EGFR c.2573T>G, p.(Leu858Arg)

Reasoning

Multiple independent, variant-specific functional studies support an abnormal EGFR effect. EGFR L858R was tested against wild-type EGFR in cell-based assays with EGF-induced activation/autophosphorylation and inhibitor-response readouts. The variant showed increased and prolonged EGF-induced activation and markedly increased sensitivity to gefitinib/erlotinib relative to wild type. The assays are biologically relevant to the EGFR tyrosine-kinase mechanism and include an appropriate wild-type comparator. The concordant results across independent studies support PS3 at strong strength under generic ACMG/AMP calibration.

Rule

Generic ACMG/AMP PS3: well-established functional studies supportive of a damaging effect; strong calibration is justified by concordant, variant-specific results from multiple independent cell-based assays with wild-type comparators and biologically relevant signaling readouts.

Evidence considered

• PMID:15118073 directly tested EGFR L858R in cultured Cos-7 cells using EGF-induced EGFR activation and gefitinib inhibition assays; compared with wild-type EGFR, L858R showed approximately two- to threefold greater EGF-induced activation, activation persisted up to three hours rather than being downregulated after 15 minutes, and the mutant was more sensitive to gefitinib.
• PMID:15329413 directly tested EGFR c.2573T>G (L858R) in transiently transfected 293T cells with wild-type comparison; inhibition of EGFR Tyr-1092 phosphorylation showed approximately tenfold greater sensitivity of L858R to gefitinib and erlotinib than wild-type EGFR.
• The functional readouts assess EGFR signaling and tyrosine-kinase inhibitor response, which are biologically relevant to the established gain-of-function mechanism for the EGFR L858R substitution.

Sources

Reasoning

The variant changes EGFR residue 858 within exon 21 of the EGFR tyrosine kinase domain, an established critical functional domain. L858R is a recurrent activating hotspot in this domain, and the supplied functional literature shows increased EGF-induced activation and increased inhibitor sensitivity of the mutant relative to wild type. No EGFR-specific domain table or VCEP specification was available; generic ACMG/AMP PM1 is therefore used.

Evidence considered

• PMID:15118073 places L858R in EGFR exon 21 and the tyrosine kinase domain and reports approximately two- to threefold greater EGF-induced activation than wild type, with persistent activation and increased gefitinib sensitivity.
• PMID:15329413 places c.2573T>G/L858R in exon 21 and reports approximately tenfold greater sensitivity to gefitinib and erlotinib than wild type in a transfected-cell assay.
• The supplied Cancer Hotspots result identifies EGFR L858 as a statistically significant hotspot.
• OncoKB provides variant-specific EGFR L858R gain-of-function and oncogenic context.

Sources

Reasoning

PM2 is met at supporting strength under the generic ACMG/AMP fallback because the exact EGFR NM_005228.5:c.2573T>G (p.Leu858Arg) variant is absent from gnomAD v2.1 and gnomAD v4.1. This is population rarity evidence only; the case bundle does not provide a disease-specific maximum credible allele frequency, ancestry-complete sampling assessment, or a validated penetrance/prevalence model, and gnomAD-Canada was unavailable. Human review is retained because the available literature and ClinVar records predominantly describe somatic lung-cancer biology or drug response rather than a defined germline EGFR disorder.

Rule

Generic ACMG/AMP PM2: the variant is absent from, or at an extremely low frequency in, population databases appropriate to the disorder. Applied here at supporting strength because the exact variant is absent from two gnomAD releases, with disease-model and ancestry-coverage limitations requiring human review.

Evidence considered

• gnomAD v2.1 direct query: the exact GRCh37 variant 7-55259515-T-G was reported as absent.
• gnomAD v4.1 direct query: the exact GRCh38 variant 7-55191822-T-G was reported as absent.
• The absence result supports rarity but does not by itself establish disease prevalence, penetrance, or a disease-specific maximum credible allele frequency.
• The gnomAD-Canada query was not available because the API request timed out, so that dataset cannot strengthen or independently confirm the rarity assessment.

Still missing

• No disease-specific maximum credible allele frequency calculation was available.
• No ancestry-specific allele counts or coverage metrics were available beyond the queried gnomAD absence results.
• gnomAD-Canada was unavailable because the query timed out.
• The bundle does not establish a clearly applicable germline EGFR disease model with known prevalence and penetrance for this variant.
• No homozygote or healthy-carrier data were available.

