• ~90% of EGFR mutations in lung adenocarcinoma can be attributed to 2 targetable mutations detectable by immunohistochemistry (IHC) (J Thorac Oncol 2017;12:612)
  • EGFR L858R point mutation in exon 21
  • EGFR E746_A750 del (15 base pair deletion) in exon 19

Images hosted on other servers:

• Positive: strong, diffuse membranous staining, variable granular cytoplasmic staining
  • Use stringent criteria to avoid false positive results; high specificity (~99%)
  • Positive IHC result for EGFR clone 43B2 or 6B6 associated with response to EGFR TKI drugs
  • ~1% false positives (cross reactivity with HER2 overexpression)
    • Usually 2+ but rarely 3+ (Mod Pathol 2013;26:1197, see Microscopic images)
• Equivocal or negative: moderate / patchy membranous staining or cytoplasmic staining only
• Negative: no staining or weak membranous staining or weak cytoplasmic staining only
• Low sensitivity (~70 - 80%)
• All results (positive, equivocal and negative) should be confirmed by molecular testing, including molecular profiling of multiple genes whenever possible
• Suboptimal fixation and processing can cause false negatives and equivocal results
• References: Cytopathology 2022;33:14, Arch Pathol Lab Med 2016;140:1331

Contributed by Fang Zhou, M.D.

Positive stain results


Equivocal stain results


Negative (nonspecific) staining

Contributed by Fang Zhou, M.D.

Positive stain results

• EGFR mutations in lung adenocarcinoma
  • White patients: ~10%
  • Asian patients: ~50%
• 2 targetable mutations comprise ~90% of all EGFR mutations in lung adenocarcinoma (J Thorac Oncol 2017;12:612)
  • EGFR L858R point mutation in exon 21: IHC clone 43B2
  • EGFR E746_A750del (15 base pair deletion) in exon 19: IHC clone 6B6
  • Mutation specific IHC assays are available
• Notes on these 2 mutation specific IHC antibodies (Arch Pathol Lab Med 2016;140:1331)
  • High specificity if using careful and conservative interpretation guidelines = positive results are associated with response to tyrosine kinase inhibitors (TKIs), specificity ~99%
    • Positive cases should have strong, diffuse membranous staining, with or without cytoplasmic staining
    • Cytoplasmic staining only = 80% do not have the mutation (PLoS One 2013;8:e59183)
    • Antibodies cross react with HER2, causing false positive results in HER2+ cases (rare, ~1%)
  • Low sensitivity for these 2 mutations (~80%)
  • They also do not detect other targetable or TKI resistance associated EGFR mutations (~10%)
  • They do not detect EGFR amplification (not targetable anyway)
  • Generally not recommended if there is access to (and adequate material for) high sensitivity molecular assays that may detect more mutations in EGFR and other genes (Arch Pathol Lab Med 2018;142:321)
  • Particularly useful in the following situations
    • Specimen is inadequate for molecular assays (low tumor cellularity or purity or decalcified)
    • There is simultaneous wild type allele amplification that obscures interpretation of EGFR molecular results (Mod Pathol 2021;34:2168)
    • Quick turnaround time is needed (1 - 2 days)
    • No access to molecular testing

• Lung, right lower lobe, biopsy:
  • Adenocarcinoma (see comment)
  • Comment: The tumor cells are positive for TTF1 while negative for p40, supporting the diagnosis. Please see synoptic report for the results of biomarker testing.
  • CAP lung cancer biomarker reporting protocol (synoptic report)
    
    

    Specimen
    Adequacy of sample for testing	Adequate
    Results
    EGFR
    EGFR L858R by immunohistochemistry (clone 43B2)	Negative
    EGFR exon 19 deletion (E746_A750del) (clone 6B6)	Positive
    Interpretation	EGFR E746_A750del immunohistochemical staining is positive, which is associated with response to EGFR tyrosine kinase inhibitors
    ALK
    ALK immunohistochemistry	Negative
    PDL1 IHC
    PDL1 IHC interpretation	Positive
    Percentage of tumor cells with staining (TPS)	40%
    Methods
    Antibody	22C3
    Controls	Internal control cells present; expected immunoreactivity
    	External controls available; expected immunoreactivity
    Assay information	Laboratory developed test

The image above depicts a positive EGFR mutation specific IHC result. What mutations in EGFR are most commonly detected in lung adenocarcinoma?

1. E746_A750del and EGFR R776C
2. EGFR G719A and EGFR S768I
3. EGFR L858R and E746_A750del
4. EGFR L858R and EGFR T790M
5. EGFR L861Q and EGFR G719X

C. EGFR L858R and E746_A750del. Among EGFR mutations in lung adenocarcinoma, these 2 mutations are the most common. Overall, KRAS is the most commonly mutated gene in lung adenocarcinoma (it is more commonly mutated than EGFR) and targeted therapies against specific KRAS mutations (such as KRAS p.G12C) are being developed. Answers A, B, D and E are incorrect because they include rare EGFR variants.

Comment Here

Reference: EGFR mutation specific antibodies

What is the current consensus recommendation for molecular testing in patients with lung adenocarcinoma?

1. A broad diagnostic IHC panel should be used in the biopsy diagnosis of lung cancer
2. ALK rearrangement should be tested by IHC only
3. Cytology specimens are never considered adequate for molecular testing
4. EGFR mutation specific IHC testing should take priority
5. Molecular profiling of multiple genes should be performed whenever possible

E. Molecular profiling of multiple genes should be performed whenever possible to detect targetable, rare and other mutations that may qualify patients for clinical trials. Answer D is incorrect because whenever possible, molecular profiling of multiple genes (including EGFR) is preferred over EGFR only testing. Answer C is incorrect because cytology cell blocks can be used successfully for molecular testing. Even smears may be used (alcohol is superior to formalin for nucleic acid preservation). Answer B is incorrect because ALK rearrangements are not only detectable by IHC but also by FISH, PCR and NGS methods. Answer A is incorrect because the current consensus recommendation is to preserve tissue for molecular detection of targetable mutations when possible. Most cases of non-small cell lung carcinoma can be classified as adenocarcinoma or squamous cell carcinoma using 2 IHC stains: TTF1 (clone 8G7G3/1) and p40. If both markers are negative or very focal, the need for additional diagnostic IHC is determined by clinical history and imaging findings (Arch Pathol Lab Med 2018;142:321, Curr Oncol 2022;29:4981, J Thorac Oncol 2019;14:377).

Comment Here

Reference: EGFR mutation specific antibodies