Pre-Assessment Review: Tools & Techniques

Dr. Jennifer Hunt

1. Formalin is which type of fixative?

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a. A cross-linking fixative b. A precipitation fixative c. A heavy metal fixative d. A decalcification fixative e. An acetic acid fixative

a. Formalin is a typical cross-linking fixative. These fixatives crosslink the proteins in cells, which includes the histones in the nucleus. The alcohol based fixatives are precipitating fixatives and the mercury and zinc containing fixatives contain heavy metals.

2. After which period of time can DNA no longer be obtained from routinely processed tissue blocks?

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a. 1 week b. 1 month c. 1 year d. 10 years e. There is no limit for properly stored material

e. Reasonable quality DNA has been obtained in tissue blocks as old and older than 25 years, with proper storage.

3. DNA in formalin fixed and paraffin embedded material is fragmented in to approximately what sized fragments?

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a. 10-20 basepairs b. 100-200 basepairs c. 300-400 basepairs d. 1000-2000 basepairs e. >3000 basepairs

c. DNA is fragmented and degraded secondary to fixation and paraffin embedding procedures. The typical fragment lengths are 300 to 400 basepairs.

4. Polymerase chain reaction was originally obtained from what source?

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a. E. coli b. Thermus aquaticus c. Streptococcus d. Candida e. It was manufactured

b. The original taq enzyme used in experiments of early PCR was from thermus aquaticus, which is an organism that thrives at high heat. This yielded a thermostable (or “heat activated”) polymerase that survived the high heat cycle denaturing the double stranded DNA.

5. In capillary electrophoresis, the product amount

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a. Can be calculated b. Can be estimated for a single peak c. Can be estimated only as a comparison between peaks d. Cannot be estimated or calculated e. Is not ever needed

c. Capillary electrophoresis is “semi-quantitative”. Two peaks within one electropherogram can be compared to one other to estimate the relative quantity of PCR product. However, since it is not truly quantitative, absolute calculations cannot be made.

6. Why is it usually difficult to use PCR from DNA to test for a translocation

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a. Because the breakpoints frequently occur in large introns b. Because the breakpoints frequently occur in large exons c. Because the breakpoints frequently involve short tandem repeats d. Because the breakpoints frequently involve many different inconsistent partners for both genes e. Because no RNA can be obtained from typical samples

a. Translocation breakpoints typically occur in large introns and may not be highly clustered. Because of this, using DNA to assess for translocations is problematic. Typically, it much better to design assays using RNA as the starting material, where the introns are spliced out.

7. In a sequencing reaction using the Sanger dideoxynucleotide chain terminator assay, what identifies each different nucleotide in the sequence?

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a. The height of the peak b. The time it takes for the reaction c. The amount of the product d. The color of the fluorescent label e. The melting temperature

d. Each nucleotide is labeled using a different fluorescent label, and thus the sequence can be read by looking at the fluorescence of each peak.

8. A significant pitfall in Sanger dideoxynucleotide chain terminator sequencing in tumor testing is

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a. A relatively low sensitivity when mixed cell populations are present b. Auto-fluorescence of the sample c. Extremely poor yield of DNA from optimally fixed samples d. PCR inhibitors, such as xylene and formalin e. Poor binding of the primer in mutated samples

a. In tumor samples, the tumor cells are rarely homogeneous. There can be significant contaminating normal cells, even with the best microdissection technique. Because oncogene mutations are usually heterozygous, sensitivity can be a major issue in traditional gene sequencing

9. Fluorescent in situ hybridization (FISH) and loss of heterozygosity (LOH) analysis can both be used to identify genes with

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a. Amplification mutations b. Translocation mutations c. Loss mutations d. Over-expression e. Point mutations

c. FISH and LOH are both used to detect allelic imbalance caused by loss mutations.

10. Amplification mutations are best detected using

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a. Polymerase chain reaction b. Reverse-transcriptase – polymerase chain reaction c. Fluorescent in situ hybridization d. Loss of heterozygosity e. Sequencing assay

c. FISH is the only assay listed that can easily be used to detect amplification mutations.

11. In a routine fluorescent in situ hybridization using routine paraffin embedded tissue sections, one significant pitfall is

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a. Truncation artifact from cutting of sections b. Artifact mutations induced by routine fixation c. Poor cell morphology caused by routine fixation d. Hybridization inhibitors, such as xylene and formalin e. Extremely poor quality DNA from optimally fixed samples

a. When tissue sections are cut at 4 or 5 microns, some nuclei are inevitably cut through and some of the chromosomes will not be represented in the section. This leads to truncation artifacts, which can be especially problematic for deletion mutation assays.

