BRAF gene mutation analysis
BRAF gene encodes a serine/threonine kinase that functions within the Ras-RafMEK-MAPK pathway, which connects extracellular signals to transcriptionalregulation. Activating mutations of BRAF are found in colorectal cancers (CRCs),
melanomas, ovarian tumors, lung and thyroid cancers. The most frequent BRAF
mutation is c.1799T>A (V600E), this mutation predisposes to apoptosis inhibition,
increases invasiveness, and occurs during carcinogenesis.
Indications for mutation analysis:
1.

2.

3.

BRAF mutation can be utilized to identify sporadic MSI-H colorectal cancer
cases and exclude them from germline mismatch repair gene testing. BRAF
V600E mutation accounts for over 90% of BRAF mutations in colorectal
cancer and is found in 30–80% of sporadic MSI-H colorectal cancer cases.
This mutation is extremely rare in tumours associated with Lynch syndrome.
The presence of the BRAF V600E mutation is strongly associated withsporadic CRCs that exhibit MSI due to somatic inactivation of MLH1 protein
expression. If either the BRAF V600E mutation or MLH1 promoter
methylation is found in a MSI-H tumor, then the tumor is probably sporadic
and further testing for Lynch syndrome may not be warranted.
BRAF mutation is an independent predictor for colorectal cancer patients’
responsiveness to EGFR inhibitors. BRAF mutations cause resistance toanti-epidermal growth factor receptor therapy in colorectal cancer patients,
therefore BRAF status is a useful biomarker for selecting patients suitable foranti-EGFR treatment.
BRAF mutation can be used to guide therapy for melanoma patients.
BRAF mutations have been detected in 70% of primary melanomas. BRAF
V600E kinase inhibitors are available for melanoma treatment, but their use
is limited to patients with metastatic melanoma with a demonstrated BRAF
V600E mutation.

Breast and Ovarian Cancer
Breast and ovarian cancers are most strongly associated with mutations of theBRCA1 and BRCA2 genes. Among women who have a clinically important BRCA
gene mutation, the lifetime risk of developing breast and/or ovarian cancer canreach 80%. Cancer-predisposing mutations in the BRCA1 and BRCA2 genes areinherited in an autosomal dominant manner. The prognosis for breast cancer
survival depends upon the stage at which breast cancer is diagnosed.
Indications for genetic testing:
1. Testing of individuals with early-age-onset of breast or ovarian cancer
2. Testing of individuals with family history of breast or ovarian cancer
3. Testing of at-risk family members for known mutations
3. Genetic counselingTesting should be performed if there is a family history of breast or ovarian cancer,
genetic alterations have been found in the family, or there is a history of breast
cancer in males in the family.

Cancer Predisposition
Determination of cancer predisposition is vital for prevention and early detectionof the disease. Early diagnosis of cancer will ensure the immediate start oftreatment, which is a key to increasing the survival and recovery. Significant
difference between the survival rates of early stage and advanced stage of cancer
points out the need for risk assessment of the disease.
Identification of genetic susceptibility to hereditary cancer syndromes enables toimplement risk-reduction strategies, estimate familial cancer risk and identify at-riskfamily members.
Cancer predisposition testing at Asper Biotech includes two next generation
sequencing based panels, allowing to analyze multiple genes associated with anincreased risk for a wide range of cancers.
Indications for genetic testing:
1.
2.
3.
4.
5.

Testing of individuals with early-age-onset of cancer
Testing of individuals with multiple primary cancers
Testing of family members of the affected individuals
Testing individuals with family history suggesting inherited pattern of cancer
but no genetic changes identified previously
Genetic counseling

Familial Adenomatous Polyposis – FAPFamilial adenomatous polyposis (FAP) is a colon cancer predisposition
syndrome characterized by the early onset of hundreds to thousands ofadenomatous polyps throughout the colon. By age 35, 95% of individuals with FAP
have polyps. If left untreated, patients with this syndrome develop colon cancer by
age 35-40. APC-associated polyposis conditions are inherited in an autosomal
dominant manner.
Indications for mutation analysis:
1. Testing of individuals with adenomatous polyps
2. Testing of first degree relatives of the affected individuals
3. Genetic counselingGlioblastoma
Glioblastomas are heterogeneous group of tumors originating from glial cells.
Glioblastomas are most frequent in pediatric age. Approximately 60% from all thecentral nervous system tumors occur in childhood. Disease prevalence is 6-7:100000 cases/year. Glioblastoma prognosis and treatment options depend on type andlocation of tumor, association to genetic disorders (e.g. neurofibromatosis) andtumor stage (localized or disseminated glioblastoma).
Indications for genetic testing:
1. Confirmation of clinical diagnosis
2. Determination of disease prognosis and recurrence risk
3. Prediction of treatment response
4. Genetic counseling

Lynch Syndrome/Hereditary Non-Polyposis
Colon Cancer – HNPCC
Lynch syndrome, also called hereditary non-polyposis colon cancer (HNPCC),
is characterized by an increased risk of colon cancer and other cancers (e.g., of theendometrium, ovary, stomach, small intestine, hepatobiliary tract, upper urinary
tract, brain, and skin). Lynch syndrome is inherited in an autosomal dominantmanner and it is associated with germline mutations in the mismatch repair (MMR)
genes MLH1, MSH2, MSH6 and PMS2. Mutation carriers have a lifetime risk of up
to 80% for colorectal cancer, 20-60% risk of endometrial cancer, as well as othertumors. Lynch syndrome is associated with early onset of cancer, the average ageof diagnosis is 45 years.
Referral for molecular testing of Lynch syndrome:1.

