EGFR c.2582T>A, p.Leu861Gln

Reasoning

PS3 is met at strong strength for EGFR p.Leu861Gln (L861Q). PMID:17177598 directly tested L861Q in several complementary functional systems: NIH-3T3 soft-agar transformation and nude-mouse tumorigenicity, Ba/F3 IL-3 independence and erlotinib sensitivity, and receptor phosphorylation/ligand-response assays. The study used wild-type EGFR and empty-vector or parental-cell controls; L861Q-expressing cells formed anchorage-independent colonies, produced tumors in all inoculated mice within 3–4 weeks while empty-vector and wild-type controls produced no measurable tumors, relieved Ba/F3 IL-3 dependence, and showed dose-dependent erlotinib-induced cell death. These orthogonal in vitro and in vivo results are biologically relevant to an activating EGFR effect and support a damaging gain-of-function effect. The evidence is strong but is not described as a validated, reproducible clinical diagnostic-laboratory assay.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• PMID:17177598, Results sections and Figures 2–4: L861Q was tested in NIH-3T3 soft-agar and nude-mouse assays, Ba/F3 signaling/IL-3-dependence assays, and erlotinib-response assays.
• PMID:17177598: wild-type EGFR and empty-vector or parental-cell controls were included; controls did not form measurable tumors, whereas L861Q cells produced tumors in all mice within 3–4 weeks.
• PMID:17177598: L861Q-expressing Ba/F3 cells were IL-3 independent, showed increased tyrosine phosphorylation, and underwent dose-dependent erlotinib-induced cell death; the paper reports mean percent control ± standard deviation for viability.
• The study provides multiple biologically relevant research assays but does not establish validation and reproducibility in a clinical diagnostic laboratory setting.

Sources

Reasoning

EGFR p.Leu861Gln lies in exon 21 of the EGFR tyrosine-kinase domain. The case evidence also identifies L861 as a statistically significant Cancer Hotspots hotspot and records 126 COSMIC observations of the variant. Under generic ACMG/AMP, this supports the mutational-hotspot route to PM1; the hotspot extraction is flagged as partial, so the underlying hotspot record warrants human confirmation.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• Cancer Hotspots reports that EGFR L861 lies in a statistically significant hotspot; the case record warns that the row extraction was partial.
• COSMIC records EGFR c.2582T>A (COSV51766344) in somatic cancers 126 times.
• Lynch et al. place L861Q in exon 21 of the EGFR tyrosine-kinase domain, and the glioblastoma study describes L861Q as an EGFR kinase-domain mutation.

Still missing

• Cancer Hotspots extraction was partial and should be confirmed against the captured hotspot record before final sign-off.

Sources

Reasoning

PM2 is met at supporting strength. NM_005228.5:c.2582T>A (EGFR p.Leu861Gln) was absent from gnomAD v2.1 and gnomAD v4.1, and the case evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets. The observed frequency is therefore 0, which is at or below the supplied generic PM2 supporting threshold of 0.0001. This is a single-nucleotide substitution, so the ACMG/AMP indel calling caveat does not apply.

Rule

PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020)

Evidence considered

• gnomAD v2.1 (exome) record for EGFR NM_005228.5:c.2582T>A: absent; observed allele frequency 0.
• gnomAD v4.1 (exome) record for EGFR NM_005228.5:c.2582T>A: absent; observed allele frequency 0.
• The case evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets.
• The supplied generic PM2 supporting threshold is <=0.0001; the observed frequency of 0 meets that threshold.

Sources

Reasoning

PP3 is met at supporting strength. NM_005228.5:c.2582T>A is a missense variant, p.(Leu861Gln), so the governing generic path is REVEL only; SpliceAI and BayesDel are not used for PP3 in this situation. The REVEL score is 0.864, which meets the supplied PP3 supporting threshold of >=0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).

