MOLECULAR PATHOLOGY IN PRACTICE CASE-BASED APPROACH. BARBORA IZRAEL VLKOVÁ

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1 MOLECULAR PATHOLOGY IN PRACTICE CASE-BASED APPROACH BARBORA IZRAEL VLKOVÁ

2 cell DNA genes chromosomes mutations growth factors cell cycle tumor suppresor genes oncogenes

3 DNA Structure

5 Intracellular Signal - receptors

7 Inhibition Estrogen II

8 Why do we need molecular pathology? The diagnosis The prognosis The therapy

10 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

11 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

12 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

13 Is CYP2C19 genotyping appropriate in this patient? rethrombosis, new antiplatelet therapy What are the limitations of the TaqMan CYP2C19 assay? designed to detect only the most common CYP2C19 allelic variants (*2, *3, and *17) associated with altered clopidogrel response

15 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

16 Further Testing? no further laboratory testing was performed based on the results the patient s antiplatelet medication was changed after coronary artery bypass recovered with no further complications

17 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 crosslinking

clinically relevant genetic variants of CYP2C19 associated with altered CYP2C19 enzymatic activity include CYP2C19*2, *3, and *17 associated with a significantly

18 clinically relevant genetic variants of CYP2C19 associated with altered CYP2C19 enzymatic activity include CYP2C19*2, *3, and *17 associated with a significantly increased risk for cardiovascular events including stroke, stent thrombosis, myocardial infarction, and death due to insufficient platelet inhibition warning on the package

19 Clinical background 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

20 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)

21 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

22 several possibilities for CF testing based on cost of testing and on timing CF sequence analysis for mother to determine if she carried 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 testing could have been ordered to test the fetus immediately

23 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

24 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?

25 CFTR sequence analysis of the fetus identified four sequence changes

26 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

27 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 fibrosis transmembrane conductance regulator (CFTR) gene defective chloride transport across membranes dehydrated secretions, resulting in tenacious mucus in the lungs, mucus plugs in the pancreas

28 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

29 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

30 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 to fetal 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

31 How could molecular testing have been used to help manage this case?

32 Reason for Molecular Testing molecular testing for paternal zygosity and prenatal testing of the fetus plays an important role in the proposed algorithms for the management of HDFN the goal is to minimize invasive procedures in these patients because additional exposure to fetal red cells can cause further sensitization

33 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

34 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

35 the fetal DNA present in maternal plasma

36 RHD zygosity analysis of the paternal sample - father was heterozygous

37 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

38 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

39 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

40 Reason for Molecular Testing 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

42 Further 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 the c.4946t>g (p.l1649r) mutation in a heterozygous state, confirming that she is a carrier of XLAS

43 Clinical background 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

44 Reason for Molecular Testing because various neoplastic myeloproliferative disorders (such as chronic myelogenous leukemia (CML), chronic neutrophilic leukemia, and chronic myelomonocytic leukemia) can have overlapping clinical and pathological features 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 response to therapy (provide baseline values)

46 Molecular Testing initial testing for a suspected case of CML may include a combination of qualitative and/or quantitative RT-PCR (reverse transcription, polymerase chain reaction) assays which target the most common BCR-ABL1 fusion transcripts associated with CML sometimes only the quantitative assay may be ordered

47 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

49 the qualitative RT-PCR assay

50 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

51 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

52 Further Testing?

kinase domain of the BCR-ABL1 fusion transcript

53 Molecular monitoring during treatment pretreatment level a resistance causing mutation found in the ABL1 kinase domain of the BCR-ABL1 fusion transcript initiation of therapy resistance to therapy response to new therapy

54 Clinical background 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 and mediastinal adenopathy cranial MRI demonstrated a 6.5-cm mass in the occipital lobe, with additional smaller cerebellar lesions a brain biopsy demonstrating metastatic adenocarcinoma consistent with a primary tumor in the lung

55 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 encodes the cytoplasmic tyrosine kinase domain of the epidermal growth factor receptor these mutations include single nucleotide missense mutations and small inframe deletions or insertions/duplications

56 What are the advantages and limitations of the available techniques? sequencing-based methods can detect any of the common mutations, including drug resistance mutations and rare novel 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)

57 Results sequence traces for part of exon 21, showing a T>G transversion at nucleotide 2573 causing a leucine to arginine mutation at codon 858 (Leu858Arg) patients with this mutation do benefit from treatment with an EGFR kinase inhibitor

59 inhibition of EGFR receptors/signaling cancergrace.org cancergrace.org

60 Why this patient/her lung tumor did not respond to EGFR inhibition? because of its location downstream of EGFR, proliferative signals 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

61 62

62 Background and Molecular Pathology 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

Blood Types and Genetics

Blood Types and Genetics Human blood type is determined by codominant alleles. An allele is one of several different forms of genetic information that is present in our DNA at a specific location on a