molecular pathology in system pathology · reason for molecular testing •molecular testing for...

MOLECULAR PATHOLOGY IN SYSTEM PATHOLOGY

HISTOPATHOLOGY - PRACTICE2018/2019

BARBORA VLKOVÁINSTITUTE OF MOLECULAR BIOMEDICINEFaculty of medicine, Comenius University

www.imbm.skbarbora.vlkova@imbm.sk

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cell

DNA

genes

chromosomes

mutations

growth factors

cell cycle

tumor suppresor genes

oncogenes

Eukaryotic cell

DNA Structure

DNA in a Chromosome

DNA Structure

The structure of genes

DNA repair

Tumor Suppressor Genes

Breast CancerSusceptibility Genes

Cell cycle control systems

The p53 Protein,a Guardian of the Genome

Cell cycle control systems

Retinoblastoma

Intracellular Signal Transduction Systems

Origin of TumorsInfluence of Growth Factors on Cell Division

Cellular Oncogenes

Colon cancer

Targeted therapies in cancer

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Targeted therapies in cancer

• Part of standard treatment

• Depend on the stage of the disease

• Tumor expresses the target of drug

• Clinical trails

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Approved targeted therapies

Adenocarcinoma of the stomach: 2Basal cell carcinoma: 2Bladder cancer: 2Brain cancer: 2Breast cancer: 12Cervical cancer: 1Colorectal cancer: 6Dermatofibrosarcoma protuberans: 1Endocrine/neuroendocrine tumors: 1Head and neck cancer: 3Gastrointestinal stromal tumor: 3Giant cell tumor of the bone: 1Kaposi sarcoma: 1Kidney cancer: 10Leukemia: 13

Liver cancer: 1Lung cancer: 13Lymphoma: 15Melanoma: 7Multiple myeloma: 6Myelodysplastic/myeloproliferativedisorders: 2Neuroblastoma: 1Ovarian epithelial cancers: 3Pancreatic cancer: 3Prostate cancer: 4Soft tissue sarcoma: 2Systemic mastocytosis: 4Thyroid cancer: 4

Types of targeted therapies

• Hormone therapies

• Signal transduction inhibitors

• Gene expression modulators

• Apoptosis inducers

• Angiogenesis inhibitors

• Immunotherapies

• Toxic monoclonal antibodies

Breast cancer & Estrogen

• 1 mil. new cases

• 0.4 mil. die

• The earliest targeted therapies

• Disrupting the activity of the hormone

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Estrogen

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Estrogen

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Breast cancer & Estrogen

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Inhibition Estrogen I

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Inhibition Estrogen II

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Inhibition Estrogen II

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Hormone therapy for prostate cancer

Breast cancer & HER2

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HER2 in normal cells

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HER2 in Signalling

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HER2 in Cancer Cells

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HER2 in Cancer Cells

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FISH

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Inhibition HER2

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Breast cancer & IGF

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IGF in cancer cells

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Inhibition IGF I

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Inhibition IGF II

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Inhibition IGF III

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IGF & Insuline

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Breast cancer & PARP

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PARP - poly(ADP ribose) polymerase 1

PARP in cancer cells

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PARP - poly(ADP ribose) polymerase 1

Inhibition PARP

53PARP - poly(ADP ribose) polymerase 1

Inhibition PARP & mutant BRCA

54PARP - poly(ADP ribose) polymerase 1

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Myeloma & NF-kB

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NF-kB in Normal Cells

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NF-kB in Normal Cells

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NF-kB in Normal Cells

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NF-kB in Cancer Cells

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Inhibiting NF-kB

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Myeloma & HDAC

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HDACs in Normal Cells

HDAC - histone acetylases

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HDACs in Normal Cells

HDAC - histone deacetylases

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HDACs in Normal Cells

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HDACs in Normal Cells

HDAC - histone acetylases

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HDACs in Normal Cells

HDAC - histone deacetylases

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HDACs in Cancer Cells

HDAC - histone deacetylases

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Inhibiting HDACs

Why do we need molecular pathology?

