. - These tests complement molecular tests undertaken on tumour tissue. - Everything is integrated at the MDT for patient management.

Question 30

What kind of information do we want from cancer genetics? (the 6 Ps of cancer genetics)

Answer 30

1) . Predisposition - gene variants informing on risk of cancer development. 2) . Profile - all gene variants present within a tumour. 3) . Prognosis - Gene variants informing on outcome. 4) . Prediction - Gene variants informing on response to therapy. 5) . Pharmacogenetic - gene variants informing on how the body handles a therapy. 6) . Pharmacotherapeutic - targetable genes. - From the germline DNA we can get information about predisposition and pharmacogenetics. - From the tumour DNA will give us the profile of the tumour and within that profile we may get prognostic information, predictive information, and pharmacotherapeutic information. - A variety of new methodologies are being developed. As always, there are technical limitations - e.g. pyrosequencing is cheap and quick but doesn't handle nucleotide repeat sequences very well. Methodologies also now allow rapid interrogation of the whole genome and the exome - comes with issues in data handling and data interpretation.

Question 31

Why is template quality important for molecular tests on harvested tumour samples? how can it be tested? What factors affect template quality?

Answer 31

- In order for molecular diagnostics to be carried out, the template must be of sufficient quality. This can be assessed using multiplex PCR. - The tissue must contain some tumour - the limit of detection of tests will vary and so there must be some correlation with histology - need to integrate different tests. - Data should be interpreted taking the whole context into account. - In the NHS the majority of tissue specimens need to be fixed in order to stop autolysis. - The most commonly used fixative is buffered formalin (10%). - The time spent in the fixative affects the integrity of the nucleic acids and proteins. - The age of the specimen also affects template quality. - If anything has been fixed in Bouin's fixative don't believe the PCR results! Contains mercury and pictic acid and degrades the DNA!

Question 32

Can we carry out molecular analysis on specimens fixed in Bouin's fixative?

Answer 32

If anything has been fixed in Bouin's fixative don't believe the PCR results! Contains mercury and pictic acid and degrades the DNA!

Question 33

What amplification can we expect to see from fixed tumour specimens? How can template quality affect this?

Answer 33

- PCR - in FFPE tissue, only about 300-500bp can be readily amplified - RT-PCR - in FFPE tissue around 100bp can be amplified but not reliable. Fresh tissue is required. - Template quality also affects post-PCR tests i.e. supplementary techniques such as SSCP/sequencing/pyrosequencing/dHPLC/HRM. - Fluorescent and chromogenic in-situ hybridisation (FISH/CISH): works in FFPE tissue.

Question 34

What are the limits of detection when using Sanger sequencing and pyrosequencing and HRM to detect tumour DNA?

Answer 34

- If there is no tumour present in the tissue or it is below the limit of detection of the method, then there is a risk of a false negative result. - Different methods will have different limits of detection e.g. Sanger sequencing requires a minimum of 20% tumour cells whilst pyrosequencing and HRM will pick up mutations in around 5% of tumour cells. - For some targets the limit of detection for HRM can go down to 2.5%. - Real time ARMS PCR can pick up a mutation frequency of 1% - but what exactly does a mutation frequency of 1% mean - is it relevant?

Question 35

Outline the stratified medicine initiative.

Answer 35

- CR-UK has launched a stratified medicine initiative. - Panel of predictive markers - the aim is to establish a panel of predictive markers which will allow patients to be stratified into the correct treatment group. - The diagnostic tests for these markers should be available in the NHS. - Inappropriate therapy (especially with biologics) costs in terms of patient morbidity and economoics - one of the aims is to prevent this.

Question 36

What gene mutations are the CR-UK biomarkers for CRC?

Answer 36

- KRAS - BRAF - TP53 - PI3KCA - UGT1A1

Question 37

What gene mutations are the CR-UK biomarkers for breast cancer?

Answer 37

- PI3KCA - TP53 - PTEN - CYP2D6

Question 38

What gene mutations are the CR-UK biomarkers for prostate cancer?

Answer 38

- PTEN - TMPRSS-ERG - TLR4

Question 39

What gene mutations are the CR-UK biomarkers for lung cancer?

Answer 39

- EGFR - EML4-ALK - XRCC2 - ERCC1 - RRMI

Question 40

What gene mutations are the CR-UK biomarkers for lung cancer?

Answer 40

- PTEN - PI3KCA - BRAF

Question 41

What gene mutations are the CR-UK biomarkers for melanoma?

Answer 41

- BRAF | - CKIT

Question 42

Overview:

Answer 42

- The features of tumour cells are the basis of investigative tests. - Genetic information and the 6 P's of cancer genetics. - The template quality: the quality of the DNA and the quality of the tumour cells. - The stratified medicine initiative.