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cell
DNA
genes
chromosomes
mutations
growth factors
cell cycle
tumor suppresor genes
oncogenes
DNA Structure
Intracellular Signal -receptors
Inhibition Estrogen II
Why do we need molecular pathology?
• The diagnosis
• The prognosis
• The therapy
Clinical background
• 57 years old male
• the emergency - substermal chest pressure, left forearm pain, dyspnea at rest
• the medical history – hypertension, hypercholesterolemia
• the social history – smoking
• ECG, laboratory results – consistent with myocardial infarction
• cardiac cathetrization revealed two stenotic coronary arteries
• metal stent placed in the coronary artery
• discharged on aspirin and antithrombotics
• one month later – similar signs and symptoms
• acute myocardial infaction
• thrombosis of the previously stented region of coronary artery
Reason for molecular testing
• a secondary coronary artery thrombosis
– despite treatment
• genetic variability in the CYP2C19 gene affects the pharmacokinetics and response to clopidogrel treatment
– some CYP2C19 variant alleles with reduced enzymatic function are associated with in-stent rethrombosis
• useful to identify resistant patients
– benefit from increased dosage or alternative drugs
• Is CYP2C19 genotyping appropriate in this patient?
– rethrombosis, new antiplatelet therapy
• What are the limitations of the TaqManCYP2C19 assay?
– designed to detect only the most common CYP2C19 allelic variants (*2, *3, and *17) associated with altered clopidogrel response
• Does the CYP2C19 assay result explain the patient’s complications?
– the patient has a reduced function CYP2C19 allele(CYP2C19*2)
– it may have contributed to the stent thrombosis and acute myocardial infarction, due to reduced efficacy of clopidogrel and ineffective platelet inhibition
Further Testing?
• no further laboratory testing was performed
• based on the results the patient’s antiplateletmedication was changed
• after coronary artery bypass recovered with nofurther complications
Background and Molecular Pathology
• genetic variants of the cytochrome p450 2C19 gene (CYP2C19) have been associated with individual variability in response to the antiplatelet medication
• a prodrug converted into an active metabolite (R130964) by CYP2C19 and other enzymes in the liver, resulting in irreversible inhibition of the platelet P2Y12 ADP receptor (P2RY12)
• results in inhibition of platelet aggregation by preventing activation of the glycoprotein IIb/IIIa receptor, which binds fibronectin and fibrinogen and is integral for fibrin cross-linking
• clinically relevant genetic variants of CYP2C19 associatedwith altered CYP2C19 enzymatic activity include CYP2C19*2,*3, and *17
• associated with a significantly increased risk forcardiovascular events including stroke, stent thrombosis,myocardial infarction, and death due to insufficient plateletinhibition
• warning on the package
• 32 years old woman, pregnant for the first time
• no history of cystic fibrosis in her family
• CF carrier screening, she tested negative for the mutations analyzed
– the mutation panel had a detection rate of 93% in Caucasians
• at 16 weeks gestation, prenatal ultrasound identified an echogenic bowel abnormality
Clinical background
• She tested negative for CF mutations; could the fetus have CF?
– yes, after carrier screening, her risk to be a carrier was reduced to 0.3%, but she was still at risk for carrying a rare mutation
– more than 1,700 CFTR sequence variants have been identified (unclear how many are pathogenic)
Reason for molecular testing
• echogenic bowel can be associated with CF
• CF is inherited, an autosomal recessive condition– if both parents are carriers of a CF mutation there is a 25% risk that the
fetus is affected
• mother may have carried a rare mutation
• father could be a carrier of CF
• carrier status - only molecular test for both parents and after it a molecular test for the fetus– the presence of two CF mutations significant can be used prenatally to
predict CF
• several possibilities for CF testing
• based on cost of testing and on timing
– CF sequence analysis for mother to determine if shecarried a rare mutation
– targeted mutation analysis for father and sequence analysis if targeted analysis were negative
– if both parents were shown to be carriers, prenatal testingcould have been ordered
– to test the fetus immediately
• an amniocentesis
– amniotic fluid was sent to the laboratory for CF sequence analysis
– DNA extracted from amniocytes, PCR, sequencing of CFTR gene
• the laboratory required a maternal sample for maternal cell contamination (MCC) studies
– a maternal peripheral blood specimen was sent also
– maternal and fetal markers were compared for evidence of MCC
• Should the parents be tested as well as the fetus?
• What happens if there are not enough fetal cells in the amniotic fluid?
• Is MCC analysis really necessary?