• The diagnosis

• The prognosis

• The therapy

1. Clinical background

• 57 years old male

• the emergency

– substermal chest pressure

– left forearm pain

– dyspnea at rest

• the medical history

– hypertension, hypercholesterolemia, peptic ulcer

• the social history – smoking

• ECG, laboratory results = myocardial infarction

• two stenotic coronary arteries

• metal stent

• antithrombotic therapy

• one month later – 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

• 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

• 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

– antiplatelet medication was changed

2. Clinical background

• a 5 year old boy

• microscopic hematuria and proteinuria

• a blood count and a metabolic panel normal

• renal ultrasound normal

• X-linked Alport syndrome (XLAS)

• urinalysis, renal function studies,audiometry, ophthalmic evaluation, and skin and/or kidney biopsy

• molecular testing for mutations in theCOL4A5 gene - defective collagen chain = changes in glomerular basement membrane

Reason for Molecular Testing

• no need for further genetic testing

• monitore kidney function for disease progression

• monitore an extra-renal manifestations

• the identification of a disease-causing mutation

– testing of at-risk family members

– targeted testing of COL4A5 exon 50 in mother and her sister to confirm a carrier status of XLAS

Further Testing?

3. Clinical background

• a 25 year old RhD-negative pregnant woman

• father of the fetus RhD-positive

• 1st pregnancy - an antibody screen negative at 15 weeks and remained negative

– treated with Rh immune globulin (RhIG) at 28 weeks

– labor at 40 weeks, the infant was RhD-positive and RhIG again

• 2nd pregnancy - anti-D detected at a titer of 1:8 at 15 weeks, 1:64 at 18 weeks

– the fetal red cells were coated with maternal alloantibodies

– intrauterine blood transfusion

– after delivered exchange transfusion and phototherapy

• 3rd pregnancy:

• What is the differential diagnosis?

– hemolytic disease of the fetus and newborn (HDFN)

– the RhD-negative mother is alloimmunized by exposure tofetal RhD-positive red cells

– maternal anti-D antibodies cross the placenta

– the antibodies lead to the destruction of the red blood cells

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

– the father is RHD-negative

• the fetus is not at risk for HDFN related to anti-D

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

Extracellular fetal DNA

Extracellular tumor DNA

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

• the chance that offspring from this father will be RHD-positive is 50%

1 2 3

• 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

• a 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

4. Clinical background

• What is the differential diagnosis?

– echogenic bowel (normal fetuses, in fetuses with CF, or in fetuses with aneuploidy, intrauterine growth retardation, congenital viral infections, ...)

• She tested negative for CF mutations; could the fetus have CF?

– at risk for carrying a rare mutation

– more than 1,700 CFTR sequence variants have been identified

Reason for molecular testing

• echogenic bowel can be associated with CF

• CF is inherited, an autosomal recessive condition

• mother may have carried a rare mutation

• father could be a carrier of CF also

• carrier status - only molecular test for both parents

• prenatal testing if both parents were shown to be carriers

• CFTR sequence analysis of the fetus identified four sequence changes

Background and Molecular Pathology

• the most common AR disorders (1/2,500)

• life expectancy has increased to the late 30s

• the cystic fibrosis transmembrane conductance regulator (CFTR)

• defective chloride transport across membranes

• a 50 year old man

• a history of hypertension and hyperlipidemia

• malaise, fatigue, and pain in the left upper quadrant

• an enlarged spleen was identified

• peripheral blood - 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

5. Clinical background

Reason for Molecular Testing

• various neoplastic myeloproliferative disorders 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 responseto therapy

Molecular Testing

• initial testing for a suspected case of CML

• RT-PCR assays which target the most common BCR-ABL1 fusion transcripts associated with CML

the qualitative RT-PCR assay

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)

– 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

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

inhibition of EGFR receptors/signaling

Why this patient 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

• 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

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