Context.—Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are molecular-targeted drugs that are innovatively effective for non–small cell lung carcinomas with EGFR mutations. Epidermal growth factor receptor is a transmembrane receptor forming dimers on ligand binding. These then stimulate signals by activating receptor autophosphorylation through tyrosine kinase activity. Autophosphorylation triggers intracellular pathways facilitating malignant conversion. The most clinically advanced EGFR inhibition strategies include small-molecule inhibition of the intracellular tyrosine kinase domain (gefitinib and erlotinib) and monoclonal antibody–mediated blockade of the extracellular ligand-binding domain (cetuximab). Lung cancers with EGFR mutations are prevalent among patients who are female, of Asian ethnicity, and nonsmokers; thus, they can obtain benefit from EGFR tyrosine kinase inhibitors.

Objective.—To survey histopathologic findings and examine correlations with EGFR mutations. We mainly focused on component cell types (hobnail, columnar, and polygonal) and presence or absence of bronchioloalveolar carcinoma elements and a micropapillary pattern. Although EGFR mutations can be detected by various methods, including polymerase chain reaction–Invader assay or direct sequencing, these are inconvenient.

Data Sources.—Review of the published literature.

Conclusion.—Detailed pathologic examination showed significant genotype-phenotype correlations between EGFR mutations and presence of a bronchioloalveolar carcinoma component, a micropapillary pattern, and the hobnail cell type. We conclude that these characteristic histologic features are good predictors of EGFR mutations, and patients with these features might be good candidates for and could benefit from therapy with EGFR tyrosine kinase inhibitors.

The epidermal growth factor receptor (EGFR), a receptor of ligands including epidermal growth factor (EGF), is a 170-kDa glycoprotein tyrosine kinase protein that straddles the cell membrane.

Expression of EGFR is prevalent in variant normal cells including cells of epidermal, mesenchymal, and neurogenic origins. When EGF binds to EGFR, signaling pathways are activated that can lead to cell proliferation and differentiation. Epidermal growth factor receptor plays an important role in cell differentiation, development, proliferation, and maintenance. With EGFR gene overexpression due to mutation or structural alteration, carcinogenesis, invasion, and metastasis are facilitated.

In recent years there has been substantial interest in developing novel therapeutic agents that specifically target growth factor pathways that are dysregulated in cancer cells. Non–small cell lung cancer (NSCLC) is the most frequent cause of cancer death in the world and targeting EGFR has played a central role in advancing NSCLC research to improve patient outcome during the last several years. With the move to personalized cancer therapy, we need to understand oncologic biology at the molecular and histopathologic levels in individual lesions. In this review article, we focus on clinicopathologic features related to EGFR change and consider their indications for clinical application.

Lung cancer is the leading cause of cancer death in men and women worldwide and identification of activating mutations of EGFR is one of the most intriguing recent discoveries in the field of lung cancer research.1,2 Epidermal growth factor receptor mutations are present in a particular subtype of lung adenocarcinomas, and cancers with this mutation have been shown to be highly sensitive to chemical inhibitors of the kinase activity of EGFR. This subtype is prevalent among patients who are female, of Japanese and other Asian ethnicity, and nonsmokers.2–4 K-ras is a downstream mediator of EGFR-induced cell signaling, and K-ras mutations confer constitutive activation of the signaling pathways without EGFR activation. Growing evidence indicates that K-ras mutations are also important in the development of lung carcinomas.5 Very recently, we found a novel transforming fusion gene resulting from linkage between the echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes in NSCLCs.6 This translocation promotes strong tyrosine kinase activity, which is a prominent feature of ALK, leading to intensive oncogenesis in NSCLCs. Cancers featuring EML4-ALK fusion thus constitute a subtype of NSCLCs that might be highly sensitive to ALK inhibitors. Interestingly, EGFR mutation, K-ras mutation, and EML4-ALK translocation are mutually exclusive.7,8 Furthermore, lung cancers with each of these alterations appear to have their own particular clinicopathologic characteristics.

In 1975, the existence of the EGF-specific receptor was first reported on the cell membrane of the fibroblast.9 Thereafter, from work with the A431 human cancer cell line, EGFR was initially defined as a 170-kDa protein.10 In 1984, the sequence of v-erbB, an oncogene of the avian erythroblastic leukemia virus, was reported to be extremely similar to that of EGFR.11 Gene products of oncogene erbB and EGFR subsequently turned out to be identical proteins. Thereafter, it was found that human genes corresponding to v-erbB were not only EGFR but also human epidermal growth factor receptor 2 (HER2), these 2 now being referred to as ERBB1 and ERBB2, respectively.

Growth factors belong to a family of polypeptides that have been shown to stimulate proliferation and/or differentiation in both normal and malignant cells. One of the first growth factors discovered was EGF. Later studies showed that this protein binds to the cell surface growth factor receptor EGFR, thereby either inducing cell proliferation or differentiation in mammalian cells.