Sources

Reasoning

EGFR NM_005228.5:c.2573T>G is a missense variant, p.(Leu858Arg). No EGFR VCEP/CSPEC PP3/BP4 specification or lookup is available; therefore the generic missense-only REVEL path applies. REVEL 0.961 exceeds the PP3 supporting threshold (>0.750). SpliceAI was not used because splice predictions do not assess this substituted amino acid's protein-level effect.

Rule

Generic ACMG/AMP fallback, missense-only computational path: REVEL >0.750 supports PP3 (supporting); calibration reference PMID:36413997.

Evidence considered

• Variant normalization identifies NM_005228.5:c.2573T>G as NP_005219.2:p.(Leu858Arg), a missense substitution.
• The REVEL lookup reports a score of 0.961. Under the generic missense calibration, REVEL >0.750 supports PP3; the calibrated REVEL threshold is from ClinGen SVI calibration work (PMID:36413997).
• No governing EGFR CSPEC/VCEP framework or gene-specific PP3/BP4 lookup was available.

Sources

Reasoning

PS1 is not met because the reviewed publications report the exact EGFR c.2573T>G (p.Leu858Arg; L858R) alteration itself, not an independently established pathogenic variant produced by a different nucleotide change resulting in the same amino-acid substitution. Exact-variant reports cannot be reused as PS1 evidence.

Evidence considered

• PMID:15118073 explicitly reports L858R in exon 21 in two gefitinib-responsive tumors.
• PMID:15118125 explicitly reports the EGFR L858R substitution in lung tumors and the H3255 cell line.
• PMID:15329413 explicitly identifies nucleotide 2573 T>G as the change producing L858R.
• PMID:15897572 reports L858R as a recurrent EGFR exon 21 substitution, but does not provide a different nucleotide change producing the same amino-acid substitution.

Still missing

• No distinct pathogenic nucleotide substitution producing the same p.Leu858Arg amino-acid change was identified in the supplied evidence bundle.

Sources

Reasoning

The exact-variant ClinVar expert-panel submission is ClinPGx 'drug response' for gefitinib-response efficacy, not Pathogenic or Likely pathogenic. The non-expert Pathogenic and Likely pathogenic submissions cannot trigger PP5.

Rule

Apply PP5 only when an exact-variant ClinVar expert-panel classification is Pathogenic or Likely pathogenic.

Evidence considered

• ClinVar variation 16609 exactly matches NM_005228.5:c.2573T>G (p.Leu858Arg). Its only expert-panel submission is ClinPGx and is classified as drug response; the Pathogenic and Likely pathogenic assertions are non-expert submissions.

Sources

Reasoning

BA1 is not met. The exact EGFR NM_005228.5:c.2573T>G (p.Leu858Arg) variant is absent from gnomAD v2.1 and gnomAD v4.1; therefore no population allele frequency exceeds a stand-alone benign threshold. The variant is also reported as a recurrent somatic cancer variant, which is not evidence for a benign population frequency.

Rule

Generic ACMG/AMP BA1: a population allele frequency is sufficiently high for stand-alone benign evidence. No such frequency is present for this exact variant.

Evidence considered

• gnomAD v2.1 direct query: the exact GRCh37 variant 7-55259515-T-G was reported as absent.
• gnomAD v4.1 direct query: the exact GRCh38 variant 7-55191822-T-G was reported as absent.
• The variant has a high somatic count in COSMIC, but somatic tumor recurrence does not establish a benign germline population frequency and was not used to meet BA1.

Still missing

• No quantitative allele-count or allele-number values were returned because the variant was absent from the queried datasets.
• gnomAD-Canada was unavailable because the query timed out.

Sources

Reasoning

BS1 is not met. The exact variant is absent from gnomAD v2.1 and gnomAD v4.1, so there is no observed allele frequency above the disease-specific benign threshold. No ancestry-specific population enrichment or frequency artifact supporting BS1 was identified.

Rule

Generic ACMG/AMP BS1: allele frequency is greater than expected for the disorder. No such observation is available for this exact variant.

Evidence considered

• gnomAD v2.1 direct query: the exact GRCh37 variant 7-55259515-T-G was reported as absent.
• gnomAD v4.1 direct query: the exact GRCh38 variant 7-55191822-T-G was reported as absent.
• No ancestry-specific observation, elevated allele frequency, or threshold artifact supporting BS1 was provided.

Still missing

• No quantitative allele-count or allele-number values were returned because the variant was absent from the queried datasets.
• gnomAD-Canada was unavailable because the query timed out.
• No disease-specific maximum credible population frequency calculation was available in the case bundle.