Evidence considered

• The normalized consequence is missense: NM_005228.5:c.2582T>A, EGFR p.(Leu861Gln), exon 21.
• Local REVEL lookup reports a score of 0.864 for this variant.
• For a missense variant, the generic rule selects REVEL only; the supplied PP3 supporting threshold is >=0.644, from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997).
• The available SpliceAI score and BayesDel score were not counted because the governing missense path does not use them for PP3.

Sources

Reasoning

PP5 requires a reputable source to recently report the variant as pathogenic when the evidence is unavailable for independent evaluation. The exact-variant ClinVar record has no expert-panel submission; its Likely pathogenic assertion is from one ordinary clinical laboratory submission with no assertion criteria, and the other submission is drug response. The available evidence therefore does not meet the required expert-panel exact-variant trigger for PP5.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-variant record NM_005228.5(EGFR):c.2582T>A (p.Leu861Gln), variation 163380: expert_panels is empty and expert_panel_submissions is 0.
• ClinVar submission audit: the Likely pathogenic submission SCV004042725 is from a clinical laboratory, has no assertion criteria, is not an expert-panel submission, and is not usable for classification.

Sources

Reasoning

BA1 is not met. NM_005228.5:c.2582T>A (EGFR p.Leu861Gln) was absent from gnomAD v2.1 and gnomAD v4.1, so its observed allele frequency was 0 and did not exceed the 5% stand-alone benign threshold. The variant is a single-nucleotide substitution, so the indel calling caveat does not apply.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 (exome) record for EGFR NM_005228.5:c.2582T>A: absent; observed allele frequency 0.
• gnomAD v4.1 (exome) record for EGFR NM_005228.5:c.2582T>A: absent; observed allele frequency 0.
• The case evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets; no subset frequency exceeds the BA1 threshold.

Sources

Reasoning

BS1 is not met. The variant was absent from gnomAD v2.1 and v4.1 and from the reported non-cancer subsets, so no elevated population frequency was observed. The generic 1% default is therefore not reached; a maximum-credible-frequency calculation is not needed to reject BS1 on the available data. There is no observed ancestry-specific enrichment to evaluate, and the variant is a single-nucleotide substitution rather than an indel.

Rule

the criterion is 'allele frequency is greater than expected for the disorder'; the 1% figure is only a default. ... BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v2.1 (exome) record for EGFR NM_005228.5:c.2582T>A: absent; observed allele frequency 0.
• gnomAD v4.1 (exome) record for EGFR NM_005228.5:c.2582T>A: absent; observed allele frequency 0.
• The case evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets; no ancestry-specific or cohort-specific elevated frequency was observed.
• The generic BS1 default supplied for this case is >=0.01, while the governing definition asks whether allele frequency is greater than expected for the disorder; the observed frequency is 0.

Sources

Reasoning

BS3 is not met. No well-established functional study in the reviewed evidence shows that EGFR p.Leu861Gln has no damaging effect on protein function or splicing. Instead, PMID:17177598 reports activating and transforming effects for L861Q across complementary cellular and in vivo assays, including tumor formation, increased signaling, IL-3 independence, and erlotinib-sensitive viability loss.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• PMID:17177598: L861Q was associated with anchorage-independent growth, tumors in all inoculated mice, increased tyrosine phosphorylation, IL-3 independence, and erlotinib-induced cell death.
• No reviewed source provided a validated benign functional or splicing result for L861Q.

Sources

Reasoning

BP4 is not met. NM_005228.5:c.2582T>A is a missense variant, so the governing generic path is REVEL only; SpliceAI cannot substitute for an indeterminate missense REVEL result, and BayesDel has no generic ACMG-strength calibration. The REVEL score is 0.864, which is above the supplied BP4 supporting threshold of <=0.29 and therefore does not support a benign computational effect.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)

Evidence considered

• The normalized consequence is missense: NM_005228.5:c.2582T>A, EGFR p.(Leu861Gln), exon 21.
• Local REVEL lookup reports a score of 0.864 for this variant.
• For a missense variant, the generic rule selects REVEL only; the supplied BP4 supporting threshold is <=0.29, from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997).
• REVEL 0.864 is outside the BP4 range and is not an indeterminate low-score result; SpliceAI was not used because its no-splice-impact result cannot establish tolerance of a missense amino-acid substitution.
• BayesDel was not used because no generic ACMG-strength calibration is established for BayesDel.