The binding of a ligand to EGFR induces conformational changes within the receptor, which increase the catalytic activity of its intrinsic tyrosine kinase, resulting in the autophosphorylation that is necessary for biologic activity. Epidermal growth factor receptor is a 170-kDa transmembrane glycoprotein that binds to specific ligands. The erbB family cell-signaling process uses EGF-like ligands that include cell-signaling transforming growth factor α (TGF-α), amphiregulin, heparin-binding EGF, epiregulin, heregulin, neuregulin, and betacellulin. Epidermal growth factor receptor is known to bind with particularly high affinity to EGF, amphiregulin, and TGF-α.

As noted above, EGFR is a member of the erbB family of receptor tyrosine kinase proteins, now known to also include HER2/neu (erbB2), HER3 (erbB3), and HER4 (erbB4). These receptors are all composed of an extracellular ligand-binding domain, a transmembrane lipophilic domain, and an intracellular tyrosine kinase domain and, with the exception of HER2, all bind to receptor-specific ligands (Figure 1). Phosphorylation of the tyrosine kinase domain followed by homodimerization or heterodimerization between receptors of the same family leads to protein activation on the cell surface. In cancer cells, this is believed to promote signaling cascades, cell growth, differentiation, cell survival, cell cycle progression, and angiogenesis.

Figure 1.

The epidermal growth factor receptor (EGFR) family proteins and their ligands. TGF, transforming growth factor; HB-EGF, heparin-binding epidermal growth factor–like growth factor; HER, human epidermal growth factor receptor

Figure 1.

The epidermal growth factor receptor (EGFR) family proteins and their ligands. TGF, transforming growth factor; HB-EGF, heparin-binding epidermal growth factor–like growth factor; HER, human epidermal growth factor receptor

Close modal

The approximately 200-kb human EGFR gene, comprising 28 exons and 27 introns, exists on the short arm of chromosome 7 (7p12). Exons 1 to 16 encode the extracellular domain, while exon 17 codes for the transmembrane domain, and exons 18 to 28 are responsible for the intracellular domains. The tyrosine kinase domain is encoded by exons 18 to 24, while the C-terminal domain is encoded by exons 25 to 28.

Receptor tyrosine kinases, such as EGFR, transmit extracellular signals of growth factors into the intracytoplasmic region and transmit their stimulus to the nuclei by signal transduction. As a result, transcriptional upregulation follows, leading to protein synthesis and transformation of cell functions or cellular architecture.

As signaling pathways of EGFR, the Ras/Raf/MAPK (mitogen-activated protein kinase) pathway, the PI3K (phosphatidylinositol-3-kinase)/Akt pathway, and the Jak (Janus kinase)/ STAT (signal transducers and activator of transcription) pathway are all important. As a result of the signal transduction, cell differentiation or cell proliferation are promoted. The Ras/Raf/MAPK pathway mainly promotes cell proliferation and survival, while the PI3K/Akt pathway is mainly associated with cell growth, inhibition of apoptosis, invasion, or migration (Figure 2).

Figure 2.

Schematic illustration of the epidermal growth factor receptor (EGFR) and downstream signaling pathways. Binding of a receptor-specific ligand leads to phosphorylation of EGFR and signaling through the mitogen-activated protein kinase (MAPK) pathway (green), signal transducers and activator of transcription (STAT) pathway (blue), and phosphatidylinositol-3-kinase (PI3K)/Akt pathway (orange). These pathways promote cell proliferation, angiogenesis, migration, adhesion, and/or invasion, while inhibiting apoptosis. SOS, son of sevenless; Grb-2, growth factor receptor–bound protein 2; Ras and Raf are well-known oncoproteins; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase

Figure 2.

Schematic illustration of the epidermal growth factor receptor (EGFR) and downstream signaling pathways. Binding of a receptor-specific ligand leads to phosphorylation of EGFR and signaling through the mitogen-activated protein kinase (MAPK) pathway (green), signal transducers and activator of transcription (STAT) pathway (blue), and phosphatidylinositol-3-kinase (PI3K)/Akt pathway (orange). These pathways promote cell proliferation, angiogenesis, migration, adhesion, and/or invasion, while inhibiting apoptosis. SOS, son of sevenless; Grb-2, growth factor receptor–bound protein 2; Ras and Raf are well-known oncoproteins; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase

Close modal

In a wide range of solid cancers, EGFR overexpression has been detected to varying degrees (Table).12 Reported values are 30% to 38% for gastric adenocarcinomas,13,14 30% to 62% for pancreatic cancers,15,16 and 100% for undifferentiated thyroid carcinomas.17 Although the prognostic significance of EGFR expression remains unclear, as reports on these issues are contradictory, a retrospective review of EGFR studies determined that EGFR expression levels are highly predictive of clinical outcome for patients with ovarian, cervical, bladder, esophageal, and head and neck cancers. They are of moderate prognostic value for gastric, colorectal, breast, and endometrial cancers and of relatively low prognostic value for NSCLCs.18 

Table 1. 