Sources

Reasoning

No validated assay evidence demonstrates normal EGFR function for L858R. The available variant-specific functional studies instead report increased EGF-induced activation, prolonged activation, and increased sensitivity to EGFR inhibitors compared with wild-type EGFR. These findings do not support a benign functional interpretation and therefore do not meet BS3.

Rule

Generic ACMG/AMP BS3: well-established functional studies showing no damaging effect are required; the available results are abnormal and therefore do not satisfy BS3.

Evidence considered

• PMID:15118073 reported increased and prolonged EGF-induced activation of L858R compared with wild-type EGFR rather than normal function.
• PMID:15329413 reported substantially increased gefitinib and erlotinib sensitivity of L858R compared with wild-type EGFR in a phosphorylation-based assay rather than evidence of a benign effect.

Still missing

• No independent, validated assay showing function comparable to wild-type EGFR was identified.

Sources

Reasoning

This is a missense variant, so BP4 is assessed only on the REVEL protein-impact path. REVEL 0.961 does not meet the benign-supporting BP4 threshold (<0.250). The low SpliceAI score was not considered because no predicted splice impact is not evidence about the amino-acid substitution.

Rule

Generic ACMG/AMP fallback, missense-only computational path: REVEL <0.250 supports BP4 (supporting); calibration reference PMID:36413997.

Evidence considered

• Variant normalization identifies NM_005228.5:c.2573T>G as NP_005219.2:p.(Leu858Arg), a missense substitution.
• The REVEL lookup reports a score of 0.961, which is not below the generic missense BP4 threshold (<0.250); calibrated REVEL thresholds are from ClinGen SVI calibration work (PMID:36413997).
• SpliceAI max delta 0.013 was deliberately not used because the variant is missense and the generic framework requires the protein-impact path only.

Sources

Reasoning

The exact-variant ClinVar expert-panel submission is drug response, not Benign or Likely benign. No expert-panel Benign/Likely benign classification is present, and non-expert assertions cannot trigger BP6.

Rule

Apply BP6 only when an exact-variant ClinVar expert-panel classification is Benign or Likely benign.

Evidence considered

• ClinVar variation 16609 exactly matches NM_005228.5:c.2573T>G (p.Leu858Arg). The only expert-panel submission is ClinPGx 'drug response'; no expert-panel Benign or Likely benign assertion is reported.

Sources

Reasoning

No proband-level germline result with confirmed parental testing is provided. The available literature describes EGFR p.Leu858Arg in somatic lung tumors and does not establish a de novo germline occurrence in this case. Therefore PS2 cannot be assessed.

Rule

Generic ACMG/AMP PS2: de novo occurrence in a patient with confirmed maternity/paternity and without evidence of a family history; assess only with documented proband phenotype, variant testing, and parental testing.

Evidence considered

• The case bundle contains no proband-level parental testing or confirmed de novo germline observation for NM_005228.5:c.2573T>G (p.Leu858Arg).
• Available papers report this alteration in lung cancer tumor tissue; matched normal tissue in the Lynch et al. report was wild type, indicating a somatic tumor event in those published cases rather than establishing a germline de novo event for this case.

Still missing

• Proband germline genotype and phenotype are not documented.
• Neither parent has a documented test result for the variant.
• Parental relationships, sample identity, and maternity/paternity are not documented.
• The available variant reports are somatic tumor studies and cannot substitute for germline de novo evidence.

Sources

Reasoning

No applicable EGFR VCEP/gene-specific specification was retrieved, so generic ACMG/AMP was used. Variant-specific observations in NSCLC cohorts do not provide a case-control comparison, control frequency, or an odds ratio for a defined germline EGFR-associated condition; PS4 cannot be assessed.

Rule

Generic ACMG/AMP PS4: prevalence in affected individuals must be significantly increased compared with controls.

Evidence considered

• No EGFR CSPEC/VCEP framework was available in the case materials; the retrieved framework specifies generic ACMG/AMP fallback.
• PMID:15897572 reported L858R in 9 of 33 EGFR-mutated tumors from a retrospective cohort of 54 Taiwanese patients with advanced NSCLC treated with gefitinib, but did not supply matched control data or a case-control enrichment statistic for this variant.

Still missing

• A defined disease context and appropriately matched affected-versus-control data for EGFR c.2573T>G (p.Leu858Arg), including case and control counts or a defensible odds ratio.

Sources

Reasoning

No applicable EGFR-specific VCEP or gene-specific framework was retrieved, so the generic ACMG/AMP PM3 rule was used. PM3 requires the variant to be observed in trans with a pathogenic variant in an affected individual with a recessive disorder. The available reports describe EGFR p.Leu858Arg as a somatic tumor mutation, including matched-normal testing showing only the wild-type sequence, but provide no affected-proband germline observation, second pathogenic allele, trans phase, or recessive inheritance context. PM3 cannot therefore be assessed from this case evidence.