12. Fluorescent in situ hybridization for translocation analysis when only one partner gene is consistent or known will likely use what type of probes?

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a. Fusion probes b. Centromeric probes only c. Viral subtype specific probes d. Break-apart probes e. None; the best assay will be reverse transcriptase – PCR

d. The most common probes for translocation analysis in tumors are break-part probes. Though fusion probes can also be designed, they would not be practical for scenarios where the second partner gene is inconsistent or not known.

13. What type of array can be used to detect allelic imbalances across the genome

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a. Tissue microarray b. Whole genome expression array c. Single nucleotide polymorphism array d. Targeted expression array e. None; the best assay will be a loss of heterozygosity panel

c. The only array on the list that can be used to detect allelic imbalances is single nucleotide polymorphism analysis. Comparative genomic hybridization could also be used (but is not listed). LOH can also be used, but constructing panels to perform across the genome would be very labor intensive and difficult.

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Pre-Assessment Review: Molecular Oncology

Dr. Hunt and Dr. Samowitz

1. Lynch syndrome is associated with

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a. Germline mutations in the APC gene b. Germline mutations in mismatch repair genes c. Sessile serrated polyps d. Thyroid cancer e. Colorectal cancers with a high percentage of BRAF mutations

b. Lynch syndrome is caused by a germline mismatch repair gene mutation, either in MLH1, MSH2, MSH6 or PMS2. Sessile serrated polyps have been hypothesized to be the precursor for sporadic, not Lynch syndrome associated, microsatellite unstable colorectal cancers. Thyroid cancers may occur in FAP, not Lynch syndrome. BRAF mutations are a fairly good indicator that an unstable colorectal cancer is sporadic; they have only rarely been reported in Lynch syndrome-associated tumors.

2. Microsatellite instability in a tumor indicates

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a. The presence of Lynch syndrome b. Defective mismatch repair c. MLH1 promoter methylation d. BRAF V600E mutation e. Loss of heterozygosity

b. Microsatellite instability, the expansion or contraction of microsatellite repeats, is caused by the normal slippage across such repeats during DNA replication plus a defective mismatch repair system so that these contractions or expansions are not corrected. It occurs in both 10-15% of sporadic colorectal cancers and associated with Lynch syndrome. MLH1 promoter methylation and/or BRAF mutations typically occur in sporadic, not Lynch syndrome-associated, colorectal cancers.

3. Copy neutral loss of heterozygosity (LOH) may be detected by

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a. FISH b. Single nucleotide polymorphism (SNP) arrays c. Comparative genomic hybridization d. Mismatch repair gene immunostaining e. Flow cytometry

b. Since copy neutral LOH results in no net loss of chromosomal material, it can only be detected by a technique, like a SNP array, which has the potential to distinguish different alleles on homologous chromosomes. Microsatellite repeat analysis can also be used to detect copy neutral LOH.

4. An endometrial cancer shows loss of MLH1 and PMS2 immunostaining. Which of the following statements is true?

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a. The presence or absence of a BRAF mutation helps determine whether the tumor is sporadic or Lynch-associated. b. If the patient is over 60, the tumor is definitely sporadic. c. MLH1 methylation would indicate that the tumor is probably sporadic. d. The most likely cause of this IHC profile is a germline PMS2 mutation. e. Endometrial cancers are not part of Lynch syndrome.

c. MLH1 methylation is the mechanism underlying most cases of sporadic microsatellite instability. Sporadic microsatellite unstable tumors of the endometrium, in contrast to those of the colorectum, usually do not harbor BRAF mutations. Older age does not necessarily rule out Lynch syndrome. A germline PMS2 mutation is most commonly associated with isolated loss of PMS2 expression. Endometrial cancers are the second most common Lynch syndrome-associated cancer

5. Long range PCR is used as a first step in sequencing PMS2 because of

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a. Numerous intronic mutations b. Numerous missense mutations c. Numerous knockout mutations d. Numerous pseudogenes e. Numerous in/del mutations

d. The presence of numerous PMS2 pseudogenes complicates evaluation of this gene. Long range PCR primers located in DNA specific to the gene is used as a first step to enrich DNA for the actual gene rather than the pseudogenes. The long range PCR products can then be used for exon by exon sequencing.