Epidermal Growth Factor Receptor (EGFR) Expression in Various Tumor Types

Epidermal Growth Factor Receptor (EGFR) Expression in Various Tumor Types
Epidermal Growth Factor Receptor (EGFR) Expression in Various Tumor Types

In 1988, it was found that human glioblastoma multiforme cells carried amplified c-erbB genes that bore short deletion mutations within the ligand-binding domain of the EGFR. The products of these mutated c-erbB genes were about 30 kDa smaller than the normal 170-kDa EGFR, and cancer cell membrane fractions containing the 140-kDa abnormal EGFR showed a significant elevation of tyrosine kinase activity without any ligand.19 This mutation type was referred to as EGFRvIII. There is no ligand binding site and the result is constant activation without any ligand binding.20 EGFRvIII is associated with cell proliferation and malignancy in various neoplasms involving breast cancers, small cell lung cancers, gliomas, and prostatic cancers.21 

In 2004, mutations of intracytoplasmic domain of EGFR gene were found in NSCLCs, and NSCLCs with such mutations were reduced in size by gefitinib, a chemical inhibitor of the kinase activity of EGFR.1,2 In the gene coding for the receptor, mutations are divided into 4 major types: point mutations in exon 18, deletions in exon 19, insertions in exon 20, and point mutations in exon 21. Particularly, the 2 most frequent mutations are deletion around codons 746 to 750 of exon 19 and transversion of T to G in codon 858 of exon 21, with an amino acid change from leucine to arginine (L858R). These 2 mutations account for approximately 90% of intracytoplasmic mutations of EGFR (Figure 3).22 They both cause conformational change in the ATP-binding domain, which results in constant activation of EGFR without ligand binding. However, affinity for gefitinib is upregulated, so that the cancer cells are susceptible to induction of apoptosis by this agent and to reduction in cancer size.23 The 2 EGFR mutations have been found to be present in normal lung tissue around cancers,24 and mice transgenic for the mutated EGFR gene develop lung cancers.25 The results thus suggest that EGFR mutation is involved at an early stage of neoplasia in the lung.

Figure 3.

Distribution of mutations in the epidermal growth factor receptor

Figure 3.

Distribution of mutations in the epidermal growth factor receptor

Close modal

In addition to the EGFR mutations increasing sensitivity to gefitinib, as mentioned above, secondary mutations can occur so that cancers become tolerant. Substitution in codon 790, with a resulting amino acid shift from threonine to methionine (T790M),1 or in codon 761, resulting in change from asparaginic acid to tyrosine (D761Y),26 are reported to be gefitinib tolerance–inducing mutations. T790M has been detected in about half of the NSCLCs exhibiting acquired gefitinib tolerance.26 Alteration of the gefitinib binding site in the EGFR cytoplasmic domain is presumably involved.

The most clinically advanced EGFR inhibition strategies include small-molecule inhibition of the intracellular tyrosine kinase domain and monoclonal antibody–mediated blockade of the extracellular ligand-binding domain. Gefitinib and erlotinib are oral anticancer drugs, inhibiting tyrosine kinase domain. Their cytoreductive effects are to some extent dependent on intracytoplasmic mutations of EGFR, as noted above, and they have been found to be useful in the treatment of NSCLCs. Cetuximab is a monoclonal antibody, binding to the ligand-binding site of EGFR and blocking its dimerization and activation. It is also effective for the wild-type EGFR, with applications in the treatment of colorectal as well as head and neck cancers.

From 2000 to 2001, 2 phase II studies of pretreated advanced NSCLCs (Iressa Dose Evaluation in Advanced Lung Cancer [IDEAL] 1 study27 and IDEAL 2 study28) were performed. The positive response rate to gefitinib was 9% to 19% and the 1-year survival rate was 21% to 36%. Cancer reduction effects were most prevalent in Asian nonsmoking females with adenocarcinomas. Cancers with EGFR mutations demonstrated significant cytoreductive effects to treatment,1,2 and this response is predominantly seen in persons with adenocarcinoma, who are nonsmokers, of female sex, and of Asian ethnicity.

From 2000 to 2001, as a first treatment for advanced NSCLCs, gefitinib was given in combination to standard treatment involving the platinum-containing drugs. Although the other drugs included gemcitabine and cisplatin29 or paclitaxel and carboplatin,30 significant combination effects were not obtained.