• What are the limitations of sequence analysis?
• CFTR sequence analysis of the fetus identified four sequence changes
• Why was mother’s first CF mutation screening result negative?
– not included in the initial carrier screening mutation panel
– it may have been a false negative result
• Are these sequence changes pathogenic?
– F508del is the most common CF mutation worldwide
– it is considered a classic CF mutation and is found in individuals with a severe form of CF
• Does this result explain the presence of echogenic bowel?
– yes
Background and Molecular Pathology
• the most common AR disorders
• 1 in 2,500 live-born children has CF
• life expectancy has increased to the late 30s
• CF is the result of mutations in the cystic fibrosistransmembrane conductance regulator (CFTR) gene
• defective chloride transport across membranes
• dehydrated secretions, resulting in tenacious mucus in the lungs, mucus plugs in the pancreas
Clinical background
• a 25 year old RhD-negative pregnant woman
• her husband RhD-positive
• her antibody screen was negative at 15 weeks and remained negative
• the patient was treated with Rh immune globulin (RhIG) at 28 weeks
• labor at 40 weeks, the infant was RhD-positive and RhIG was administered to the mother
• one year later, the patient became pregnant again and anti-D was detected at a titer of 1:8 at 15 weeks, the titer increased to 1:64 at 18 weeks.
• testing indicated that the fetal red cells were coated with maternal alloantibodies
• the fetus was treated with intrauterine blood transfusion
• at 36 weeks gestation, the fetus was delivered and was treated with exchange transfusion and phototherapy
• In the following year, the patient became pregnant for the third time.
• What is the differential diagnosis?
– a typical presentation of hemolytic disease of the fetus and newborn (HDFN)
– the RhD-negative mother is alloimmunized by exposure tofetal RhD-positive red cells
– in subsequent pregnancies, maternal anti-D antibodies cross the placenta into the fetal circulation
– the antibodies may lead to the destruction of the red blood cells in an antigen-positive fetus, leading to hemolytic disease
• How could molecular testing have been used to help manage this case?
Reason for Molecular Testing
• molecular testing for paternal zygosity and prenataltesting of the fetus plays an important role in the proposed algorithms for the management of HDFN
• the goal is to minimize invasive procedures in thesepatients because additional exposure to fetal red cells can cause further sensitization
• paternal zygosity is used to predict the risk of HDFN in each pregnancy
– homozygous for the RHD gene (RHD/D)
• the fetus is predicted to be RhD positive, the fetus can be monitored and invasive procedures may be avoided
– heterozygous (RHD/d) for the RHD gene
• fetal DNA testing through amniocentesis, chorionic villus sampling (CVS) or the testing of free fetal DNA in maternal plasma can be used to determine whether the fetus is RHD-positive or RHD-negative
– the father is RHD-negative
• the fetus is not at risk for HDFN related to anti-D
Laboratory test
• testing on direct amniotic fluid
• a backup culture
• testing of parental samples
• possibility – testing the fetal DNA present in maternal plasma
• testing more regions of the RHD gene in order to recognize allelic variants
• the fetal DNA present in maternal plasma
• RHD zygosity analysis of the paternal sample -father was heterozygous
• RHD zygosity analysis of the paternal sample -father was heterozygous
• RHD zygosity analysis of the paternal sample - father was heterozygous
• the chance that offspring from this father will be RHD-positive is 50%
• the fetus tested positive for all of the RHD-specific targets, indicating that the sample was RHD-positive• the fetus is at risk for hemolytic disease of the newborn related to anti-D
Clinical background
• a 5 year old boy
• microscopic hematuria and proteinuria on routine screening
• a blood count and a metabolic panel normal
• renal ultrasound normal and without hydronephrosis
• a diagnosis of X-linked Alport syndrome (XLAS)
• the diagnosis requires urinalysis, renal function studies,audiometry, ophthalmic evaluation, and skin and/or kidney biopsy
• molecular testing for mutations in the COL4A5 gene
• other may be inconclusive in the early stages
• molecular testing - for prognosis, for family testing
Reason for Molecular Testing
• no need for further genetic testing
• kidney function should be monitored closely for disease progression to allow timely treatment and intervention
• an ophthalmologist and audiologist - extra-renal manifestations
• the identification of a disease-causing mutation – testing of at-risk family members
• targeted testing of COL4A5 exon 50 in mother revealed thec.4946T>G (p.L1649R) mutation in a heterozygous state, confirming that she is a carrier of XLAS
Further Testing?
