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Colon carcinoma and three polyps were resected in a right hemicolectomy of a 33-year-old man. Without a family history, the man's age and the location of the tumor warranted testing for hereditary nonpolyposis colorectal carcinoma. Histo-logical staining was negative for MSH2 protein. Paraffin sections and a blood sample were submitted

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to the molecular pathology laboratory for microsatellite instability testing. DNA was isolated from tumor cells dissected from four paraffin sections and from the patient's white blood cells. Both were amplified at the five microsatellite loci recommended by the National Cancer Institute. Results are shown below:

BAT25 BAT26 D17S250 D2S123 D5S346

■ MSI analysis of normal cells (N, top row) and tumor cells (T, bottom row) at the five NCI loci.

A 23-year-old college student was sent to the student health office with a painful bruise that persisted for several weeks. He was referred to the local hospital, where the physician ordered a bone marrow biopsy and a CBC. His white blood cell count was 28,000/^L; red blood cells, 2 million/^L; hemoglobin 8, hematocrit 20, platelets, 85,000/^L. Neu-trophils were 7%, lymphocytes were 5%, and blasts were 95%. The pathologist who examined the bone marrow biopsy requested flow cytometry analysis of the aspirate. The cells expressed 84% CD20, 82% CD34, 92% HLA-DR, and 80% CD10/CD19. The results of these tests indicated a diagnosis of acute lymphoblastic leukemia. A blood specimen was sent to the cytogenetics laboratory for chromosomal analysis. Twenty metaphases examined had a normal 46,XY chromosomal complement. Interphase FISH was negative for t(9;22) in 500 nucleii.

1. What are the two important checkpoints in the cell division cycle that are crossed when the regulation of the cell division cycle is affected?

2. An EWS-FLI-1 mutation was detected in a solid tumor by RT-PCR. Which of the following does this result support?

a. Normal tissue b. Ewing's sarcoma c. Inherited breast cancer d. Microsatellite instability

3. Mutation detection, even by sequencing, is not definitive with a negative result. Why?

4. A PCR test for the bcl-2 translocation is performed on a patient with suspected follicular lymphoma. The results show a bright band at about 300 bp for this patient. How would you interpret these results?

5. Which of the following misinterpretations would result from PCR contamination?

a. False positive for the t(15;17) translocation b. False negative for the t(15;17) translocation c. False negative for a gene rearrangement

6. After amplification of the t(12;21) breakpoint by RT-PCR, the PCR products along with the proper molecular weight standard were loaded and resolved on an agarose gel. What might be the explanation for each of the following observations when the gel is exposed to ultraviolet light? (Assume that positive and amplification controls and a reagent blank control are included in the run.)

a. The gel is blank (no bands, no molecular weight standard)

b. Only the molecular weight standard is visible c. The molecular weight standard is visible; there are bands in every lane at 200 bp, even in the reagent blank lane

7. What is observed on a Southern blot for gene rearrangement in the case of a positive result?

a. No bands b. Germline bands plus rearranged bands c. Smears d. Germline bands only

8. Cyclin D1 promotes passage of cells through the G1 to S checkpoint. What test detects translocation of this gene to chromosome 14?

a. t(14;18) translocation analysis (BCL2, IGH)

b. t(15;17) translocation analysis (PML/RARA)

c. t(11;14) translocation analysis (BCL1/IGH)

d. t(8;14) translocation analysis (MYC/IGH)

9. Why is the Southern blot procedure superior to the PCR procedure for detecting clonality in some cases?

a. Southern blot requires less sample DNA than does PCR

b. The PCR procedure cannot detect certain gene rearrangements that are detectable by Southern blot c. Southern blot results are easier to interpret than PCR results d. PCR results are not accepted by the College of American Pathologists

10. Interpret the following results from a translocation assay.

Target -

Are the samples positive, negative, or indeterminate? Sample 1: Sample 2: Sample 3:

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