Sources

Reasoning

BP6 requires a reputable source to recently report the variant as benign when the evidence is unavailable for independent evaluation. The exact-variant ClinVar record has no expert-panel submission and contains no benign or likely benign assertion; it contains one Likely pathogenic clinical-laboratory submission and one drug-response submission. The required exact-variant expert-panel benign trigger is absent, so BP6 is not met.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-variant record NM_005228.5(EGFR):c.2582T>A (p.Leu861Gln), variation 163380: expert_panels is empty and neither submission is benign or likely benign.
• ClinVar submission audit: the two submissions are Likely pathogenic and drug response, both without assertion criteria, and expert_panel_submissions is 0.

Sources

Reasoning

The variant is EGFR p.Leu861Gln (L861Q). The reviewed literature documents this same amino-acid change, but does not establish a previously established pathogenic variant with the same amino-acid change caused by a different nucleotide change. The available ClinVar submissions lack assertion criteria and are somatic/drug-response records, so they cannot supply the required pathogenic comparator.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• ClinVar variation 163380 exactly matches NM_005228.5:c.2582T>A (p.Leu861Gln), but the two submissions have no assertion criteria and are somatic/drug-response records rather than an established pathogenic germline comparator.
• Lynch et al. reported L861Q in Patient 7 and the glioblastoma study reported L861Q, but neither source documents a different nucleotide change producing the same amino-acid change as an established pathogenic variant.

Still missing

• A validated pathogenic comparator with the same p.Leu861Gln amino-acid change caused by a different nucleotide change is not documented.

Sources

Reasoning

The available report identifies EGFR p.(Leu861Gln) in a lung-cancer tumor, including Patient 7 in a gefitinib-response series, but provides no evidence that the variant was germline, no confirmed maternity and paternity, no parental testing, and no documented absence of family history. Therefore the required de novo evidence cannot be assessed.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• Lynch et al. 2004 (PMID:15118073), Results and Tables 1-2: EGFR L861Q was reported in Patient 7's primary lung tumor, but the source does not report parental testing, confirmed maternity or paternity, germline origin, or family history.
• The case data identify the variant as a somatic lung-cancer finding and provide no proband trio or family segregation dataset.

Still missing

• Germline testing of the proband is not documented.
• Both maternity and paternity confirmation are not documented.
• Parental variant testing is not documented.
• Family history and disease phenotype documentation are not provided.

Sources

Reasoning

Generic ACMG/AMP PS4 requires a statistically significant increase in variant prevalence in affected individuals versus controls, with OR or RR >5.0 and a confidence interval excluding 1.0. The reviewed variant-specific paper reports EGFR p.(Leu861Gln) in one gefitinib-responsive lung-cancer patient, but it does not provide an unaffected or population control comparison, variant case/control counts, an odds ratio, a relative risk, or a confidence interval. Therefore no valid case-control estimate can be calculated. The literature_pass criteria index also lists PMID:15118125 and PMID:24627688 for PS4, but ai_classified_papers is empty and neither paper has a paper_extracts entry or available full text in this case, so those leads do not establish PS4.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• Lynch et al. 2004 (PMID:15118073), Results and Tables 1-2: EGFR L861Q was reported in Patient 7, one of nine gefitinib-responsive NSCLC patients; the paper provides no unaffected or population control comparison for this exact variant.
• No case/control counts, odds ratio, relative risk, or confidence interval for EGFR c.2582T>A/p.Leu861Gln are present in the supplied evidence.
• The literature_pass PS4 index lists PMID:15118125 and PMID:24627688, but no verified variant-specific full-text finding is available for either source in this case.

