Tumor markers Petronini

Question 1

Tumour markers are found in ______________?

Answer 1

serum of patients, and they are useful for diagnosis, evaluation of
efficiency of the therapy and also important to monitor the progression of the disease.

Question 2

Classification of markers:

Answer 2

❖ Oncofetal Antigens: oncofetal means present in the fetus, for example, AFP has the same
role in the fetus and detects albumin in the adult.
❖ Antigens associated with tumors;
❖ Hormones secreted by cancer tissues;
❖ Proteins secreted by tumor tissues;
❖ Enzymes secreted by tumor tissues;
❖ Tumour markers in urine: PCA3 is dosed in a urine sample after prostate hyperplasia.
The first five are present in serum, but the last one is
present in urine.

Question 2

The theoretical features of a marker are:

Answer 2

➢ Exclusive and early production by the cancer cells.
➢ Changes in serum concentration concerning the effectiveness of therapy and the course of
the disease.
➢ Not detectable in subjects without cancer

Question 3

Clinical tumour markers important for the diagnosis of the
most important tumors are:

Answer 3

• for lung cancer we have CA and CEA.
• for liver cancer we have AFP.
• for prostate cancer we have PSA.
• for breast cancer we have CA 15.3, CEA.
• for stomach cancer we have CEA.
• for pancreas cancer we have CA125 and CEA.
• for colon cancer we have CEA.
• for ovaries cancer we have CA125 and CEA.

Question 3

You can obtain a sample from a tumor by:

Answer 3

• biopsy: for example, in lung tumors and breast tumors.
• fine needle aspiration: it’s a typical way to obtain a sample of thyroid cancer, but you can also use
  it for breast and lung, depending on tumor localization. For example, in the case of a lung tumor, you
  can reach it by bronchoscope, so you have to use fine needle aspiration under tomography for the
  localization of the tumor. For breast cancer, you can use mammotomy which permits taking a sample of tissue.
• cytological smear

Question 3

• Prostate cancer:

Answer 3

The very important antigen is PSA (Prostatic Specific Antigen). There is a quiz asking: “What is
PSA?” It’s Prostatic Specific Antigen, a marker useful for the diagnosis of prostate
cancer.
The normal value of this antigen is less than 4 ng/ml. If the PSA is between 4
and 10 ng/ml is useful for the diagnosis of prostatic hypertrophy or
hyperplasia, is a benign condition; in this case, you have to detect not only the total
PSA, but the ratio of free PSA and PSA bound to protein.
If you have a benign condition, the frequency of free PSA increases.
If you have cancer the bound of PSA increases.
If PSA is higher than 10 ng/ml is very commonly related to cancer.

Another important aspect is related to the time course of the dosage; it’s very
important to measure the PSA value not only once, but all the time. If you have a value of 8 ng/ml,
you have to repeat the measure every month to see if you have an increase of the PSA value.

Question 3

markers of Breast cancer?

Answer 3

The marker is Antigen Carbohydrate 15.3.

Question 3

Markers of lung cancer?

Answer 3

marker specific for small cell lung cancer, a tumor particularly present in
heavy smokers: NSE (Neuronal Specific Enolase). Enolase is an enzyme involved in
glycolysis, so it’s very useful for small-cell cancer and neuroendocrine tumors.

For non-small cell cancer is more important CEA (Carcino Embryonic Antigen): this marker is a
glycoprotein with a molecular weight of about 200 kilo Dalton and it’s the most important marker for
gastrointestinal tumors. Colon cancer is associated with an increase in CEA.

If you have a subject with a value of CEA very high in the serum, it’s very probable the presence of
metastases in the lung, so high CEA values are associated with lung metastases. Another site where one can have metastases due to colon tumors is the liver.
This marker is not specific for the lung or colon, but you can see that its value increases also in other tumors such as pancreas, stomach, and breast tumors.

Question 4

When the total PSA is less than 4 ng/ml, is not important to determine the ______ and _______ PSA
ratio. If the total PSA is between ___________ng/ml, a free total ratio less or equal to 10% indicates prostate cancer. In almost 50% of cases, a ratio above 25% indicates a lower risk of cancer.

Answer 4

free, bound, 4 and 10
Another important aspect is the patient’s age, because the risk increases with age.
To summarize, it’s important:
- the ratio free/bound;
- the time course of the determination;
- the age of the subject.

Question 4

markers of Colorectal cancer?

Answer 4

CEA is important also in this tumor. For this marker there is an important aspect: you can have an increasee in the marker value without the presence of tumor in heavy smokers, so it’s better to quit
smoking for at least one week before the test. So false positive is related to smoke.

Question 5

two types of lung cancer?

Answer 5

• non-small cell lung cancer.
• small cell lung cancer, also called microcytoma.

Question 6

________ marker is used for many tumors, very useful in colon cancer to access the follow-up of the
patient and it’s also important for the diagnosis. CEA increases in lung cancer, gastrointestinal
tumor, breast cancer and ovary cancer (so it’s not specific to a tumor).

Answer 6

• CEA

Question 7

Markers of Liver cancer?

Answer 7

The marker is Alpha FetoProtein (AFP); sometimes is produced also in normal conditions.

Question 8

The importance of tumour immunohistochemistry is relevant in?

