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Prostate Cancer Sequencing Panel with CNV Detection

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing and CNV

Test Code Test Copy GenesCPT Code Copy CPT Codes
5459 ATM 81408,81479 Add to Order
BRCA1 and BRCA2 81162
BRIP1 81479,81479
CHEK2 81479,81479
EPCAM 81479,81403
HOXB13 81479,81479
MLH1 81292,81294
MSH2 81295,81297
MSH6 81298,81300
NBN 81479,81479
PALB2 81406,81479
PMS2 81317,81319
RAD51C 81479,81479
RAD51D 81479,81479
TP53 81405,81479
Full Panel Price* $540
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
5459 Genes x (16) $540 81162, 81292, 81294, 81295, 81297, 81298, 81300, 81317, 81319, 81403, 81405, 81406, 81408, 81479(x16) Add to Order

New York State Approved Test

Pricing Comments

We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available.

This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.

Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

This panel analyzes genes that have been associated with hereditary prostate cancer. It is difficult to estimate the clinical sensitivity of this test due to the paucity of large cohort studies. However, in a recent study of 692 men with documented metastatic prostate cancer, pathogenic variants in these genes were identified: ATM (11 pathogenic variants [1.6%l]), BRCA1 (6 [0.9%]), BRCA2 (37 [5.3%]), CHEK2 (10 [1.9%]), and PALB2 (3 [0.4%]) (Pritchard et al. 2016. PubMed ID: 27433846).

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Clinical Features

Prostate cancer is the most frequently diagnosed cancer in males in the United States (Siegel et al. 2017. PubMed ID: 28055103). A man’s lifetime risk of developing invasive prostate cancer is 1 in 8 (Siegel et al. 2017. PubMed ID: 28055103). This disorder is typically considered a cancer of the elderly, and the median age of onset is ~68 years. However, recent studies suggest an increase in the incidence of early onset of prostate cancer (Gupta et al. 2017. PubMed ID: 28413383). Initially prostate cancer may be asymptomatic, but more advanced prostate cancers usually cause symptoms, which include problems urinating, blood in the urine, trouble getting an erection, and pain in the hips, spine and chest (American Cancer Society. 2014). When prostate cancer is limited to the prostate gland itself, it may be curable. However, cancer cells may spread to other parts of the body, particularly the lymph nodes and bones (American Cancer Society. 2014). Early diagnosis is critical to successful treatment.

Genetics

Prostate cancer is highly heritable, with an overall estimated heritability of 40% - 60% (Lichtenstein et al. 2000. PubMed ID: 10891514; Hjelmborg et al. 2014. PubMed ID: 24812039). Approximately 5% -10% of prostate cancer is caused by rare pathogenic variants in susceptibility genes (Steinberg et al. 1990. PubMed ID: 2251225; Carter et al. 1992. PubMed ID: 1565627; Pritchard et al. 2016. PubMed ID: 27433846). The mode of inheritance appears to be autosomal dominant (AD) (Schaid et al. 1998. PubMed ID: 9585590).

This prostate cancer next generation sequencing panel assesses genes that have been shown to be causative when mutated for disorders that have prostate cancer as a clinical feature.

ATM: Ataxia-telangiectasia is an autosomal recessive disorder caused by pathogenic variants in the ATM gene. ATM encodes a serine protein kinase (ATM) that is involved in DNA repair via phosphorylation of downstream proteins. It senses double-strand DNA breaks, coordinates cell-cycle checkpoints prior to repair, and recruits repair proteins to damaged DNA sites (Taylor et al. 2004. PubMed ID: 15279810). Pathogenic variants in ATM result in defective checkpoint cycling. Previous studies suggest an association between ATM heterozygous carriers and prostate cancer susceptibility. For example, a retrospective case series of patients with prostate cancer found that 1.6% (11 of 692) had an ATM pathogenic variant (Pritchard et al. 2016. PubMed ID: 27433846). Specific variants, such as ATM p.Ser49Cys, have been found to be associated with an increased risk of prostate cancer (Dombernowsky et al. 2008. PubMed ID: 18565893).

BRCA1 and BRCA2: BRCA1 is a tumor suppressor gene that is involved in a number of cellular processes including DNA damage repair, cell cycle progression, gene transcription and ubiquitination. BRCA2 is a tumor suppressor gene that along with RAD51 has a large role in DNA repair processes and genome stability. Male carriers of BRCA1 and BRCA2 pathogenic variants have a higher risk of developing cancers, including prostate cancer (Thompson and Easton. 2002. PubMed ID: 12237281; Liede et al. 2004. PubMed ID: 14966099). Particularly, prostate cancer has been observed at higher rates in BRCA2 carriers than in the general population (Mersch et al. 2015. PubMed ID: 25224030).

