Hereditary Endometrial Cancer Sequencing and Deletion/Duplication Panel
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and Del/Dup via NGS
|Test Code||Test Copy Genes||CPT Code Copy CPT Codes|
|5457||BRCA1 and BRCA2||81162||Add|
|Full Panel Price*||$790.00|
|Test Code||Test Copy Genes||Total Price||CPT Codes Copy CPT Codes|
|5457||Genes x (12)||$790.00||81162, 81292, 81294, 81295, 81297, 81298, 81300, 81317, 81319, 81321, 81323, 81403, 81405, 81406, 81479(x7)||Add|
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The great majority of tests are completed within 28 days.
Compared to other familial cancers, there have been relatively few studies investigating the familial risk of endometrial cancer (Gayther and Pharoah. 2010. PubMed ID: 20456938). Overall, Lynch syndrome is attributed to pathogenic variants predominantly in the MLH1, MSH2, MSH6, and PMS2 genes in approximately 50%, 40%, 7%-10% and < 5% of cases, respectively (Kohlmann and Gruber. 2012). The majority of these variants are single nucleotide substitutions or small insertions and deletions. Missense, nonsense and splicing EPCAM mutations are involved in congenital tufting enteropathy (Human Gene Mutation Database), whereas EPCAM deletions account for 1-3% of Lynch syndrome cases (Kohlmann and Gruber. 2012). Large deletions and genetic rearrangements account for 20%, 5%, 20%, 7%, and 100% of identifiable pathogenic variants in the MSH2, MLH1, PMS2, MSH6, and EPCAM genes respectively (Kohlmann and Gruber. 2012).
Endometrial cancer (EC) is the 6th most common diagnosed cancer among women worldwide. The vast majority of cases are sporadic, however it is estimated that 2%-5% are familial and linked to germline variants in genes associated with mismatch repair and Lynch syndrome (O'Hara and Bell. 2012. PubMed ID: 22888282). Approximately 75% of ECs are diagnosed in the early stages of tumor development, with a reported average survival rate of 75% (Siegel et al. 2013. PubMed ID: 23335087). Epidemiological studies have reported Caucasian women have a 2.9% lifetime risk of developing EC, compared to their African-American counterparts with a reported 1.7% lifetime risk (Burke et al. 2014. PubMed ID: 24905773). In women with a family history of Lynch Syndrome (LS), EC incidence is greater than or equal to that of colorectal cancer. In many cases, endometrial or ovarian cancer presents as the woman’s first malignancy (Aarnio et al. 1999. PubMed ID: 10188721; Vasen et al. 1999. PubMed ID: 10348829; Watson et al. 2008. PubMed ID: 18398828; Gayther and Pharoah. 2010. PubMed ID: 20456938).
The predominant EC histotypes are endometrioid, serous, and clear cell; however more rare carcinosarcomas, mucinous carcinomas, squamous cell carcinomas, and transitional cell carcinomas histotypes have been reported (O'Hara and Bell. 2012. PubMed ID: 22888282; Acharya et al. 2005. PubMed ID: 16321764; Dedes et al. 2011. PubMed ID: 21221135). Endometrioid ECs are estrogen-dependent tumors (Type I tumors) that are low-grade and diagnosed early in tumor progression. Surgical intervention is curative in most cases (5 year survival rate is ~90%). Established risk factors for endometrioid ECs are associated with unopposed estrogen exposure associated with obesity, nulliparity, early age of menarche, late age at menopause, and estrogen therapy in post-menopausal women (O'Hara and Bell. 2012. PubMed ID: 22888282). Serous and clear cell ECs are estrogen-independent (Type II tumors) that predominantly arise in post-menopausal women with no clear associated risk factor(s) besides increased age. Serous and clear cell histotypes have a worse prognosis than their endometrioid EC counterparts, accounting for 40-50% of all deaths from endometrial cancer, but only represent ~10-20% of cases (O'Hara and Bell. 2012. PubMed ID: 22888282; Hamilton et al. 2006. PubMed ID: 16495918, Hamilton et al. 2008. PubMed ID: 18197002; Setiawan et al. 2013. PubMed ID: 23733771).
Women with Lynch syndrome (LS) or hereditary nonpolyposis colorectal cancer, both autosomal dominant conditions, are at an increased risk for the development of colon, ovarian, and endometrial cancers (Burke et al. 2014. PubMed ID: 24905773). Lynch syndrome is characterized by germline variants within mismatch repair pathway genes, which include (but are not limited to) MLH1, MSH2, MSH6, and PMS2. Single stranded DNA breaks are repaired via the base excision repair or nucleotide excision repair systems with components encoded by the genes described above. Inheritance of a pathogenic germline variant in any of these genes results in a LS diagnosis, with a second variant occurring somatically later in life. LS-associated endometrial cancers are usually diagnosed after menopause, with only 15% diagnosed before the age of 50 and only 5% before the age of 40 (Gallup and Stock. 1984. PubMed ID: 6462572; Burke et al. 2014. PubMed ID: 24905773).