Answer 8

1) classification of anaplastic malignancy: anaplastic means no
differentiation, so you do not know the origin. You can use
monoclonal antibodies directed to cytokeratin: if the cells in the
the sample is stained (observe the brown painting), it indicates that it
has an epithelial origin.
2) Classification of leukemias and lymphomas to establish the
origin, so if it is B or T cell lymphoma.
3) Determination of the site of origin of metastatic tumors: you have metastasis in the brain but do not know where the primary tumor is, for example, melanoma. If you can obtain a tissue sample, you can determine the presence of a specific tissue antigen.
4) Identification of molecules of prognostic and predictive importance for example estrogen receptor (ER), which is a predictive factor for hormone therapy and also a prognostic factor. Tumors expressing the receptor are less aggressive than tumors not presenting the receptor

Question 9

Can you give me an example of hormone therapy?

Answer 9

Tamoxiphene, Letrozol, inhibitor of
aromatase or anti-estrogenic therapy

Question 10

Why is microarray technology useful?

Answer 10

you can study the expression of the genes
(transcriptome, so mRNA) or evaluate the mutation in some genes
This means that you can study cells at:
- gene level (the sequence of the gene to see if there is a point mutation).
- expression level (mRNA).
- protein level.

Question 11

The cDNA that is the DNA complementary to DNA:

Answer 11

1. Are labeled with florescent dyes;
2. Are introduced into the array;
3. Fluorescence is an indicator of binding;
4. And binding means gene expression

With this technique you can study:
- cells treated with a drug compared with untreated
cells;
- tumour tissue compared with one healthy: this will
show different expression between treated and not
treated cells or normal and neoplastic cells

Question 12

It’s easier to treat a tumour that has an activated oncogene or a tumour that has lost the oncosuppressor?

Answer 12

It’s easier to cure a tumor with an activated oncogene because we can use
drugs that block that pathway. The only possibility to cure a tumor that has lost an oncosuppressor is through gene therapy. For example, if you have deleted p53, these cells do not die, they do not
undergo apoptosis

Question 12

DNA Microarrays is used to?

Answer 12

define gene expression
profiles of cancer cells: after mRNA isolation, you
have to retro-transcribe the mRNA into cDNA and during this process, you label it with red or green fluorescent dye the messenger from cancer or normal
cell respectively. Then you make the hybridization: if you obtain the red color, it means that these genes are
over-expressed in cancer cells. If you obtain the green color, it means that the genes are over-expressed in normal cells. Yellow and black colors mean that the genes are equally expressed. This is a useful technique to establish in that tumor the oncogenic driver. When you have a gene over-expressed in tumor cells, it means that the gene is a proto-oncogene becoming an oncogene. It may be also bcl 2, but the gene is related to cancer.
If you obtain an over expression in normal cells, this may be a sign of an oncosuppressor because
it’s lost in cancer cells.

Question 12

Why is FISH so important?

Answer 12

This technique is very important for the determination of the translocation of bcr and abl, present in 95% of chronic myeloid leukemia. Translocation from chromosome 9 to 22 generates the Philadelphia chromosome, a chromosome 22 shorter than normal. When abl and bcr are fused, the fluorescences red and green overlap and you obtain a yellow fluorescence. Green means normal, so the gene is not translocated, red means translocation, and yellow means fusion. In this case, there is a drug called Imatinib (the first target drug) which inhibits this kinase; it’s very important because the patient assuming this drug every day, survives.

Question 13

FISH (Fluorescence In Situ
Hybridization):

Answer 13

it’s a process that paints chromosomes or
portions of chromosomes with fluorescent dyes. It’s useful
for:
* identification of chromosomal abnormalities (can you
give me an example of chromosomal abnormalities in
cancer? Translocation, amplification, gene fusion).
* Aiding in gene mapping or toxicological studies to
determine the ploidy of chromosomes.
This is the procedure:
1. Label the probe DNA with fluorescence dyes.
2. Denaturation.
3. Hybridization.

Question 14

Different methods for the isolation of cells:

Answer 14

• one is based on the density gradient
  centrifugation: you create the gradient and red cells move to the bottom of the tumor, leukocytes in the middle, and with a particular density, you can find these cells.
• another technique is the use of immunomagnetic beads.
• another technique is based on the use of
  fluorescent monoclonal antibodies directed to Epithelial Cell Adhesion Molecules (epCAM): there is a machine called Veridex that isolates CTCs by using antibodies directed to epCAMs. In this case,
  a problem arises, EMT: if a cell has the epithelial-mesenchymal transition, it does not express the epCAMs, so you can’t isolate this cell. Now
  researchers are moving to study other methods for the CTCs’ isolation

Question 15

Blood cells, for example white blood cells, express ______, a
marker identifying leukocytes. In the case of red cells problems arise, because they do not have the
nucleus, so the nucleus can’t interfere with the monoclonal antibodies and they are very easy to
isolate. The real problem is white blood cells.

Answer 15

CD45

Question 16

an example of how we can identify the best cancer therapy?

Answer 16

the nude mice do not have immune
system so they can accept a translocation and not reject it. We can implant a piece of tumor, obtained by biopsy or by surgical strategies, and the tumor develops. You can remove it and propagate it in different animals. You obtain the hospital of mice: you can treat different groups of animals with different drugs and decide which is the best one; in this case, you have a complete regression of the tumor by using monoclonal antibodies directed to Met. Met is an oncogene that is mutated and over-expressed in many tumors; when it is expressed, the tumor is very aggressive. The only limitation is that in this case, you do not have the immune system effect, but
it’s not so important for accessing the effects of the drug. Of course, in this case, you can’t study the immuno-checkpoint inhibitors, so the drugs are used to potentiate the immune system.