CHEK2: CHEK2 encodes a protein kinase that protects the genome from ionizing radiation and genotoxic insults. To date, approximately 90 pathogenic variants have been reported throughout the CHEK2 gene, and >90% are detectable by sequencing (Human Gene Mutation Database). It has been suggested that CHEK2 may be a potential prostate cancer susceptibility gene (Wang et al. 2015. PubMed ID: 26629066).

HOXB13: HOXB13 is involved in embryonic development and regulation of androgen receptor target genes. It is a tumor suppressor, and germline variants have been associated with prostate cancer susceptibility with an odds ratio of 3-8 (Zhen et al. 2018. PubMed ID: 29669169).

MLH1, MSH2, MSH6, PMS2, and EPCAM: Germline pathogenic variants in these genes have been associated with Lynch syndrome. Previous studies have suggested that prostate cancer may be observed in patients harboring a mismatch repair (MMR) gene pathogenic variant (Grindedal et al. 2009. PubMed ID: 19723918; Haraldsdottir et al. 2014. PubMed ID: 24434690: Rosty et al. 2014. PubMed ID: 25117503).

NBN: Nijmegen breakage syndrome (NBS) has been associated with an elevated risk of prostate cancer (Cybulski et al. 2013. PubMed ID: 23149842; Cybulski et al. 2004. PubMed ID: 14973119). The NBN protein normally associates with the MRE11A and RAD50 proteins to form the MRN complex. The MRN complex, upon DNA damage, is involved in DNA repair and cell cycle arrest via the ATM kinase. Pathogenic variants in NBN lead to faulty DNA repair and improper cell cycle control. In Eastern European with NBS, c.657_661del5 is the most common pathogenic variant and accounts for more than 90% of all mutant alleles in NBN (Varon et al. 1998. PubMed ID: 9590180).

TP53: Li-Fraumeni syndrome (LFS) is inherited in an autosomal dominant manner and is caused by heterozygous germline pathogenic variants in the TP53 gene (Malkin et al. 1990. PubMed ID: 1978757; Srivastava et al. 1990. PubMed ID: 2259385). TP53 encodes the often studied cellular tumor p53 antigen (Soussi. 2010. PubMed ID: 20930848). p53 is a ubiquitously expressed DNA-binding protein that plays a major role in the regulation of cell division, DNA repair, programmed cell death, and metabolism. More than 200 pathogenic variants have been reported throughout the TP53 gene, and nearly all are detectable by DNA sequencing (Human Gene Mutation Database). Several gross deletions encompassing one or more exons of the TP53 gene have been described (Human Gene Mutation Database), but these account for less than 1% of all LFS patients (Schneider et al. 2013. PubMed ID: 20301488). The average risk of developing cancer for carriers of TP53 pathogenic variants has been estimated to be ~73% for men (Chompret et al. 2000. PubMed ID: 10864200). In a Dutch case series of 180 families with Li-Fraumeni syndrome or Li-Fraumeni-like syndrome, a TP53 pathogenic variant was identified in 24 families (Ruijs et al. 2010. PubMed ID: 20522432). Li-Fraumeni syndrome is responsible for multiple cancers including prostate cancer. In a French case series of 214 families with TP53 pathogenic variants, 4 prostate cancer cases were reported (Bougeard et al. 2015. PubMed ID: 26014290).

A few other genes such as PALB2, RAD51C and RAD51D are included in this NGS panel based on preliminary evidence of their involvement in prostate cancer (Pritchard et al. 2016. PubMed ID: 27433846).

Testing Strategy

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.

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This panel typically provides ≥98% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing.

This test also includes analysis of the inversion of exons 1-7 in MSH2.

Indications for Test

This test is suitable for individuals with a personal and/or family history of prostate cancer. Individuals with or without a family history of prostate cancer with early onset (<50 years) should be assessed with this panel. This test especially aids in a differential diagnosis of similar phenotypes, rules out particular syndromes, and provides analysis of multiple genes simultaneously. This test is specifically designed for heritable germline variants and is not appropriate for the detection of somatic variants in tumor tissue.