Still missing

• A study-specific unaffected or population control denominator and variant count are needed.
• An odds ratio or relative risk with a 95% confidence interval cannot be calculated from the available evidence.
• The PS4-indexed PMID:15118125 and PMID:24627688 require primary-text verification if full text becomes available.

Sources

Reasoning

PM3 cannot be assessed because the available record concerns a somatic EGFR tumor variant and provides no recessive-disease context, affected-proband genotype, pathogenic partner variant, parental or offspring testing, or evidence establishing trans phase. The ClinVar record identifies the variant origin as somatic, which does not establish the germline allelic observations required for PM3.

Rule

For recessive disorders, detected in trans with a pathogenic variant...This requires testing of parents (or offspring) to determine phase.

Evidence considered

• ClinVar variation 163380 records NM_005228.5(EGFR):c.2582T>A (p.Leu861Gln) as a somatic-origin submission; this does not provide germline phase or a pathogenic variant in trans.
• The available case evidence contains no parental or offspring genotypes, no cis/trans phasing result, and no affected-proband observation with a second pathogenic allele.

Still missing

• A defined recessive EGFR-associated disorder and inheritance model are not established.
• No affected-proband observation with a second pathogenic variant is documented.
• No parental or offspring testing establishes trans phase.

Sources

Reasoning

The variant is p.Leu861Gln. The available same-residue candidate review found no confirmed comparator candidates, and the reviewed papers document L861Q itself rather than a different missense substitution at Leu861 that was determined to be pathogenic. The available records therefore do not establish the required pathogenic alternate missense change.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• The same-residue review recorded zero candidates and no confirmed pathogenic comparator for residue 861.
• The reviewed papers report L861Q but do not report a different missense change at Leu861 determined to be pathogenic.

Still missing

• A validated pathogenic alternate missense variant at Leu861 is not documented.

Sources

Reasoning

The available report documents EGFR p.(Leu861Gln) in a lung-cancer tumor but does not establish germline origin or provide evidence that the variant is de novo. There is no parental testing and no information about maternity, paternity, or family history, so assumed de novo status cannot be assessed.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• Lynch et al. 2004 (PMID:15118073), Results and Tables 1-2: the variant was identified in a primary lung-cancer tumor from Patient 7; no parental testing or assumed-de novo evidence is reported.
• The case data provide no germline trio or parental testing results.

Still missing

• Germline testing of the proband is not documented.
• Parental testing is not documented.
• Evidence supporting assumed de novo occurrence is not documented.

Sources

Reasoning

No affected relatives, meioses, genotype results, or family segregation analysis are reported for EGFR p.(Leu861Gln). The available publication describes a single tumor finding and treatment response rather than familial disease, so co-segregation cannot be assessed.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• Lynch et al. 2004 (PMID:15118073), Results and Tables 1-2: reports the variant in one lung-cancer patient but contains no familial segregation analysis or affected relatives.
• The case data contain no family-level genotype-phenotype observations for this variant.

Still missing

• No affected family members are documented.
• No informative meioses or segregation count is provided.
• No genotype and phenotype data for relatives are provided.

Sources

Reasoning

The evidence supports that EGFR missense mutations can have gain-of-function effects and occur in the EGFR tyrosine-kinase domain, but it does not provide the required gene-level demonstration that benign missense variation is rare. Absence of this variant from gnomAD is not a gene-wide benign-missense-rate estimate, so PP2 cannot be assessed.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• The variant is absent from gnomAD v2.1 and v4.1, but single-variant absence does not establish a low gene-wide rate of benign missense variation.
• The glioblastoma study reports oncogenic and signaling effects for EGFR L861Q, supporting a missense disease mechanism but not the required gene-wide benign-missense-rate condition.

Still missing

• A gene-level benign missense constraint or equivalent low-rate assessment for EGFR is not available.

