Libre Pathology talk:Study Group

Michael's thoughts on the exam

• I wrote it and passed it in 2012. I also did the American exam the same year and passed that.
• The pass rate for the FRCPC exam is pretty high.
  • 2009-2011 it was 96+/-3.9% for Canadian medical school grads on their first attempt.

Written

• I though it was picking at details. Some things are very relevant to practise... other less so.
  • The pocketbook version of Robbins covers most of it.

Practical (slide) exam

• You should know the answer almost immediately.
  • If you don't know, write something down and move on.
• It is set to broadly cover everything.
• If it isn't a spot diagnosis... it should not be on.
• Somethings are PGY2/PGY3 stuff. One should not overthink things.
• Anecdotally, the first impression is usually the right one.
  • I think one should stick with the first impression.

Gross exam

• Go with the most probable if you're uncertain.
• I worked through the Atlas of Gross Pathology with Histologic Correlation (see Pathology books for the reference).
  • I am not sure this is necessary... but I thought it was useful.
• Flickr.com/Google images has a lot to offer in this respect.
• Gross spot diagnosis.

Forensic exam

• I thought this was tricky... and I liked forensics.
• Residents that took the exam prior to me said the same.

Cytology exam

• Some of the cases have several images.
• I remember being confused... the first three images were from one case. I remember thinking... I have the same diagnosis three times.
• Like the forensics and gross sections - this section isn't too long. From an exam strategy point-of-view, this makes it less likely that a diagnosis is repeated.

Oral exam

• I think this is to test if you are safe and useful.
  • By "safe" I mean: knowing your limits and consulting with a colleague when appropriate.
  • By "useful" I mean: you don't need to consult on everything.
• The examiners ask a pre-determined list of questions.
  • Questions may depend on one another and, in fairness, they are told to redirect you.
    • Example: You see a lung biopsy with hyaline material... and you go down the fibrosis route-- but it is really amyloidosis.
      • The examiners will say something like "how would one work-up suspected amyloid?" or "lets assume this is amyloid..."
• If you're a Canadian resident, you cannot be examined by someone within your residency program.
• As far as I know, examiners are told to be stone-faced, i.e. show no emotion.
• Some of the cases were very straight forward.
• I didn't think anything was really exotic.

Michael (talk) 23:43, 25 October 2014 (EDT)

Short answer questions on genetics and molecular pathology.

These are some questions I came up with that are plausible to me... let me know if they are out to lunch.

Molecular Pathology Rotation Notes

UNIT 1

List three differences between DNA and RNA.
DNA (double stranded, thymine, deoxyribose, more stable; RNA single stranded, ribose, uracil

What are the three stop codons?
[UAA, UGA, UAG]]

Where does transcription begin?
promoters at the 5' end before the coding region

List 2 enzymes necessary for transcription and their function.
helicase, polymerase

List and describe three post transcription modifications of RNA.
Splicing, cappping, 3'polyadenylation,

Why is alternative splicing important?
Using the basic construction blocks of coding sequences allows a large variety of recombinations, more efficient coding (e.g. creating functions to call))

List three differences between somatic and germline mutations.
Somatic: not passed on to progeny, only tumour or particular tissue cells with mutation, Germline: passed onto progeny, all cells have mutation * unless mosaicism or chimerism

What is the difference between a missense and a non-sense mutation?
Missense the new base pair does not change the amino acid found in the protein at that location, non-sense changes the amino acid in the protein at that location

Define a frameshift mutation.
deletion of a non-multiple of 3 which causes all further trinucleotide combinations to no longer code for the correct amino acid, often results in a premature stop codon

Why are inversion mutations difficult to detect?
When the are smal, e.g. only a few base pairs

Describe the potential sequelae of a translocation mutation.
when a segment on one chromosome is transferred to another, make a gene non-functional or can result in a fusion gene

UNIT 2

Translate the following: c.1524_1527delCGTA.
a small deletion of CGTA between the 1524 and 1527 base pairs

List 5 features of SNPs.
[[Most common DNA sequence variation in humans, must occur in >=1% of a particular population, frequency of SNPs varies by groups, but responsible for >90% of human genetic variation, an can be found in any region of genome]]

Define a regulatory SNP versus a synonymous SNP?

What is the difference between a microstalellite and a minisattelite?
Microsatellite = stretches of DNA with sequences of 2-4 base pairs repeated a few dozen times (STRP), minisatellite = variable number of tandem repeats 10-100bp in lenght

Describe Hardy-Weinberg Equilibrium?
Mathematical probability function to describe allelic and genotype frequency in a random mating scenario

What factors can disrupt the H-W equilibrium?
non random mating, migration, genetic drift, founder effects, mutation, natural selection

What is linkage disequilibrium?
The closer two genes are together on the chromosome the more likely they are to be found toghether in a population, during meiosis some exchange of material happens between the two chromosomes

UNIT 3

What are the three major steps of PCR?
denaturing, primer annealing, strand extending

What is the hallmark of PCR?
The cycling at different temperatures, in the presence of key reaction components to traget and exponentially amplify a specific DNA target sequence