6. MSH2 methylation is

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a. Commonly associated with sporadic microsatellite unstable tumors

b. Typically due to a germline three prime deletion of EPCAM

c. Associated with isolated loss of MSH2 expression in an associated tumor

d. Usually not inherited e. Commonly associated with BRAF mutations.

b. A three prime deletion of EPCAM leads to transcription read through into MSH2 and subsequent promoter hypermethylation. This would be associated with loss of MSH2 and MSH6 in an associated tumor. Since this is a germline mutation, it is inherited (as opposed to most cases of germline MLH1 methylation). Acquired MLH1 methylation is the basis for most sporadic microsatellite unstable tumors and is commonly associated with BRAF mutations.

7. EGFR inhibitors should not be used in an individual whose colon cancer shows

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a. Microsatellite instability b. MLH1 methylation c. A KRAS codon 12 mutation d. EGFR overexpression by FISH e. Wild type BRAF

c. Codon 12 and 13 mutations in KRAS lead to constitutive activation of the EGFR pathway downstream of EGFR. Such tumors will not respond to EGFR inhibitors. There is some evidence that mutated BRAF may also lead to a lack of response. EGFR expression in colorectal cancer is probably not relevant to inhibitor therapy. MLH1 methylation and microsatellite instability are also not relevant to this treatment.

8. Which of the following statements regarding GISTS is false?

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a. The most common site of mutation for both KIT and PDGFRA is the juxtamembrane domain.

b. KIT and PDGFRA mutations are mutually exclusive. c. Homozygous KIT mutations are typically associated with more aggressive tumor behavior d. Exon 9 mutations require a higher dose of imatinib e. KIT deletions and insertions are in frame

a. The juxtamembrane domain of KIT (exon11) is the most common site of mutation for this gene. The most common site of mutation for PDGFRA is exon 18, the kinase II domain.

9. Which of the following statements regarding GISTS is true?

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a. If the initial mutation is in KIT, secondary resistance develops due to a mutation in PDGFRA.

b. Secondary resistance often develops in the tyrosine kinase domain of KIT. c. Secondary mutations most commonly develop in tumors

with primary exon 9 KIT mutations. d. Secondary mutations commonly occur in wild type tumors. e. Wild type GISTS or GISTs with PDGFRA p.Asp842Val mutations respond well to imatinib.

b. After therapy with imatinib, a tumor with a KIT mutation often develops secondary resistance due to a mutation in the tyrosine kinase domain. Secondary resistance develops in the same gene (and the same allele) as the originally mutated gene. Secondary mutations most commonly develop in tumors with exon 11 KIT mutations. Secondary mutations are not common in treated wild type tumors. Neither wild type GISTS nor GISTs with PDGFRA p.Asp842Val mutations respond well to imatinib.

10. KIT mutations are associated with

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a. GIST in NF1 b. GISTS in Carney’s triad c. GISTS in Carney-Stratakis syndrome d. Familial GISTS e. Most pediatric GISTS

d. Most pediatric GISTS and GISTS associated with NF1, Carney’s triad, and Carney-Stratakis syndrome are wild type for KIT and PDFGRA; GISTs associated with Carney-Stratakis syndrome have mutations in succinate dehydrogenase subunits SDHB, SDHC or SDHD. Familial GISTS may be associated with KIT or PDGFRA mutations.

11. With respect to melanomas, BRAF mutations

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a. Are most common in mucosal melanomas b. Can be used to determine whether a melanocytic

lesion is benign or malignant. c. Are most commonly the V600E alteration d. Indicate sensitivity to imatinib therapy e. Are commonly associated with microsatellite

instability

c. The most common BRAF mutation in melanoma is the V600E alteration; this mutation is not associated with MSI in melanomas, as melanomas do not typically exhibit MSI. Both benign nevi and malignant melanomas commonly harbor BRAF mutations, so BRAF mutations cannot be used to determine malignancy. BRAF mutations occur in about one half of cutaneous melanomas; they are less common in acral lentiginous and mucosal melanomas. BRAF-mutated melanomas are sensitive to vemurafenib, not imatinib. KIT mutated melanomas can respond to imatinib therapy.

12. Select the false statement: BRAF-directed therapy

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a. Works against melanomas, regardless of the mutational status of BRAF

b. Commonly leads to resistance c. May lead to cutaneous squamous cell carcinomas d. Is contraindicated for BRAF wild type tumors e. Is most commonly used for the BRAF V600E

mutation. a. BRAF-directed therapy is contraindicated for BRAF wild type tumors, as it does not work and may lead to paradoxical activation of MAPK signaling and accelerate disease progression. The BRAF V600E mutation is the most common mutation seen in melanoma and probably responds best to anti-BRAF therapy. The development of resistance to a BRAF inhibitor and the side effect of cutaneous squamous cell carcinomas are common.