Sources

Reasoning

PP4 requires a patient phenotype or family history highly specific for a disease with a single genetic etiology. The available record concerns a somatic EGFR alteration in a lung-cancer tumor and a treatment-response report, not a documented constitutional phenotype or family history. No phenotype details establishing a highly specific single-gene disorder are provided, so PP4 cannot be assessed.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• Lynch et al. 2004 (PMID:15118073): reports a somatic EGFR L861Q tumor finding and gefitinib response, but does not provide a highly specific constitutional phenotype or family history.
• The case record identifies the finding in lung adenocarcinoma and does not document a phenotype specific to a disease with a single genetic etiology.

Still missing

• A detailed patient phenotype or family history is not provided.
• A single-gene disease context applicable to this variant is not established.

Sources

Reasoning

BS2 is not assessed. The available population records show absence of the variant, but they do not document an identified healthy adult carrying the variant in the genotype required for this criterion. No homozygote, heterozygote healthy-adult observation, hemizygote observation, disease prevalence estimate, or penetrance assessment applicable to BS2 was provided.

Rule

BS2: Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v2.1 and v4.1 report the variant as absent; absence is not evidence that a healthy adult individual carried the variant.
• The available case evidence contains no qualifying healthy-adult carrier, homozygote, or hemizygote observation and no penetrance/age-of-onset data for BS2.

Still missing

• A documented healthy adult carrying the variant in the inheritance pattern relevant to the disorder.
• Applicable disease penetrance and age-of-onset information for interpreting that observation.

Sources

Reasoning

No affected family members with negative variant testing are reported. The available evidence concerns a tumor from one patient and does not provide relatives' phenotypes, genotypes, or a segregation analysis; therefore lack of segregation cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• Lynch et al. 2004 (PMID:15118073), Results and Tables 1-2: reports a single patient tumor finding and does not report affected relatives or negative variant testing in relatives.
• The case data contain no family non-segregation observations.

Still missing

• No affected relatives are documented.
• No relatives' variant-testing results are provided.
• No family phenotype and genotype comparison is available.
• Phenocopy and multiple-pathogenic-variant considerations cannot be evaluated.

Sources

Reasoning

The available evidence describes EGFR kinase-domain missense mutations and gain-of-function biology, but it does not establish that disease-causing variants in the relevant gene-disease context are primarily truncating. The criterion therefore cannot be assessed from the supplied evidence.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• OncoKB characterizes EGFR L861Q as an oncogenic gain-of-function variant, and the reviewed papers describe kinase-domain missense mutations; neither source establishes a primarily truncating disease mechanism for EGFR.

Still missing

• A gene-disease review demonstrating that disease-causing EGFR variants are primarily truncating is not available.

Sources

Reasoning

BP2 cannot be assessed because no pathogenic variant in trans or cis with NM_005228.5:c.2582T>A is documented, and no phase, affected-proband, or inheritance evidence is available. The ClinVar record identifies the available observations as somatic rather than providing the constitutional allelic data required for BP2.

Rule

Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• ClinVar variation 163380 records NM_005228.5(EGFR):c.2582T>A (p.Leu861Gln) as a somatic-origin submission; it does not document a pathogenic variant in cis or trans.
• The available case evidence contains no paired pathogenic variant, phase result, parental or offspring testing, or affected-proband allelic observation relevant to BP2.

Still missing

• No pathogenic partner variant is documented in the same proband or sample.
• No parental, offspring, or molecular phasing data establish cis or trans configuration.
• The applicable fully penetrant dominant inheritance context is not established.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The supplied record documents EGFR L861Q in a somatic lung-cancer context but does not document a separate pathogenic molecular cause in the same case. The reported tumor finding and treatment response are not sufficient to establish the BP5 alternate-basis scenario.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• Lynch et al. 2004 (PMID:15118073): reports EGFR L861Q in a lung-cancer tumor and clinical response to gefitinib, but does not report a separate alternate molecular basis for that case.
• The case evidence contains no documented second pathogenic molecular finding that could satisfy BP5.

Still missing

• No separate pathogenic variant or other alternate molecular basis is documented for the same case.
• Complete case-level molecular findings and disease attribution are needed to evaluate BP5.

Sources