• a 50 year old man
• a history of hypertension and hyperlipidemia
• presented to his primary care physician for malaise, fatigue, and pain in the left upper quadrant
• an enlarged spleen was identified
• laboratory analysis of the peripheral blood revealed a marked leukocytosis consisting of increased granulocytic precursors at various stages of maturation
• a bone marrow biopsy showed increased granulocytic precursors with maturation
• family history was negative for any hematologic disorders
Clinical background
Reason for Molecular Testing
• because various neoplastic myeloproliferative disorders (such as chronic myelogenous leukemia (CML), chronic neutrophilic leukemia, and chronic myelomonocyticleukemia) can have overlapping clinical and pathologicalfeatures
• the molecular testing has diagnostic significance
• testing for the BCR-ABL1 fusion transcript - identified at the chromosomal level as t(9;22)(q34;q11)
• other conditions will be negative for BCR-ABL1, while CML will be positive
• molecular testing in CML monitor the patient’s responseto therapy (provide baseline values)
Molecular Testing
• initial testing for a suspected case of CML mayinclude a combination of qualitative and/or quantitative
• RT-PCR (reverse transcription, polymerase chainreaction) assays which target the most common BCR-ABL1 fusion transcripts associated with CML
• sometimes only the quantitative assay may be ordered
• the qualitative RT-PCR assay
– a rapid, simple, and inexpensive assay that can detect BCR-ABL1 fusion transcripts in CML
– detect the presence or absence of the BCR-ABL1 fusion transcripts
– it does not allow for quantitative monitoring of the response to therapy
the qualitative RT-PCR assay
• the quantitative RT-PCR assay
– to detect the presence of a BCR-ABL1 fusion transcript and also quantitate its level relative to an internal control transcript
– helpful - it can be used to monitor response to therapy over time
the quantitative RT-PCR assay
• the patient was positive for a high level of BCR-ABL1 fusion transcript with an e14a2 (b3a2):GUSB ratio of >100%
• together with the clinical findings is consistent with a diagnosis of CML
Further Testing?
Molecular monitoring during treatment
pretreatment level
initiation of therapy resistance to therapy response to new therapy
a resistance causing
mutation found in the
ABL1 kinase domain
of the BCR-ABL1
fusion transcript
• a 45 year old female nonsmoker
• complaining of dry cough, pleuritic pain, and headache
• chest X-ray revealed an opacity in the left lower lobe of the lung
• chest CT scan showed a 3.7-cm mass in the left lower lobe andmediastinal adenopathy
• cranial MRI demonstrated a 6.5-cm mass in the occipital lobe, with additional smaller cerebellar lesions
• a brain biopsy demonstrating metastatic adenocarcinomaconsistent with a primary tumor in the lung
Clinical background
• What is the role of molecular testing in this clinical context?
– to guide therapy selection, specifically with regards to the use of an EGFR tyrosine kinase inhibitor (EGFR-TKI)
– ffpe tumor samples to detect activating mutations in exons 18 through 21 of the EGFR gene, the region that encodesthe cytoplasmic tyrosine kinase domain of the epidermalgrowth factor receptor
– these mutations include single nucleotide missense mutations and small inframe deletions or insertions/duplications
• What are the advantages and limitations of the available techniques?
– sequencing-based methods can detect any of the commonmutations, including drug resistance mutations and rarenovel variants
– however, this method is fairly laborintensive and slow
– DNA from wt cell can interfere with the ability to detect the mutant sequence
– benign elements commonly outnumber malignant cells in biopsy specimens (2nd sampling require)
Results
• sequence traces for part of exon 21, showing a T>Gtransversion at nucleotide 2573 causing a leucine to argininemutation at codon 858 (Leu858Arg)
• patients with this mutation do benefit from treatment with an EGFR kinase inhibitor
• cancergrace.org
cancergrace.org
inhibition of EGFR receptors/signaling
Why this patient/her lung tumor did not respond to EGFR inhibition?
• because of its location downstream of EGFR, proliferativesignals from a mutated KRAS protein will not be inhibited by EGFR blockade
• as a result, KRAS mutant lung cancers do not respond to EGFR inhibition
http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/dna-sequencing-fragment-analysis/reagents-kits/kras.printable.html
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• lung cancer – the most lethal cancer
• the outcomes typically poor
• 2003 – treatment targeting EGFR tyrosine
kinase
• confirmed association between EGFR mutation
status and response to the TKI therapy
• female nonsmokers, asian ethnicity – sustained
responses
Background and Molecular Pathology