What factors affect the method of genotyping chosen?
throughput, type of variants that can be genotyped, equipment and costs, TAT, technical expertise, and multiplex ability

Define sensitivity, specificity, positive predictive value and negative predictive value.
Sensitivity = probability of a positive test in a disease, specificity = probability of a negative dest in a non-diseased patient

Define reproduciblity and accuracy of an analytical test.
Reproducability = probability of the test repeatedly producing the same reults in the same person, Accuracy = the degree to which the observed genotype matches the true genotype

Describe briefly Sanger sequencing.
DIdeoxynucleotides are used in a mix with deoxynucleotides, the Di*** terminate the chain, and so you get all possible lengths of chains so then you put them all in order and you can read (based on weight) which one is at each position

Describe briefly how Taqman automated genotyping is used for allele detection.
Microsatellite instability

How are DNA microarrays used to identify drug disposition or responses?
Microsatellite instability

UNIT 4

Describe the procedure for submitting FFPE slides for KRAS for colorectal cancer.
Microsatellite instability

Compare and contrast uniplex versus multiplex genotyping.
Microsatellite instability

Compare and contrast conventional vs massively parallel sequencing.
Microsatellite instability

What is multiplex ligation-dependent ligation (MLPA)?
Microsatellite instability

What is fragment analysis?
Microsatellite instability

Compare and contrast RT-PCR vs qRTPCR.
Microsatellite instability

What is MSI?
Microsatellite instability

What is methylation analysis?
Microsatellite instability

UNIT 5

What are the four test features required to be documented by the CLIA?
Microsatellite instability

What are "in vitro diagnostics" vs "laboratory developed tests"?
Microsatellite instability

What does validation mean?
Microsatellite instability

What are the four performance characteristics that need to be verified for FDA cleared/approved tests?
Microsatellite instability

What are the six performance characteristics that need to be verified for FDA cleared LDTs or modified FDA cleared/approved tests?
Microsatellite instability

UNIT 6

List the components of a molecular pathology report.
Microsatellite instability

Define analytical sensitivity and clinical sensitivity.
Microsatellite instability

What should be said in a report of a molecular test on a patient for residual disease if no previous positive assay was confirmed?
Microsatellite instability

Define ammended report versus addendum report.
Microsatellite instability

Whose responsibility is it to sythesize the test results with other clinico-pathological information?
Microsatellite instability

How long are cytogenetic reports required to be kept by CAP?
Microsatellite instability

What is the recommended process to use test results if an assay is not yet validated for clinical use?
Microsatellite instability

Give three examples of "grey areas" which warrant discretion of professionals involved to use a non-validated test?
Microsatellite instability

What reference standard is available for gene nomenclature?
Microsatellite instability

Create a table of the most common gene rearrangements associated with heme and soft tissue diseases.
Microsatellite instability

What is a "DNA fingerprint" and what can it be used for?
A method that examines multiple areas of short tandem repeats to identify paternity, mosaicism, chimerism, and identity in forensics cases

Robbins and Cotran Chapter 5 9th Edition:

MC cause of spontaneous abortion is ?
A demonstrable chromosomal abnormality.

1% of all newborn infants possess a gross chromosomal abnormality and 5% of people <25y present with
a genetic disease.

Mutation
permanent change in the DNA, if affect germ cells are transmitted to the progeny

List and describe 4 broad categories of human genetic disorders:

[[Disorders related to mutation sin single genes with large effects i. Usually follow classic Mendelian pattern of inheritance ii. Often highly penetrant (large proportion of pop with gene has disease) b. Chromosomal disorders i. Structural or numerical alterations in autosomes and sex chromosomes ii. Uncommon, high penetrance c. Complex multigenic disorders i. Interactions between multiple variant forms of genes and environmental factors (polymorphisms), poly genic means disease when many polymorphism present d. Single gene disorders with nonclassic patterns of inheritance (not mendelian) i. Disorders resulting from triplet repeat mutations ii. Mutations in mitochondrial DNA iii. Those influenced by genomic imprinting iv. Those influenced by gonadal mosaicism]]

List and describe the possible outcomes of a point mutation in a coding region?
[[a. Missense mutation – pt mutation changes amino acid code, conservative when the amino acid is preserved, non conservative when replaced with another amino acid, b. Nonsense mutation – makes a stop codon ]]

List and describe the possible outcomes of point mutation or deletion in a non-coding region.
[[a. Promoters/enhancers – interfere with binding of transcription factors, marker reduction or total lack of transcription, b. Introns – defective splicing > failure to make mature RNA > no translation]]

List and describe the possible outcomes of deletions and insertions.
[[a.Small coding: not multiple of three = frameshift, if multiple of 3 than add or del amino acids accordingly, often premature stop codon i. Tay Sachs disease: 4 base pair insertion in Hexosaminidase A gene ]]

List and describe the possible outcomes of trinucleotide repeat mutations.
[[a. Usually G&C, dynamic and increase during gametogenesis, “RNA stutters”,b. Fragile X – CGG 250-4000, Huntinton’s Disease ]]