Rule

Generic ACMG/AMP 2015 PM3: pathogenic variant observed in trans with a pathogenic variant in an affected individual with a recessive disorder.

Evidence considered

• PMID:15118073 reports EGFR L858R in gefitinib-responsive lung-cancer tumors and states that matched normal tissue from tested patients showed only the wild-type sequence, supporting a somatic tumor observation rather than a germline trans configuration.
• PMID:15118125 reports L858R as a heterozygous mutation in lung adenocarcinoma tumors and a tumor-derived cell line, but gives no second pathogenic EGFR allele, trans/cis phase, affected-proband recessive phenotype, or inheritance analysis.
• The case bundle contains no segregation, family, phase, or allelic observations establishing PM3.

Still missing

• No germline affected-proband case data were provided.
• No pathogenic variant in the same gene was documented in trans.
• No phase information or recessive inheritance context was provided.

Sources

Reasoning

PM5 cannot be assessed from the supplied evidence because no alternate missense substitutions at EGFR residue 858 with an established pathogenic classification were identified. The available papers describe L858R itself, not qualifying alternate substitutions at the same residue.

Evidence considered

• The paper extracts for PMID:15118073, PMID:15118125, PMID:15329413, and PMID:15897572 report L858R, the case variant, rather than an alternate amino-acid change at residue 858.
• The supplied PM5 candidate summary reports zero same-residue candidates and states that classic PM5 semantics could not be safely confirmed.

Still missing

• A curated, independently classified pathogenic alternate missense variant at EGFR residue 858 is required to assess PM5.

Sources

Reasoning

No proband-level de novo observation without confirmed parental relationships is documented. The available evidence concerns somatic lung tumors and does not provide an unconfirmed-parentage germline de novo observation. Therefore PM6 cannot be assessed.

Rule

Generic ACMG/AMP PM6: assumed de novo occurrence when maternity/paternity has not been confirmed; requires a documented de novo germline observation and an appropriate phenotype, but does not permit treating somatic tumor observations as germline evidence.

Evidence considered

• The case bundle provides no unconfirmed-parentage de novo germline observation for NM_005228.5:c.2573T>G (p.Leu858Arg).
• The extracted literature reports the variant in somatic lung cancer specimens and therefore does not establish PM6 evidence.

Still missing

• No germline proband observation is documented.
• No parental testing or evidence that parents were unavailable/unconfirmed is documented.
• No phenotype and de novo case report linking this variant to the relevant inherited disorder is documented.
• The available tumor literature is not applicable as PM6 evidence.

Sources

Reasoning

No affected or unaffected relatives, informative meioses, pedigree, familial variant testing, or phenotype-segregation data are provided. The available publications are tumor cohorts and do not constitute familial segregation evidence. Therefore PP1 cannot be assessed.

Rule

Generic ACMG/AMP PP1: co-segregation with disease in multiple affected family members; strength depends on the number and informativeness of meioses and phenotype specificity. Tumor cohorts are not segregation evidence.

Evidence considered

• The case bundle contains no family segregation data for NM_005228.5:c.2573T>G (p.Leu858Arg).
• The extracted publications describe unrelated lung cancer tumor samples and clinical response cohorts, not co-segregation of a germline variant with a phenotype.

Still missing

• No pedigree or number of informative meioses is available.
• No variant results in affected relatives are available.
• No variant results in unaffected relatives are available.
• No phenotype consistency assessment across relatives is available.

Sources

Reasoning

PP2 cannot be assessed because the bundle does not provide a validated gene-level analysis showing that benign missense variation is uncommon and pathogenic missense variation is a common disease mechanism for the relevant EGFR clinical context. The variant-specific oncogenic and functional evidence does not establish the PP2 gene-level rule.

Evidence considered

• No EGFR-specific VCEP framework, gene-level missense rule, or validated benign/pathogenic missense distribution analysis is present in the supplied bundle.
• Variant-specific functional and oncogenic reports were not used as a substitute for the gene-level PP2 requirement.

Still missing

• A validated EGFR missense-constraint and disease-mechanism assessment, applicable to this clinical context, is required.

Reasoning

No applicable EGFR-specific PP4 specification or case phenotype was provided. The available sources describe somatic NSCLC and treatment response, not a phenotype established as highly specific for a germline EGFR-related disorder.