13. Select the false statement.

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a. Uveal melanomas are commonly associated with GNAQ or GNA11 mutations.

b. KIT mutations may occur in uveal melanomas c. KIT mutations are seen in acral lentiginous and

mucosal melanomas d. PDGFRA mutations are common in mucosal

melanomas e. Most melanomas with KIT mutations respond to

imatinib

d. KIT mutations, not PDGFRA mutations, are relatively common in mucosal melanomas.

14. Isocitrate dehydrogenase (IDH) mutations

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a. Indicate astrocytic lineage b. Are useful diagnostically to identify an

infiltrative glioma c. Are mostly seen in primary GBM’s d. Are usually in IDH 2 in gliomas e. Are associated with a shorter survival for

gliomas

b. In an astrocytic proliferation, the presence of an IDH mutation indicates an infiltrative glioma. Both astrocytomas and oligodendrogliomas commonly have IDH mutations, and mutations are most commonly seen in IDH1. These mutations are seen in >90% of secondary GBM’s and are not commonly seen in primary GBM’s. Mutations are associated with a longer survival.

15. Attenuated FAP is

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a. Associated with missense rather than nonsense or frameshift mutations

b. Due to bi-allelic APC mutations c. Associated with mutations at the extreme

five prime or three prime ends of the gene. d. Associated with serrated polyposis e. Secondary to promoter methylation

c. Attenuated FAP is characterized by mutations at the extreme five prime or three prime ends of the gene. The mutations, like all (or nearly all) APC mutations are nonsense or frameshift mutations. It is thought that a restart after the five prime mutation or a slightly shortened protein due to the three prime mutation leads to a partly functional APC protein and, therefore, the attenuated phenotype. Attenuated FAP is associated with adenomas, not sessile serrated polyps.

16. All of the following are true regarding bi-allelic germline mismatch repair mutations except

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a. PMS2 and MSH6 are over-represented. b. Childhood hematologic malignancies and brain

tumors may occur c. NF-1 like skin findings are often present d. Relatives with mono-allelic mutations may not have

to be screened as closely as individuals with typical Lynch syndrome.

e. Affected individuals are at no increased risk for typical Lynch syndrome tumors.

e. Individuals with bi-allelic germline mismatch repair syndrome who survive long enough are at risk for typical Lynch syndrome tumors, such as colon cancer at a mean age of 16 (30 years earlier than the average onset for typical Lynch syndrome).

17. Select the false statement. MYH-associated polyposis is associated with

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a. An attenuated adenomatous polyposis phenotype b. Homozygous or compound heterozygous hotspot

missense mutations. c. Microsatellite instability d. G:C to T:A transversion mutations e. A recessive pattern of inheritance

c. MYH-associated tumors are microsatellite stable.

18. Gardner’s syndrome is associated with mutations in

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a. APC b. MUTYH c. Bi-allelic mismatch repair genes d. PTEN e. SMAD4

a. Gardner’s syndrome is due to germline APC mutations.

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1. Knudson’s hypothesis helped explain what type of tumor related genes

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a. Oncogene point mutations b. Tumor suppressor gene mutations c. Tumor associated viruses d. Gene amplifications e. Translocation mutations

b. Knudson’s hypothesis was the first description of tumor suppressor genes and their mutations. It was based on Knudson’s work with hereditary retinoblastoma. He proposed that these genes required two genetic hits for tumorigenesis to occur.

2. The most common two hits to the genes that are described by Knudson’s hypothesis have long been considered to be:

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a. A point mutation on one copy and an amplification on the second copy

b. Promoter methylation on one copy and a translocation on the second copy

c. A point mutation on one copy and a deletion on the second copy

d. Both copies with point mutations e. Both copies with promoter methylation

c. In tumor suppressor genes, tumor related alterations include point mutations, loss mutations and promoter methylation. But a point mutation and loss mutation are thought to be the most common combination.

3. Which of the following is not an assay that can detect allelic imbalance

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a. Sequencing b. Loss of heterozygosity analysis c. Fluorescence in situ hybridization d. Comparative genomic hybridization e. Single nucleotide polymorphism array

a. Allelic imbalance can be either due to a deletion or to amplification. The only assay that cannot detect these imbalances of the above is the sequencing reaction.

4. Which of the following genes has been described with mutations in both sporadic and hereditary parathyroid carcinomas?

