MOLECULAR PATHOLOGY IN PRACTICE CASE-BASED … ·  · 2015-12-11MOLECULAR PATHOLOGY IN PRACTICE CASE-BASED APPROACH ... dyspnea at rest ... •What is the differential diagnosis?

MOLECULAR PATHOLOGY IN PRACTICECASE-BASED APPROACH

BARBORA IZRAEL VLKOVÁ

[email protected]

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