Genes

Official Gene Symbol OMIM ID
ATM 607585
BRCA1 113705
BRCA2 600185
BRIP1 605882
CHEK2 604373
EPCAM 185535
HOXB13 604607
MLH1 120436
MSH2 609309
MSH6 600678
NBN 602667
PALB2 610355
PMS2 600259
RAD51C 602774
RAD51D 602954
TP53 191170
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Ataxia-Telangiectasia Syndrome AR 208900
Breast-Ovarian Cancer, Familial 1 AD 604370
Breast-Ovarian Cancer, Familial 2 AD 612555
Breast-Ovarian Cancer, Familial 3 AD 613399
Breast-Ovarian Cancer, Familial 4 AD 614291
Diarrhea 5, With Tufting Enteropathy, Congenital AR 613217
Familial Cancer Of Breast AD 114480
Hereditary Nonpolyposis Colorectal Cancer Type 4 AD 614337
Hereditary Nonpolyposis Colorectal Cancer Type 5 AD 614350
Hereditary Nonpolyposis Colorectal Cancer Type 8 AD 613244
Li-Fraumeni Syndrome AD 151623
Lynch Syndrome I AD 120435
Lynch Syndrome II AD 609310
Muir-Torre Syndrome AR 158320
Nijmegen Breakage Syndrome AR 251260
Pancreatic Cancer 2 AD 613347
Pancreatic Cancer 3 AD 613348
Pancreatic Cancer 4 AD 614320
Prostate Cancer AD 176807
Turcot Syndrome AR 276300

Related Test

Name
PGxome®

CONTACTS

Genetic Counselors
Geneticist
Citations
  • American Cancer Society- www.cancer.org.
  • Bougeard et al. 2015. PubMed ID: 26014290
  • Carter et al. 1992. PubMed ID: 1565627
  • Chompret et al. 2000. PubMed ID: 10864200
  • Cybulski et al. 2004. PubMed ID: 14973119
  • Cybulski et al. 2013. PubMed ID: 23149842
  • Dombernowsky et al. 2008. PubMed ID: 18565893
  • Grindedal et al. 2009. PubMed ID: 19723918
  • Gupta et al. 2017. PubMed ID: 28413383
  • Haraldsdottir et al. 2014. PubMed ID: 24434690
  • Hjelmborg et al. 2014. PubMed ID: 24812039
  • Human Gene Mutation Database (Bio-base).
  • Lichtenstein et al. 2000. PubMed ID: 10891514
  • Liede et al. 2004. PubMed ID: 14966099
  • Malkin et al. 1990. PubMed ID: 1978757
  • Mersch et al. 2015. PubMed ID: 25224030
  • Pritchard et al. 2016. PubMed ID: 27433846
  • Rosty et al. 2014. PubMed ID: 25117503
  • Ruijs et al. 2010. PubMed ID: 20522432
  • Schaid et al. 1998. PubMed ID: 9585590
  • Schneider et al. 2013. PubMed ID: 20301488
  • Siegel et al. 2017. PubMed ID: 28055103
  • Soussi. 2010. PubMed ID: 20930848
  • Srivastava et al. 1990. PubMed ID: 2259385
  • Steinberg et al. 1990. PubMed ID: 2251225
  • Taylor et al. 2004. PubMed ID: 15279810
  • Thompson and Easton. 2002. PubMed ID: 12237281
  • Varon et al. 1998. PubMed ID: 9590180
  • Wang et al. 2015. PubMed ID: 26629066
  • Zhen et al. 2018. PubMed ID: 29669169
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TEST METHODS

Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes

Test Procedure

NextGen Sequencing

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 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.

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 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

Copy number variants (CNVs) such as deletions and duplications are detected from next generation sequencing data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution, and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as PCR, aCGH or MLPA before they are reported.
Analytical Validity

NextGen Sequencing

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.

Deletion and Duplication Testing via NGS
 
In general, sensitivity for single, double, or triple exon CNVs is ~80% and for CNVs of four exon size or larger is close to 100%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.
Analytical Limitations

NextGen Sequencing

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 ~10 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.

Deletion and Duplication Testing via NGS
 
This CNV calling algorithm used in this test detects most deletions and duplications; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g. pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g. single vs. two or more exons), and inadequate coverage. 
 
Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.
 
In nearly all cases, our ability to determine the exact copy number change within a targeted gene is limited. In particular, when we find copy excess within a targeted gene, we cannot be certain that the region is duplicated, triplicated etc. In many duplication cases, we are unable to determine the genomic location or the orientation of the duplicated segment with respect to the gene. In particular, we often cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we may not be able to determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.
 
Our ability to detect CNVs due to somatic mosaicism is limited.
Order Kits

Ordering Options


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.
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.

SPECIMEN TYPES
WHOLE BLOOD

(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.

DNA

(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.

CELL CULTURE

(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.
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