Rule

Generic ACMG/AMP PP4: the patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• No EGFR CSPEC/VCEP framework was available in the case materials; the retrieved framework specifies generic ACMG/AMP fallback.
• The exact ClinVar expert-panel assertion is for gefitinib-response efficacy (drug response), not a Pathogenic/Likely pathogenic assertion for a germline disease phenotype.

Still missing

• The proband phenotype, inheritance context, and a disease-specific demonstration that the phenotype is highly specific for an EGFR-related germline disorder.

Sources

Reasoning

BS2 was not assessed because the case bundle contains no evidence that the exact variant was observed in healthy adults, no homozygote observations, and no disease-specific penetrance or age-of-onset analysis. Absence from population databases cannot establish BS2.

Rule

Generic ACMG/AMP BS2: the variant is observed in a healthy adult individual, or in multiple healthy adults for a recessive disorder, or in a homozygous state in a dominant disorder. No qualifying observation is available.

Evidence considered

• The population evidence reports absence from gnomAD v2.1 and v4.1, but does not report healthy heterozygotes or homozygotes for this exact variant.
• Somatic tumor reports and treatment-response studies were not used for BS2 because they do not establish healthy germline carriage or homozygosity.

Still missing

• No healthy-adult carrier observations were provided.
• No homozygote observations were provided.
• No disease-specific penetrance, prevalence, age-of-onset, or ascertainment analysis was provided.

Reasoning

No affected family members tested for the variant and no informative unaffected relatives are documented. Consequently, there is no reliable non-segregation evidence to support BS4.

Rule

Generic ACMG/AMP BS4: lack of segregation in affected members of a family; apply only when family structure, phenotype, testing, and informativeness support a valid non-segregation assessment.

Evidence considered

• The case bundle contains no familial testing or non-segregation observations for NM_005228.5:c.2573T>G (p.Leu858Arg).
• Wild-type matched normal tissue reported in somatic lung cancer studies is not an unaffected relative and cannot be used as BS4 evidence.

Still missing

• No pedigree or familial phenotype information is available.
• No testing of affected relatives is available.
• No testing of informative unaffected relatives is available.
• No penetrance, age, or phenotype-specificity information is available to interpret a negative familial result.

Sources

Reasoning

BP1 cannot be assessed because the bundle does not establish that truncating variants are a well-established predominant disease mechanism for the relevant EGFR disorder, such that missense variants would be relatively less likely to be pathogenic. The available evidence concerns EGFR somatic activating missense biology and does not establish the BP1 gene-level premise.

Evidence considered

• No applicable EGFR VCEP specification or validated gene-level evidence establishing truncating-variant predominance is present in the supplied bundle.
• The supplied literature describes activating EGFR missense biology and therefore cannot by itself establish BP1.

Still missing

• A disease-context-specific demonstration that truncating variants predominate as the pathogenic mechanism is required for BP1.

Reasoning

No applicable EGFR-specific VCEP or gene-specific framework was retrieved, so the generic ACMG/AMP BP2 rule was used. BP2 requires observation in trans with a pathogenic variant for a fully penetrant dominant disorder or in cis with a pathogenic variant in any inheritance model. The available evidence is limited to somatic tumor reports of EGFR p.Leu858Arg and contains no documented pathogenic allele in the same individual, cis/trans phase, or germline inheritance context. BP2 cannot therefore be assessed.

Rule

Generic ACMG/AMP 2015 BP2: observed in trans with a pathogenic variant for a fully penetrant dominant disorder or in cis with a pathogenic variant in any inheritance model.

Evidence considered

• PMID:15118073 describes L858R in tumor samples and matched-normal testing showing only wild-type sequence in tested patients, without reporting a second pathogenic allele or cis/trans relationship.
• PMID:15118125 describes L858R in lung tumors and a tumor-derived cell line, without reporting a second pathogenic variant or allelic phase.
• The case bundle contains no germline compound-variant, phase, or inheritance evidence establishing BP2.

Still missing

• No second pathogenic variant in the same individual was documented.
• No cis or trans phase information was provided.
• No germline inheritance or affected-proband context was provided.

Sources

Reasoning

No proband phenotype or independent molecular diagnosis was supplied. Therefore, there is no evidence that the observed phenotype is explained by an alternate molecular cause.

Rule

Generic ACMG/AMP BP5: a variant is found in a case with an alternate molecular basis for disease.

Evidence considered

• The case materials contain variant-level somatic cancer and drug-response evidence but no proband-level alternate molecular diagnosis.

Still missing

• Proband-level phenotype and molecular testing results establishing an alternate diagnosis that fully explains the presentation.