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a. KRAS gene b. BRAF gene c. RET gene d. HRPT2 gene e. APC gene

d. Hyperparathyroidism-jaw-tumor syndrome includes parathyroid carcinoma and facial bone tumors. The gene responsible for this is the HRPT2 gene. This is a tumor suppressor gene, where one copy has an inherited mutation and the second copy acquires a second hit. In sporadic parathyroid carcinoma, mutations in this gene are also seen, and are responsible for loss of expression of the protein product.

5. Tumor oncogenes typically have all of the following tumor-related mutation except

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a. Amplification mutations b. Translocation mutations c. Point mutations d. Deletion mutations e. Intrachromosomal rearrangements

d. Tumor oncogene mutations are not usually deletion mutations. The other types of events are all common in oncogenes.

6. In lung adenocarcinomas, which of the following exons of the ErbB1 (EGFR) gene are most often involved in known mutations?

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a. 16, 17, 18, 19 b. 17, 18, 19, 20 c. 18, 19, 20, 21 d. 19, 20, 21, 22 e. 20, 21, 22, 23

c. The most common mutations in ErbB1 (EGFR) gene in adenocarcinoma of the lung occur in the tyrosine kinase domains, which are encoded in the region of exons 18-21.

7. Which of the following are mutations in lung cancer that are associated with responsiveness to specific targeted agents?

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a. Translocation involving the ALK gene b. Amplification in the MET gene c. A point mutation in exon 19 of the ErbB1 (EGFR)

gene d. A point mutation in codon 600 of the BRAF gene e. A point mutation in codon 12 of the KRAS gene

a. The EML4/ALK translocation is the only one listed that confers sensitivity or responsiveness to a targeted agent. The other mutations listed are typical resistance mutations.

8. What is the most common mutation found in papillary thyroid carcinoma

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a. KRAS mutation b. BRAF mutation c. RET mutation d. RET/PTC translocation e. PPAR /PAX8 translocation

b. The most common mutation in papillary carcinoma is the V600E mutation in the BRAF gene. It is found in approximately 50% of conventional papillary carcinomas.

9. In cytology specimens which are interpreted using the Bethesda classification, which category of tumor is most likely to have the mutation associated with papillary carcinoma?

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a. Benign b. Atypia of undetermined significance (AUS) for

follicular lesion of undetermined significance (FLUS) c. Suspicious for malignancy d. Non-diagnostic or inadequate e. None of the above

c. The rate of positivity for BRAF varies by diagnostic category. It is lowest the benign and the AUS/FLUS categories and highest in the suspicious for malignancy category.

10. All of the following salivary gland tumors have been shown to have translocations, except:

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a. Mucoepidermoid carcinoma b. Adenoid cystic carcinoma c. Mammary analog secretory carcinoma d. Acinic cell carcinoma e. Hyalinizing clear cell carcinoma

d. Acinic cell carcinoma is not currently known to have any recurrent or consistent translocations. The other tumors in this list have all been shown to have specific translocations.

11. Which anatomical subsites is most common for HPV-associated head and neck squamous carcinoma?

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a. Oral tongue b. Tonsil and tongue base c. Nasopharynx d. Glottis e. Supraglottis

b. HPV-associated head and neck squamous carcinomas occur most frequently in the oropharynx, which includes the tonsil and tongue base. The other sites are very uncommon for this tumor.

12. Which subtype of HPV is most commonly found in HPV-associated head and neck squamous carcinoma?

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a. HPV 6 b. HPV 11 c. HPV 16 d. HPV 18 e. HPV 33

c. HPV 16 accounts for most of the HPV-associated head and neck squamous carcinoma.

13. Which of the following immunohistochemical stains can be used as a strong surrogate marker for HPV-associated head and neck squamous carcinoma?

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a. p53 b. p63 c. p16 d. Retinoblastoma e. Cyclin D1

c. p16 is over-expressed in HPV-associated head and neck squamous carcinomas, secondary to transcriptionally active virus and the expression of viral proteins. Although cyclin D1 and p53 can be over-expressed in head and neck squamous carcinomas, these are typically smoking related tumors, not virus induced tumors. .

14. Molecular subtyping of breast cancers

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a. Is currently used in routine clinical diagnostic practice

b. Is not currently used in routine clinical diagnostic practice

c. Is used for deciding therapy d. Cannot be approximated by any other diagnostic

modality e. Has no clinical significance

b. Although molecular subtyping of breast cancer may have clinical significance, it has not become a routine clinical test in diagnostic practice. There is a set of protein markers that can be used to approximate the molecular subtype.

Association for Molecular Pathology 9650 Rockville Pike

Bethesda, MD 20814

AMPEducation@amp.org

www.amp.org

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