TP53 SEQUENCING FOR LI-FRAUMENI SYNDROME AND SOMATIC MU
Label Name: TP53 LI-FRA
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6871 
Last Review:  3/27/2017 11:03:03 PM
Specimen Type
  Blood
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 3  ML
Label Reminders
  Be sure to include patient's name, history #, date and time of collection, and collector's initials.
Collection Notes
  All:
  • Formalin Fixed Paraffin Embedded Tissue: The laboratory can receive either a paraffin embedded tissue block or four freshly cut (within one week)5uM thick unstained slides containing 3 to 20 square mm of tissue. Unstained slides should be accompanied by an H&E stained slide for histologic evaluation (acquired mutation detection).

    Peripheral Blood: One lavender-top EDTA tube (minimum of 3 mls) is required for testing. Forward unprocessed peripheral blood promptly to the laboratory at ambient temperatures (germline mutation detection). THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.
 
Transport
  Deliver peripheral blood specimens to lab at ambient temperature. If there is a delay of more than 24 hours in delivery, refrigerate the sample. DO NOT FREEZE.

Formalin fixed paraffin embedded tissue blocks and slides can be sent to the lab at ambient temperature.
Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values
TP53 LI-FRA
Methodology
  This assay uses PCR amplification followed by Sanger DNA sequencing to detect mutations in the TP53 tumor suppressor gene. The coding regions and intron/exon junctions of TP53 exons 4 through 11 are amplified from purified genomic DNA by PCR. The primers used for PCR contain M13 universal primer "tails" at their 5' ends, and have 3' ends that are homologous to their genomic target sequence. PCR products are treated with an exonuclease/phosphatase mixture (ExoSAP-IT) and sequenced using universal M13 forward and reverse primers with the Big Dye Terminator v3.1 Cycle Sequencing Kit. These products are purified with the Big Dye XTerminator Purification Kit and resolved using the ABI 3130xl Genetic Analyzer. Data is analyzed by the ABI Data Collection software v3.0, Sequencing Analysis software 5.2 and SeqScape software v2.6.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
   
   
Clinical Significance and Interpretive Data
    BACKGROUND:

Mutations of the TP53 tumor suppressor gene are the most common genetic alteration in human cancer. They are found in approximately 50% of all tumors. The spectrum of TP53 mutations consists predominantly of missense mutations, but also includes nonsense mutations, deletions, insertions, duplications, and splice site mutations. Greater than 99% of these mutations fall between exons 4 and 11. Three hot spots at amino acids 175 in exon 5, 248 in exon 7, and 273 in exon 8 represent the most frequent sites of mutation across various types of cancer. Sequencing of exons 4 through 11 (and their intron/exon borders) makes it possible to detect missense and nonsense mutations, as well as small deletions, insertions, duplications, and splice site mutations in a single assay.

CLINICAL SIGNIFICANCE AND UTILITY:

Characterization of the mutation status of TP53 can aid in clinical diagnosis, provide prognostic value, and influence treatment for cancer patients. More specifically, in CNS tumors derived from glial cells, the presence of a mutated TP53 is predictive of an astrocytic phenotype and a poor prognosis for patients. The presence of acquired mutations in TP53 in patients with chronic lymphocytic leukemia (CLL) is an independent predictor of rapid disease progression. Patients without acquired TP53 mutations tend to have a better prognosis.

Finally, sequencing for germline mutations in TP53 can be used to diagnose Li-Fraumeni syndrome (An autosomal dominant tumor syndrome associated with an increased risk of breast cancer, sarcomas and brain tumors).

INTERPRETATION:

Coding and flanking intronic sequences of the TP53 gene are compared to a reference sequence (GenBank accession no. U94788). Sequence changes found in these regions will be reported as (1) known disease-causing mutations, (2) a mutation previously unreported, but of the type expected to be disease causing (3) sequence variation of uncertain clinical significance, or (4) benign polymorphisms. Variants of uncertain clinical significance may require additional studies including gene sequencing of other family members or other functional studies. All sequence changes, with the exception of benign polymorphisms, are confirmed by reamplification and resequencing of the relevant exon(s). Several TP53 gene mutation databases exist, including:

• International Agency for Research on Cancer (IARC)
• Human Genome Mutation Database
• The Universal Mutation Database
• Sanger Catalogue of Somatic Mutations in Cancer (COSMIC)

In the event that a sequence change is detected, these databases are searched to determine whether the sequence change is a previously reported mutation or polymorphism or a novel sequence change with unclear significance. At the discretion of the director, the significance of previously unreported sequence change may be inferred based on the type of the variant, its position in the gene, and its effect on the amino acid sequence. For additional information, or for help interpreting the results of this test, please contact the Clinical Molecular Diagnostics Laboratory.

REFERENCES:

Dicker F et al. The detection of TP53 mutations in chronic lymphocytic leukemia independently predicts rapid disease progression and is highly correlated with a complex aberrant karyotype. Leukemia. 2009; 23:117-124

Mclendon RE, Herndon II JE, West B, et. al. Survival analysis of presumptive prognostic markers among oligodendroglioma. Cancer. 2005;104:1693-9.

Soussi T, Be´roud C. Significance of TP53 mutations in human cancer: a critical analysis of mutations at CpG dinucleotides. Hum Mutat. 2003;2:192–200.
   
Indications
    To identify somatic TP53 mutations to aid in the classification and management of brain tumors, CLL, and AML.

Prognostic testing in patients with chronic lymphocytic leukemia with an intact chromosome 17.

Testing for for germ-line TP53 gene mutations in patients suspected of having Li-Fraumeni syndrome.
   
Limitations
    The sensitivity and specificity of DNA sequencing is high for the detection of nucleotide base changes, small deletions, and insertions in the regions analyzed. However, this assay may not detect an acquired mutation which is present below the 30% detection limit (i.e., mutant cell population of < 30%). Only the coding regions of the TP53 gene and immediate flanking intronic sequences were examined. Changes in the promoter region, farther into the introns, or in other non-coding regions of the gene would not be detected. Mutations in genes other than TP53 would not be identified. Large deletions, duplications, multiple exon insertions, sequence alterations adversely affecting primer binding, and complete deletion of one allele may not be identified using these methods. Poor genomic DNA quality or quantity, the presence of PCR inhibitors, and mutations or polymorphisms in the PCR primer binding sequences can result in un-interpretable or (rarely) inaccurate results.

Multiple factors can contribute to prognosis in patients with CLL including, clinical stage, chromosomal deletions (del 17p), IgH mutation status and expression of prognostic markers including ZAP-70 and CD38. Thus, this assay is intended for use as an aid in determining patient prognosis, but is not a substitute for a complete pathologic and clinical evaluation. Similarly, these results are not intended to be used as sole criteria for the diagnosis of Li-Fraumeni Syndrome and are not a substitute for a physician's judgment and clinical experience.
   
Test Synonyms
  Synonym(s): CLL
Synonym(s): Li-Fraumeni
Synonym(s): P53
Synonym(s): TP53
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: