ALK c.3586C>A, p.Leu1196Met

Reasoning

PS3 is met at strong strength. The exact ALK p.Leu1196Met variant was evaluated in multiple complementary functional systems. In engineered EML4-ALK Ba/F3 cells, L1196M increased baseline ALK phosphorylation, supported IL-3-independent growth, and markedly reduced inhibitor sensitivity; the reported PF-1066 IC50 was 1815 nM. An independent accelerated-mutagenesis study reproduced the resistance phenotype after site-directed reintroduction, measured a crizotinib IC50 greater than 1000 nM for ALK phosphorylation inhibition, and found complete insensitivity of L1196M Ba/F3 xenografts to crizotinib at 100 or 200 mg/kg daily. A separate patient-derived resistance study also demonstrated high-level crizotinib resistance in engineered Ba/F3 cells. These orthogonal biochemical, cellular, reproducibility, and in vivo results are biologically relevant to ALK kinase function and support a damaging gain-of-function effect, although they do not establish loss of protein function or a splicing defect.

Rule

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

Evidence considered

• Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors, Results/Figures 4B–4E and Table 2: engineered EML4-ALK L1196M increased baseline phosphorylation, supported IL-3-independent Ba/F3 growth, and showed PF-1066 IC50 1815 nM.
• Crizotinib-resistant mutants of EML4-ALK identified through an accelerated mutagenesis screen, Results/Table 2 and xenograft studies: L1196M had crizotinib viability IC50 981 nM, ALK-phosphorylation inhibition IC50 >1000 nM, reproduced resistance after site-directed reintroduction, and produced xenografts completely insensitive to crizotinib at 100 or 200 mg/kg daily.
• Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer, Results and Discussion: L1196M was identified in two patients and showed greater crizotinib resistance than G1269A in engineered EML4-ALK Ba/F3 assays without a growth disadvantage in the absence of drug.
• Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers, Results/Figures 1–2: L1196M was identified in a crizotinib-resistant tumor and conferred high-level resistance in engineered EML4-ALK Ba/F3 cells, with reduced but retained activity of other ALK inhibitors.

Sources

Reasoning

PM2 is met at supporting strength. The variant is absent from gnomAD v2.1 and gnomAD v4.1, and the available non-cancer gnomAD v2.1 and v3.1 records also report absence. With no VCEP-specific population-source requirement, the all-comers datasets are the default; the absence is consistent with the supplied PM2 supporting threshold of <=0.0001.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 reports the variant as absent.
• gnomAD v4.1 reports the variant as absent.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer records also report absence; no VCEP rule requires those sources for this generic interpretation.
• The supplied generic PM2 calibration threshold is <=0.0001, with PM2 downgraded to supporting strength under the ClinGen SVI recommendation identified in the case prompt (PMID:25741868).

Sources

Reasoning

The governing rule is: "The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls." ACMG 2015 Note 1 specifies: "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." The reviewed variant-specific papers report ALK p.L1196M in crizotinib-resistant affected NSCLC patients, including two patients in PMID:22235099 and one patient/resistant tumor in PMID:22277784, but provide no unaffected or population-control carrier counts or matched-ancestry control frequency for this exact variant. Therefore no OR/RR or 95% CI can be computed: the required inequality is OR > 5.0, but no OR value is available to substitute, and the CI criterion also cannot be evaluated. The evidence demonstrates case observation but not the required statistically significant case-control enrichment.

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

• PMID:22235099, Results: ALK L1196M was identified in two patients with acquired crizotinib resistance; the paper does not provide unaffected-control carrier counts for this exact variant.
• PMID:22277784, Results and Table 1: L1196M was identified in a crizotinib-resistant tumor; the study reports affected resistant patients but no unaffected or population-control comparison for this exact variant.
• PMID:22034911 and PMID:21613408 were checked because literature triage classified them as variant-specific; both report experimental resistance/functional assays rather than a case-control prevalence comparison.
• No OR/RR can be calculated from the available data; the governing operator is >5.0, but there is no numeric OR/RR to substitute and no 95% CI to test against 1.0.

Still missing

• Exact-variant unaffected or population-control carrier counts, or a matched-ancestry control frequency, are unavailable.
• No computable OR/RR and 95% confidence interval are available.

Sources

Reasoning

The variant is the missense substitution NM_004304.5:c.3586C>A, p.(Leu1196Met), so the generic PP3/BP4 missense path requires REVEL and does not use SpliceAI. REVEL is 0.598, below the generic PP3 supporting threshold of >=0.644 calibrated by Pejaver et al. 2022 (PMID:36413997); therefore PP3 is not met. BayesDel is not used because no generic ACMG-strength calibration is established, and the SpliceAI max delta of 0.001 is outside the applicable missense path.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.) ... PP3 (supporting): >= 0.644 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• The normalized consequence is missense: NM_004304.5:c.3586C>A, NP_004295.2:p.(Leu1196Met).
• Local REVEL predictor lookup reports a score of 0.598 for the variant (source_registry key: revel).
• Published generic calibration: PP3 supporting requires REVEL >=0.644 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997).
• Under the generic missense path, REVEL is the sole applicable predictor; the available SpliceAI max delta score of 0.001 and BayesDel score are not used for PP3.

Sources

Reasoning

The governing rule is: "Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation." Under the required generic rule for this interpretation, PP5 is restricted to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. ClinVar variation 376135 has one exact-variant Likely pathogenic clinical-laboratory submission with no assertion criteria, zero stars, and no expert-panel submission. Thus the required expert-panel source is absent and PP5 is not met.

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 variation 376135 exactly matches NM_004304.5(ALK):c.3586C>A (p.Leu1196Met), but the only submission is Likely pathogenic from one clinical laboratory, with no assertion criteria, zero review stars, and no expert-panel submission.
• The ClinVar classification is therefore not eligible to trigger PP5 under the required exact-variant expert-panel-only rule.

Sources

Reasoning

BA1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, so no filtering allele frequency or grpmax FAF is available and the >=0.05 stand-alone benign threshold is not reached. The non-cancer gnomAD v2.1 and v3.1 records are also absent; no population-specific frequency supports BA1.

Rule

BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v2.1 reports the variant as absent; therefore no filtering allele frequency or grpmax FAF is available.
• gnomAD v4.1 reports the variant as absent; therefore no filtering allele frequency or grpmax FAF is available.
• The available gnomAD non-cancer records also report absence, and no governing VCEP rule selects an alternative population source.
• The generic BA1 calibration threshold is >=0.05, supplied in this case as ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).

Sources

Reasoning

BS1 is not met on the available population evidence. The variant is absent from gnomAD v2.1 and v4.1, so no filtering allele frequency or grpmax FAF exceeds the generic >=0.01 threshold. No disease-specific maximum credible allele-frequency calculation is available, but the absence itself provides no support for BS1.

Rule

BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v2.1 reports the variant as absent; no filtering allele frequency or grpmax FAF is available for comparison with the BS1 threshold.
• gnomAD v4.1 reports the variant as absent; no filtering allele frequency or grpmax FAF is available for comparison with the BS1 threshold.
• The generic BS1 default calibration threshold is >=0.01; the prompt requires frequency to be evaluated against disorder prevalence, penetrance, and allelic heterogeneity, with Whiffin et al. 2017 preferred for a per-gene derivation (PMID:28518168).

Still missing

• A gene- and disorder-specific maximum credible allele-frequency calculation is unavailable because a validated germline ALK disorder prevalence, penetrance, and allelic-heterogeneity model is not established in the case materials.

Sources

Reasoning

The variant is a missense substitution, so the generic PP3/BP4 missense path requires REVEL only; SpliceAI is not an alternative path for this variant. REVEL is 0.598, which is above the generic BP4 supporting threshold of <=0.29 and therefore does not support a benign computational effect. The score is not in a BP4 range, so BP4 is not met. BayesDel is not used because no generic ACMG-strength calibration is established.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• The normalized consequence is missense: NM_004304.5:c.3586C>A, NP_004295.2:p.(Leu1196Met).
• Local REVEL predictor lookup reports a score of 0.598 for the variant (source_registry key: revel).
• Published generic calibration: BP4 supporting requires REVEL <=0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997).
• Under the generic missense path, SpliceAI is not used to substitute for a non-supportive REVEL result; the available SpliceAI max delta score of 0.001 is therefore not BP4 evidence for this missense variant.

Sources

Reasoning

The governing rule is: "Variant found in a case with an alternate molecular basis for disease." The available evidence reports ALK L1196M in ALK-positive or ALK-rearranged cancer contexts and in acquired crizotinib resistance, but does not document a case in which an alternate molecular basis explains the disease instead of this variant. No case-specific alternate pathogenic molecular finding is available to satisfy BP5.

Rule

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

Evidence considered

• The available literature describes ALK-positive cancer and acquired resistance settings, but does not document an alternate molecular basis for the disease in a case carrying this exact variant.
• No case-specific alternate molecular diagnosis was supplied.

Still missing

• A case-level alternate pathogenic molecular finding and evidence that it explains the disease are not documented.

Sources

Reasoning

The governing rule is: "Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation." Under the required generic rule for this interpretation, BP6 is restricted to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. ClinVar variation 376135 has one exact-variant Likely pathogenic clinical-laboratory submission, no assertion criteria, zero stars, and no expert-panel submission; there is no qualifying benign expert-panel classification. Therefore 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 variation 376135 exactly matches NM_004304.5(ALK):c.3586C>A (p.Leu1196Met), but its sole submission is Likely pathogenic from one clinical laboratory, with no assertion criteria, zero review stars, and no expert-panel submission.
• No exact-variant ClinVar expert-panel Benign or Likely benign classification is present, so BP6 cannot be applied.

Sources

Reasoning

The variant is ALK p.Leu1196Met (c.3586C>A). The reviewed literature documents this same nucleotide and amino-acid change in cancer resistance studies, but does not establish a previously pathogenic ALK missense variant caused by a different nucleotide change at the same codon. The ClinVar submission is a single somatic, zero-star likely pathogenic submission without assertion criteria and cannot establish the required comparator. Therefore, the same-amino-acid/different-nucleotide evidence needed for PS1 is not available.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• PMID:21613408, Results/Figures 4B-4E and Table 2: experimentally studies ALK L1196M, the queried amino-acid change.
• PMID:22034911, Results/Table 2: experimentally studies ALK L1196M and reports its resistance phenotype.
• PMID:22235099, Results and Discussion: reports L1196M in two patients and in engineered EML4-ALK.
• PMID:22277784, Results/Table 1/Figures 1-2: reports L1196M as a secondary resistance mutation.
• ClinVar variation 376135: one somatic likely pathogenic submission, no assertion criteria, zero review stars; this is not an established pathogenic comparator for PS1.

Still missing

• A validated pathogenic ALK variant caused by a different nucleotide change but producing p.Leu1196Met is not documented.

Sources

Reasoning

No proband-level clinical evidence, family-history assessment, or parental testing results are provided. Therefore, it cannot be determined whether the variant is de novo with both maternity and paternity confirmed in a patient with the disease and no family history.

Rule

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

Still missing

• Proband diagnosis and phenotype consistent with the relevant disease
• Family-history assessment
• Maternity confirmation
• Paternity confirmation

Reasoning

ALK p.Leu1196Met lies in the ALK tyrosine-kinase domain and at the gatekeeper position in the ATP/crizotinib-binding pocket. The cited studies provide strong functional evidence that this is a critical functional region. However, the generic PM1 rule also requires the domain to be without benign variation, and the available population evidence only shows that the queried variant is absent from gnomAD v2.1 and v4.1; it does not establish absence of benign missense variation throughout the ALK kinase domain. No VCEP domain table or approved hotspot rule is available. The complete generic PM1 determination therefore remains unresolved.

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

• PMID:21613408, Results section on ALK variants: L1196M is studied in the ALK kinase fusion and alters kinase activity and inhibitor sensitivity.
• PMID:22034911, Results/Discussion: L1196M is described as a gatekeeper mutation in the ALK kinase domain that impairs crizotinib binding/inhibition.
• PMID:22235099, Results/Discussion: L1196M is located in the ATP/crizotinib-binding pocket and is characterized as a gatekeeper resistance mutation.
• PMID:22277784, Results: L1196M is reported among secondary mutations within the ALK tyrosine-kinase domain.
• gnomAD v2.1 and gnomAD v4.1: the queried ALK c.3586C>A variant is absent; these records do not provide a domain-wide benign-variation analysis.
• Cancer Hotspots: no statistically significant hotspot row was found for ALK L1196; no VCEP-specific hotspot route exists for this generic interpretation.

Still missing

• Domain-wide benign-variation assessment for the ALK tyrosine-kinase domain is not available.

Sources

Reasoning

PM3 cannot be assessed because the available evidence does not document a recessive disorder context, an affected proband with a pathogenic variant in trans, or parental/offspring testing establishing phase. The reported ALK p.Leu1196Met functional and somatic-cancer literature does not provide the required germline allelic observation or phase information.

Rule

PM3: For recessive disorders, detected in trans with a pathogenic variant ... Note: This requires testing of parents (or offspring) to determine phase.

Evidence considered

• Case evidence identifies ALK NM_004304.5:c.3586C>A (NP_004295.2:p.(Leu1196Met)) but provides no family, affected-proband, parental, offspring, or phase observations.
• Variant-specific publications PMID:22034911, PMID:22235099, PMID:22277784, and PMID:21613408 describe somatic NSCLC resistance or engineered EML4-ALK functional assays, not germline allelic observations establishing a pathogenic variant in trans.

Still missing

• A defined recessive ALK disorder context is not documented.
• No affected proband with a known pathogenic variant in trans is documented.
• No parental or offspring testing establishes phase.

Reasoning

The available same-residue review did not identify a distinct missense substitution at ALK residue 1196 that had been determined pathogenic. The cited papers report the queried L1196M substitution, but do not provide a different pathogenic missense change at residue 1196. The collected ClinVar record is for the queried variant itself and is a single somatic submission without assertion criteria, so it cannot supply the required comparator.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• pm5_candidates: no same-residue comparator candidates were collected; the artifact does not itself establish PM5.
• PMID:21613408, PMID:22034911, PMID:22235099, and PMID:22277784: each reports/studies L1196M, not a different pathogenic missense substitution at residue 1196.
• ClinVar variation 376135: the record is for the queried L1196M variant and contains one somatic likely pathogenic submission without assertion criteria.

Still missing

• A different missense variant at ALK residue 1196 with a validated pathogenic determination is not documented.

Sources

Reasoning

No proband-level clinical evidence, family-history assessment, or parental testing results are provided. Therefore, there is no basis to assess an assumed de novo occurrence without confirmation of paternity and maternity.

Rule

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

Still missing

• Proband diagnosis and phenotype consistent with the relevant disease
• Family-history assessment
• Parental testing results or documented unavailability of maternity and paternity confirmation

Reasoning

No affected relatives, family genotypes, or segregation data are provided. The available evidence concerns a somatic cancer resistance variant and does not establish co-segregation with disease in affected family members.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... May be used as stronger evidence with increasing segregation data.

Still missing

• Affected family members and their disease phenotypes
• Genotypes for affected relatives
• Pedigree and informative meioses

Reasoning

The available evidence describes ALK missense mutations in cancer and the queried variant's kinase-domain activity, but does not establish both gene-level prerequisites for PP2: a low rate of benign missense variation and missense variants as a common disease mechanism in the applicable germline disease context. No gene-wide benign-missense analysis or validated disease-mechanism summary meeting both requirements is available.

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 available ALK literature is primarily somatic cancer and drug-resistance research and does not establish the two gene-level PP2 prerequisites.

Still missing

• A validated gene-wide benign missense-rate assessment is not available.
• A validated germline disease-context assessment that missense variation is a common ALK disease mechanism is not available.

Reasoning

The governing rule is: "Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology." The available case materials identify the variant and literature observations in resistant ALK-positive NSCLC, but do not provide the present patient's phenotype, family history, or a phenotype-specific diagnostic context demonstrating a disease with a single genetic etiology. The published studies are not sufficient to substitute for the patient's phenotype assessment.

Rule

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

Evidence considered

• The reviewed papers describe acquired crizotinib-resistant ALK-positive NSCLC cohorts, not the present patient's phenotype or family history.
• PMID:22235099 and PMID:22277784 therefore cannot establish that this patient's phenotype is highly specific for a disease with a single genetic etiology.

Still missing

• Patient phenotype and diagnostic features are not provided.
• Patient family history and evidence for a single genetic etiology are not provided.

Sources

Reasoning

BS2 cannot be assessed from the available evidence. No healthy adult carrier, homozygote, or hemizygote observation is reported, and the case does not establish a specific germline ALK disorder with early-onset full penetrance or its inheritance model. Absence of a reported observation is not evidence that the BS2 healthy-adult condition has been satisfied.

Rule

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.

Still missing

• A defined germline disorder, inheritance model, expected age of onset, penetrance, and documented healthy adult observation are needed to assess BS2.

Reasoning

BS3 is not assessed. The available studies do not show that ALK p.Leu1196Met has no damaging effect on protein function or splicing. Instead, they report increased baseline ALK phosphorylation, preserved oncogenic cellular growth, and resistance to crizotinib. Retained sensitivity to alternative ALK inhibitors does not establish normal protein function and cannot support BS3.

Rule

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

Evidence considered

• The reviewed functional studies show altered ALK phosphorylation, drug resistance, or preserved oncogenic growth rather than demonstrating absence of a damaging effect; retained activity of alternative inhibitors is not evidence of normal ALK function.

Still missing

• No well-established functional or splicing study demonstrates normal ALK p.Leu1196Met function under the BS3 definition.

Sources

Reasoning

No affected family members, family genotypes, or pedigree information are provided. Consequently, lack of segregation cannot be evaluated, and no conclusion about phenocopies or multiple pathogenic variants can be made.

Rule

Lack of segregation in affected members of a family.

Still missing

• Affected family members and their disease phenotypes
• Genotypes for affected relatives
• Pedigree and assessment for phenocopies or multiple pathogenic variants

Reasoning

The available evidence does not establish that ALK disease is caused primarily by truncating variants. The reviewed ALK studies instead focus on activating or drug-resistance missense substitutions in the kinase domain, but those somatic cancer observations do not by themselves establish the complete germline variant spectrum needed to rule BP1 in or out. Therefore BP1 is not assessed.

Rule

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

Evidence considered

• PMID:21613408, PMID:22034911, PMID:22235099, and PMID:22277784: describe ALK kinase-domain missense mutations in somatic cancer and treatment resistance, but do not establish the germline disease variant spectrum required for BP1.

Still missing

• A comprehensive, validated disease-context variant spectrum showing whether ALK disease is primarily truncating is not available.

Sources

Reasoning

BP2 cannot be assessed because no evidence documents this variant in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant. The available literature concerns somatic or engineered ALK contexts and does not establish cis/trans phase for this variant in an affected individual.

Rule

BP2: 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

• Case evidence identifies ALK NM_004304.5:c.3586C>A (NP_004295.2:p.(Leu1196Met)) but provides no cis/trans phase data or pathogenic partner variant.
• Variant-specific publications PMID:22034911, PMID:22235099, PMID:22277784, and PMID:21613408 concern somatic NSCLC resistance or engineered EML4-ALK assays and do not establish the BP2 allelic configuration.

Still missing

• No pathogenic partner variant is documented.
• No cis/trans phase testing is documented.
• A fully penetrant dominant ALK disorder context is not established for this observation.