Several other genes have been implicated in the development of hereditary endometrial cancers. While BRCA1 and BRCA2 pathogenic variants are known to contribute to elevated risks of breast and ovarian cancer, studies support that pathogenic variants in BRCA1 particularly contribute to increased risk of endometrial cancer (Segev et al. 2015. PubMed ID: 25838159). Approximately 5% of women with uterine serous carcinoma have germline pathogenic variants in BRCA1, CHEK2, or TP53 (Pennington et al. 2013. PubMed ID: 22811390). 3’ end deletions of the EPCAM gene can result in LS via silencing of the adjacent MSH2 promoter and subsequently MSH2-deficiency. Carriers of EPCAM deletions have a 12% cumulative risk for endometrial cancer development, particularly when the deletion extends close to the MSH2 promoter region (Kempers et al. 2011. PubMed ID: 21145788). PTEN pathogenic variants are associated with autosomal dominant Cowden syndrome, which is characterized by hamartomatous tumors in multiple organ systems and an increase risk of multiple cancers. Lifetime risk of endometrial cancer in women with Cowden syndrome is approximately 10%-28%, and variants within PTEN account for a small proportion of endometrial cancer cases (Shai et al. 2014. PubMed ID: 24838932). Germline variants in POLD1 have been identified in European families with a history of colorectal cancer, colonic adenomas, and predispose individuals to endometrial and brain cancers. However, to date the incidence of POLD1 germline variants in endometrial cancer cases is unclear (Shai et al. 2014. PubMed ID: 24838932).
There is little information regarding the role of MUTYH in endometrial cancer. At least one MUTYH compound heterozygote with endometrial cancer has been identified (Barnetson et al. 2007. PubMed ID: 17956577), however other reports have failed to identify a genetic basis for MUTYH in endometrial cancer (Ashton et al. 2009. PubMed ID: 19338676).
Cumulative risk for endometrial cancer rises significantly after the age of 40, and by 70 varies depending on the gene in which a pathogenic variant is inherited. Women with endometrial cancer under the age of 50 having a first-degree relative diagnosed with a LS-related malignancy have a 43% chance of carrying a germline pathogenic variant in a mismatch repair gene and a 9% chance if two or more first-degree relatives have had an EC diagnosis (Gayther and Pharoah. 2010. PubMed ID: 20456938). For MLH1, the cumulative risk is reported to be 54%, while for MSH2 and MSH6 the cumulative risk is 21% and 16%-71%, respectively (Bonadona et al. 2011. PubMed ID: 21642682; Burke et al. 2014. PubMed ID: 24905773; Gayther and Pharoah. 2010. PubMed ID: 20456938).
For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
This panel provides full coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
Individuals with a clinical presentation of endometrial cancer, hereditary nonpolyposis colorectal cancer, Lynch syndrome, or a family history including endometrial cancer are candidates for this test. A positive test does not mean that a currently unaffected individual will develop endometrial cancer and a negative test does not mean that an individual will not develop endometrial cancer. Furthermore, this test is specifically designed for germline variants and is not appropriate for the detection of somatic variants in tumor tissue.
|Official Gene Symbol||OMIM ID|
|BRCA1 and BRCA2||600185|
- Genetic Counselor Team - email@example.com
- Jerry Machado, PhD, DABMG, FCCMG - firstname.lastname@example.org
- Aarnio et al. 1999. PubMed ID: 10188721
- Acharya et al. 2005. PubMed ID: 16321764
- Ashton et al. 2009. PubMed ID: 19338676
- Barnetson et al. 2007. PubMed ID: 17956577
- Bonadona et al. 2011. PubMed ID: 21642682
- Burke et al. 2014. PubMed ID: 24905773
- Dedes et al. 2011. PubMed ID: 21221135
- Gallup and Stock. 1984. PubMed ID: 6462572
- Gayther and Pharoah. 2010. PubMed ID: 20456938
- Hamilton et al. 2006. PubMed ID: 16495918
- Hamilton et al. 2008. PubMed ID: 18197002
- Kempers et al. 2011. PubMed ID: 21145788
- Kohlmann and Gruber. 2012. PubMed ID: 20301390
- O'Hara and Bell. 2012. PubMed ID: 22888282
- Pennington et al. 2013. PubMed ID: 22811390
- Segev et al. 2015. PubMed ID: 25838159
- Setiawan et al. 2013. PubMed ID: 23733771
- Shai et al. 2014. PubMed ID: 24838932
- Siegel et al. 2013. PubMed ID: 23335087
- Vasen et al. 1999. PubMed ID: 10348829
- Watson et al. 2008. PubMed ID: 18398828
Sequencing and Deletion/Duplication Testing via NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).
(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants
Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.
Deletion and Duplication Testing via NGS
As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.
In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.
Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.
When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).
We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.
In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.
Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.
We cannot be certain that the reference sequences are correct.
Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.
We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.
myPrevent - Online Ordering
- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.