Lung Cancer Susceptibility via the EGFR Gene
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test||Individual Gene Price||CPT Code Copy CPT Codes|
Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.
For ordering targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 28 days.
The clinical sensitivity of this test is unknown due to the few reported cases of lung cancer susceptibility and EGFR mutations.
Deletion/Duplication Testing via aCGH
|Test Code||Test||Individual Gene Price||CPT Code Copy CPT Codes|
# of Genes Ordered
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The great majority of tests are completed within 28 days.
The clinical sensitivity of this test is unknown because thus far, no pathogenic germline deletions or deletions in the EGFR gene have been reported.
Lung cancer is the leading cause of cancer deaths in the United States (Siegel et al. 2013). There are two major forms of lung cancer, nonsmall cell lung cancer (NSCLC) and small cell lung cancer, which represent 85% and 15% of lung cancers, respectively. The major histological subtypes of NSCLC include squamous cell carcinoma, adenocarcinoma, large cell lung cancer, and bronchioloalveolar carcinoma (Ramalingam et al. 2011). While cigarette smoking accounts for the majority of lung cancers (Bunn. 2012), there are likely individuals who have a predisposition to lung cancer compared to the general population (Thomas et al. 2013).
Lung cancer is associated with pathogenic variants in the EGFR gene, which encodes the epidermal growth factor receptor that acts through a tyrosine kinase pathway leading to cellular proliferation. Somatic mutations in the EGFR gene are associated with lung cancer, and because of this, individuals with lung cancer may respond to tyrosine kinase inhibitors (Ramalingam et al. 2011). Germline variants in the EGFR gene have also infrequently been associated with lung cancer predisposition, which appear to occur in an autosomal dominant manner. Cellular studies have shown increased autophosphorylation levels of EGFR by some of these germline variants (Centeno et al. 2011). In addition, a mouse model harboring these variants has been developed that is susceptible to lung adenocarcinomas (Regales et al. 2007). It has been suggested that a pathogenic germline variant in the EFGR gene may act in concert with a somatic EGFR mutation, and possibly lead to the induction of lung cancer and may cause resistance or sensitivity to therapy depending on the combination of variants (Centeno et al. 2011; Ohtsuka et al. 2011; van Noesel et al. 2013; Chung et al. 2010). Due to the lower oncogenic activity of some of these germline EGFR variants, induction of lung cancer may also be caused by combinations with other somatic pathogenic variants in other genes (e.g. KRAS) (Thomas et al. 2013). The relatively few pathogenic variants that have been reported in the EGFR gene are missense variants (Human Gene Mutation Database).
For this NextGen test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for the gene listed below. 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 any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.
Indications for Test
No specific testing guidelines for germline testing for EGFR pathogenic variants exists for lung cancer patients. Individuals who have lung cancer at an early age or who never have been smokers and/or have a family history of lung cancer may be considered.
***This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic EGFR mutations in tumor tissue.***
|Official Gene Symbol||OMIM ID|
|Hypomagnesemia Sequencing Panel|
- Genetic Counselor Team - firstname.lastname@example.org
- Jerry Machado, PhD, DABMG, FCCMG - email@example.com
- Bunn Jr PA. 2012. Worldwide Overview of the Current Status of Lung Cancer Diagnosis and Treatment. Archives of pathology & laboratory medicine 136: 1478–1481.
PubMed ID: 23194039
- Centeno I, Blay P, Santamaría I, Astudillo A, Pitiot A, Osorio F, González-Arriaga P, Iglesias F, Menéndez P, Tardón A. 2011. Germ-line mutations in epidermal growth factor receptor (EGFR) are rare but may contribute to oncogenesis: A novel germ-line mutation in EGFR detected in a patient with lung adenocarcinoma. BMC cancer 11: 172. PubMed ID: 21575252
- Chung K-P, Shih J-Y, Yu C-J. 2010. Favorable response to gefitinib treatment of lung adenocarcinoma with coexisting germline and somatic epidermal growth factor receptor mutations. Journal of Clinical Oncology 28: e701–e703. PubMed ID: 20823418
- Human Gene Mutation Database (Bio-base).
- Ohtsuka K, Ohnishi H, Kurai D, Matsushima S, Morishita Y, Shinonaga M, Goto H, Watanabe T. 2011. Familial lung adenocarcinoma caused by the EGFR V843I germ-line mutation. Journal of Clinical Oncology 29: e191–e192. PubMed ID: 21172876
- Ramalingam SS, Owonikoko TK, Khuri FR. 2011. Lung cancer: New biological insights and recent therapeutic advances. CA: A Cancer Journal for Clinicians 61: 91–112. PubMed ID: 21303969
- Regales L, Balak MN, Gong Y, Politi K, Sawai A, Le C, Koutcher JA, Solit DB, Rosen N, Zakowski MF, Pao W. 2007. Development of New Mouse Lung Tumor Models Expressing EGFR T790M Mutants Associated with Clinical Resistance to Kinase Inhibitors. PLoS ONE 2: e810. PubMed ID: 17726540
- Siegel R, Naishadham D, Jemal A. 2013. Cancer statistics, 2013. CA: A Cancer Journal for Clinicians 63: 11-30. PubMed ID: 23335087
- Thomas A, Xi L, Carter CA, Rajan A, Khozin S, Szabo E, Dennis PA, Giaccone G, Raffeld M. 2013. Concurrent Molecular Alterations in Tumors With Germ Line Epidermal Growth Factor Receptor T790M Mutations. Clinical Lung Cancer 14: 452–456. PubMed ID: 23540867
- van Noesel J, Ven WH van der, Os TAM van, Kunst PWA, Weegenaar J, Reinten RJA, Kancha RK, Duyster J, Noesel CJM van. 2013. Activating germline R776H mutation in the epidermal growth factor receptor associated with lung cancer with squamous differentiation. J. Clin. Oncol. 31: e161–164. PubMed ID: 23358982
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.
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.
Deletion/Duplication Testing Via Array Comparative Genomic Hybridization
Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.
PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.
Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.
This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.
aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.
Breakpoints, if occurring outside the targeted gene, may be hard to define.
The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.
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.