PATH 310 Mod 3 & TBL Content - Final Copy

hematopoietic stem cells - defining principles

long-lived cells that give rise to all blood cells - capable of self-renewal and multipotency

location of HSCs

start in the yolk sac and migrate to liver and spleen; after birth, most HSCs are in bone marrow

Neoplasms

when stem cell differentiation becomes pathological; a disorder of cell growth; can be benign or malignant

Four Characteristics of Neoplastic Cells

exceeds normal growth, autonomous, result of mutation, monoclonal

oncogene

cancer-inducing gene responsible for encoding oncoproteins

proto-oncogene

a normal gene that upon mutation acquires the ability to function as an oncogene

oncoprotein

protein that causes the transformation of a cell into a tumour cell

tumour suppressor gene

a gene whose inactivation contributes to cancer development

leukemia definition

a type of cancer primarily affecting the body’s blood-forming tissues; can be acute or chronic

how does leukemia disrupt hematopoiesis?

dominated by a single clone (disease stem cell has competitive advantage)

etiology of leukemia

often idiopathic; related to somatic mutations (NOT in germ cells)

CML Signs & Symptoms

general fatigue, night sweats, weight loss, abdominal ‘heaviness’, low hemoglobin/RBC count

Genetic Etiology of CML

cells derived from CML bone marrow had an abnormal, shortened chromosome 22 (the Philadelphia chromosome)

how does the Philadelphia chromosome form?

reciprocal translocation of genetic material between chromosomes 9 and 22 = t(9;22); it is found in 100% of CML cases but not entirely specific to CML

impact of somatic chromosomal translocation

the genetic change does not exist in ALL cells of the CML patient, only in leukemia cells

benefits of karyotyping

gold-standard, can identify variations of the translocation, can quantify how many cells are carrying the transformation

limitations of karyotyping

requires viable dividing cells, labour intensive, expensive, requires expert analysis, invasive sampling procedure, sensitivity

Philadelphia Chromosome Gene Fusion

BCR gene (from chromosome 22) + ABL gene (from chromosome 9) = BCR-ABL gene

Normal function of BCR protein

coiled-coil domain is most important; involved in homotypic protein-protein interactions

Normal ABL function

highly regulated, non-receptor tyrosine kinase which regulates transcription; inhibits apoptosis

functional consequences of BCR-ABL gene fusion (3)

addition of coiled-coil domain (allows for homodimerization, can stimulate OWN catalytic activity), addition of kinase domain (retains ability to signal for cell growth AND inhibit apoptosis), loss of ABL N-terminus (causes BCR-ABL to be constitutively active)

what is the ONLY biomarker for CML?

the Philadelphia chromosome

what are three alternative diagnostic approaches for CML besides karyotyping?

Fluorescence in situ hybridization (FISH), Polymerase Chain Reaction (PCR), Nested PCR

FISH to detect translocation

denature chromosomes to yield single strands - large single-stranded DNA probes are synthetically generated with fluorescent tags that hybridize to chromosomes; a colour change to YELLOW indicates gene fusion

benefits of FISH

easy to interpret, quantitative (how often is signal observed), more sensitive than karyotyping, can be done on interphase cells

limitations of FISH

expensive, time consuming, need to find enough cancer cells to analyze

PCR to detect gene fusion

produce forward BCR and reverse ABL primer - if the genes are fused, a product will form

benefits of PCR

easy to read output, sensitive, rapid and economical, can use bone marrow or blood, does not require dividing cells

limitations of PCR

requires isolation of RNA (unstable), may miss some rare breakpoints

purpose of nested PCR

can increases sensitivity of regular PCR BUT also decreases specificity

What is the ‘poster child’ for CML treatment?

the drug imatinib mesylate

Imatinib Mechanism of Action

acts as a selective tyrosine kinase inhibitor (targets ABL catalytic site to limit cell growth)

qRT-PCR

used to identify the AMOUNT of biomarker in the CML patient; used when levels drop below detectable level for qualitative testing and for treatment monitoring

Complete Hematologic Response

no longer clinical evidence of disease

Complete Cytogenic Response

karyotyping and FISH are not sufficient anymore

Complete Molecular Response

can no longer detect via PCR (most sensitive test)

why does CML recurrence occur?

initial translocation occurs in HSCs, which are relatively resistant to treatment and capable of self-renewal

imatinib resistance mechanisms

missense mutations in the BCR-ABL catalytic site, which prevents imatinib binding site

Sensitivity Formula

True Positives/(True Positives + False Negatives)

Specificity Formula

True Negatives/(False Positives + True Negatives)

PPV Formula

True Positives/(True Positives + False Positives)

NPV Formula

True Negatives/(False Negatives + True Negatives)

Positive LR Formula

sensitivity/(1-specificity)

Negative LR Formula

(1-sensitivity)/specificity

Sensitivity Definition

the ability of a test to correctly identify people who have a specific disease/condition; uses columns

Specificity Definition

the ability of a test to correctly identify people who do not have the disease/condition

PPV Definition

the probability that a person who receives a positive test result actually has the disease

NPV Definition

the probability that a person who receives a negative test result truly does not have the disease