Tumour tissue analysis to evaluate the MMR proteins expression byimmunohistochemical analysis (IHC) and DNA microsatellite instability (MSI)
testing is suggested for the individuals meeting Amsterdam II/Bethesda
criteria.
2. If absence of the MLH1/PMS2 proteins expression is observed by IHC,
methylation analysis of the MLH1 gene promoter and/or testing of thesomatic BRAF V600E mutation is recommended in order to exclude sporadic
colorectal cancer cases.
3. If tumour with MMR deficiency and MSI high is detected, further mutation
analysis from peripheral blood/normal tissue of the MMR genes is indicated.
Indications for mutation analysis:
1.
2.
3.

Testing of individuals meeting Amsterdam II/Bethesda criteria
Testing of individuals with family history of colorectal cancer or other Lynch
syndrome-related cancers
Testing of at-risk family members for known mutations

4.

Genetic counseling

Lynch Syndrome and Polyposis Syndrome
Lynch syndrome is characterized by an increased risk of colon cancer and othercancers (e.g., of the endometrium, ovary, stomach, small intestine, hepatobiliary
tract, upper urinary tract, brain, and skin). Lynch syndrome is inherited in an
autosomal dominant manner and most commonly it is associated with germline
mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2.
Mutation carriers have a lifetime risk of up to 80% for colorectal cancer, 20-60% riskof endometrial cancer, as well as other tumors. Lynch syndrome is associated withearly onset of cancer, the average age of diagnosis is 45 years.
In addition to Lynch syndrome the following syndromes are included in the testing:
Familial adenomatous polyposis (FAP), MUTYH-associated polyposis,
BMPR1A-Related Juvenile Polyposis, SMAD4-Related Juvenile Polyposis,
PTEN hamartoma tumor syndrome, Peutz-Jeghers Syndrome and othercancers.
Indications for genetic testing:
1.
2.
3.
4.
5.
6.

Confirmation of clinical diagnosis
Testing of individuals meeting Amsterdam II/Bethesda criteria
Testing of individuals with family history of polyposis syndromes and Lynch
syndrome-related cancers
Differentiation of FAP from MUTYH-associated polyposis
Differentiation of juvenile polyposis from other hamartomatous polyposis
syndromes
Genetic counselingMicrosatellite instability – MSI
Microsatellite instability (MSI) is the mutational signature found in colorectal
cancers that evolve as a result of inactivation of the DNA mismatch repair system.
Defects in the genes involved in mismatch repair lead to an accumulation ofsomatic mutations in a cell, which may result in the cell becoming malignant.
Germline mutations in mismatch repair genes are associated with developing
hereditary non-polyposis colon cancer. Approximately 90% of colon cancers fromfamilies meeting Amsterdam criteria have MSI.
Indications for genetic testing:
1.
2.
3.
4.

Testing of individuals with colorectal or other Lynch-associated cancers toestimate the probability of mutations in mismatch repair genes
Determination of disease prognosis
Prediction of tumor response to chemotherapeutic agents
Genetic counselingMUTYH-associated polyposis
MUTYH-associated polyposis (MAP) is an autosomal recessive disordercharacterized by a variable number of colorectal adenomas with a high risk ofdeveloping colorectal cancer. MAP is caused by biallelic germline mutations in
MUTYH gene, but there is also evidence that monoallelic mutation carriers have anincreased risk for developing colorectal cancer. The clinical symptoms of MAP areoften undistinguishable from that of familial adenomatous polyposis (FAP) or
attenuated FAP (AFAP) caused by mutations in adenomatous polyposis coli (APC)
gene, but the age of onset is usually later compared to FAP patients. The two mostcommon mutations in Caucasians, accounting for about 80% of mutant MUTYH
alleles, are p.Y179C and p.G396D (also known as Y165C and G382D).
Indications for genetic testing:
1.
2.
3.

Testing of individuals with clinical symptoms similar to FAP or AFAP but inwhom no APC gene mutation has been identified
Testing of first degree relatives of the affected individuals
Genetic counseling

Thiopurine S-Methyltransferase Deficiency –TPMT
Thiopurine drugs such as 6-mercaptopurine and azathioprine are used as
chemotherapeutic agents and their active metabolites have bothimmunosuppressive as well as antiproliferative effects.Thiopurine smethyltransferase (TPMT) is an enzyme that catalyzes the s-methylation of
thiopurine-type compounds and mediates the formation of inactive metabolites.
Genetic polymorphisms that affect this enzymatic activity can result in lifethreatening toxicity. Drug-induced bone marrow toxicity may cause
myelosuppression, anemia, bleeding tendency, leukopenia, and infection.
There are three main genetic changes in the TPMT gene that determine
metabolization efficiency of the thiopurines (rs1800462, rs1800460, andrs16880254) in 80-95% of cases.
Indications for genetic testing:
Genetic testing of variations in the TPMT gene allows determining the activity of theTPMT enzyme, and makes it possible to choose an individual’s optimal dose before
starting treatment. In principal, this test should be done for all patients who areundergoing treatment with thiopurine drugs.