List and describe three examples of inheritance of single gene mutations

[[a. AD – manifested in the heterologous state, one parent of index case is usually affected, males and females affected and both can transmit conditioni. De novo cases may not have affected parentii. Penetrance = fraction of people with gene who have the traitiii. Variable expressivity = those with mutant gene have variety of phenotypesiv. Often age of onset is delayed so can reproduce before die from diseasev. Biochem mechanisms1. Reduced production of a protein or dysfunctional/inactive protein2. Involved in regulation of complex metabolic pathyway subject to feedback inhibition3. Key structural proteins (collagen and cytoskeleton of RBC)a. May be a dominant negative , e.g. osteogenesis imperfecta4. Gain of function are rare, 2 formsa. Increased in proteins normal function (excess enzyme activity)b. Huntinton’s diseas (abn protein accumulates, toxic to neurons)b. ARi. Largest category – both alleles at a locus are mutated1. Expression is uniform, complete penetrance common, early onset, unaffected carrier family members, mostly enzymesc. X Linkedi. All sex linked, and almost all are recessive , if Y Chromosome affected usually infertile males > no progenyii. Male expression b/c hemizygous, daughter carriers with variable phenotype because of lionization of 2nd X e.g G6DPiii. Dominant . vitamin D resistant rickets]]

Stopped at P142

Molecular Genetic Diagnosis

List three basic molecular diagnostic techniques
[[a. Karyotyping, b. Southern blot, c. Sanger DNA sequencing, d. Polymerase chain reaction]]

CAP Molecular Diagnosis of Lung Cancer

List 5 treatment defining molecular transformation, the neoplasm, and the genetic alteration
[[1. 100% of CML: BRR-ABL > Imatinib, 2. 20% of Lung Adenocarcinoma: EGFR > Erlotinib/Gefitinib, 3. 25% Infiltrative ductal carcinoma of breast HER2>Trastuzumab, 4. 50% of Melanoma, BRAF v600E > PLX4032, 5. 4% of Lung Adenocarcinoma: ALK > Crizotinib]]

List and describe 5 areas of Genetic characaterization of tumours for personalized medicine
DNA mutations, DNA chromosomal alterations, mRNA and MiRNA profiling, Proteomics, DNA epigenetics

What fraction of Lung adenocarcinomas have no known detactable mutations
42%

What are the three most common molecular alterations of Lung Adenocarcinoma
KRAS 23%, EGFR 15%, TP53 5%

What is the two most common molecular alteration makes patients with EGFR mutations resistant to targetted therapies?
KRAS (primary) and T790M (primary and acquired)

List two EGFR kinase inhibitors.
Gefitinib/Iressa, Erlotinib/Tarceva

What are the three most common cancers associated with KRAS mutations?
Pancreatic 90%, Colon 50%, Lung NSCLC 30%

Why don't KRAS + tumours respond to Anti EGFR therapies?
KRAS is downstream from EGFR, so changing the function of EFGR would not have any effect on mutated KRAS

Explain the cost effectiveness of genetic testing for targetted therapies?
Most molecular tests cost $200-1000, vs one month of targetted therapy $2000-10000/month

What are the three most common cancers associated with BRAF mutations?
Melanoma 70%, Papillary Thyroid Carcinoma 50%, Ovarian serious carcinoma 30%, Colon cancer 10%, Hint Papillary architecture

Beta catenin/CTNNB1 expression is found with which histological pattern of lung adenocarcinoma?
[[Low grade adenocarcinoma of fetal type, poor px, <40yo, and has glycogen rich glandular formations, may occur in FAP patients]]

What is the most common ALK rearrangement found in NSCLC?
EML4-ALK (90% of the 13% of lung cancers found to due to ALK fusions)

List some pros and cons of ALK FISH.
[[Pros: commercial FDA approved probes available, not too expensive, moderately easy to disseminate screening, clinically validated, and failed tests on poorly preserved tissues are not reported as negative. Cons: need fish lab expertise (including pathologist and PhD), can be tricky if genes are close]]

List some pros and cons of ALK IHC.

CAP Molecular Diagnosis of AML

List 6 genes associated with Acute Myeloid Leukemia that have been identified by cloning translocation break points
RUNX1, RUNX1T1, PML, CBFB, ETV6, MLL

List the 5 main categories of classification of Acute Myeloid Leukemia
1. AML with recurrent genetic abnormalities, 2. AML with myelodysplasia-related changes, 3. Therapy related myeloid neoplasms, 4. AML, NOS, 5. Myeloid sarcoma

Give any three translocations identified in AML.
t(8,21), inv (16), t(15,17), t(9,11), t(6,9), inv(3), t(1,22), mutated NPM1, mutated CEBPA

What entities are fall under the AML, NOS classification?
[[ AML with minimal differentiation, AML without maturation, AML with maturation, Acute myelomonocytic leukemia, Acute monoblastic/monocytic leukemia, Acute erythroid leukemias (pure erythroid, erythroleukemia, erythroid/myeloid), Acute Megakaryoblastic leukemia, Acute basophilic leukemia, Acte panmyelosis with myelofibrosis]]