In 28 countries, not including Japan, a phase III study has been performed for 1692 cases of posttreatment advanced NSCLCs (Iressa Survival Evaluation in Lung Cancer).31 For either all lung cancers or lung adenocarcinomas, gefitinib (versus placebo) could not significantly prolong survival time for patients. However, on subset analysis, gefitinib did significantly enhance survival in Asian persons and nonsmokers.

Major clinical problems caused by gefitinib are acute lung damage and interstitial pneumonia, the latter being the most significant side effect.32 An epidemiologic investigation by the West Japan Thoracic Oncology Group, which used approximately 2000 cases, revealed an incidence rate of 3.2% to 3.5% and a death rate of 1.2% to 1.4%. Generally, ineffectiveness of steroid therapy makes the condition serious. Male sex, the existence of lung fibrosis before treatment, and a smoking habit were identified as risk factors for development of interstitial lung diseases related to gefitinib therapy. Thus, the effective treatment group for gefitinib and the high-risk group for interstitial lung disease with gefitinib are widely dissociated. This means that it is essential to preselect patients for gefitinib therapy.

Lung cancers with EGFR mutations are prevalent among patients who are young, of female sex, never-smokers, and of East Asian ethnicity.2–4,33–35 

Correlations between morphology and EGFR mutations in lung adenocarcinomas have been investigated previously. Concerning histopathology, a bronchioloalveolar carcinoma (BAC) histologic feature and well-differentiated to moderately differentiated grades were earlier reported to predict responsiveness to the EGFR tyrosine kinase (TK) inhibitor and the presence of EGFR mutations33,34 The finding that the hobnail cell type and a micropapillary morphology can predict a higher incidence of EGFR mutations in lung adenocarcinomas has been reported more recently.36 

Cell type classification of lung adenocarcinomas was originally performed by Hashimoto et al,37 describing hobnail, columnar, polygonal, goblet, and mixed cell types. They combined the Clara (nonciliated bronchiolar) cell type and type II cell type as the hobnail cell type because these types have the same cytologic features and are usually found to be mixed. This classification was applied with slight modification to a series of lesions in our hospitals. We divided lung cancers into hobnail, columnar, and polygonal cell types, focusing on the most frequent cell type rather than using the mixed cell category. Also, we merged the goblet and columnar cell types because of similarity in histologic and etiologic features and because the goblet cell type is present in minority (Figure 4, A through C).

Figure 4.

Microscopic appearance of the 3 cytologic subtypes. A, Hobnail cell type. Apical portions of carcinoma cells containing nuclei protrude or bulge into the lumen. B, Columnar cell type. Nonciliated columnar or cuboidal cells, with or without mucus in their cytoplasm, have flat apical portions. C, Polygonal cell type. Note polygonal cells showing sheet-like growth (hematoxylin-eosin, original magnifications ×400)

Figure 4.

Microscopic appearance of the 3 cytologic subtypes. A, Hobnail cell type. Apical portions of carcinoma cells containing nuclei protrude or bulge into the lumen. B, Columnar cell type. Nonciliated columnar or cuboidal cells, with or without mucus in their cytoplasm, have flat apical portions. C, Polygonal cell type. Note polygonal cells showing sheet-like growth (hematoxylin-eosin, original magnifications ×400)

Close modal

As a result, the hobnail cell type was found to be significantly more associated with EGFR mutations than any of the other groups (P < .001). The cell type classification also relates to differences in mutation frequency and pattern of TP53 (which codes for p53 protein).37 The hobnail cell type, characterized by cytoplasmic protrusions and a tadpole or hobnail appearance, shows a low TP53 mutational frequency, mainly of spontaneous transition type at CpG nucleotides. In contrast, the columnar cell type shows a high TP53 mutational frequency, with G to T transversions, considered to be caused by exogenous carcinogenic agents like those found in tobacco smoke. We identified a significant difference in EGFR mutation rates between the hobnail cell type and the other 2 types. This finding provided further evidence of differences in the genetic background of EGFR mutations.

Additionally, we have focused on the presence of BAC component, as well as micropapillary pattern (MPP), defined as papillary structures with tufts lacking a fibrovascular core (Figure 5, A and B).38 The micropapillary component belongs to moderately differentiated structures because of the lack of stroma.38 When a cancer comprises more than 5% MPP, the prognosis has been shown to be poor, even with pathologic stage I disease.38 

Figure 5.

Histologic features of the micropapillary pattern in pulmonary adenocarcinomas. A, Note diffuse distribution of tufts in alveolar spaces. B, Papillary tufts lack central fibrovascular cores (hematoxylin-eosin, original magnifications ×40 [A] and ×400 [B])

Figure 5.

Histologic features of the micropapillary pattern in pulmonary adenocarcinomas. A, Note diffuse distribution of tufts in alveolar spaces. B, Papillary tufts lack central fibrovascular cores (hematoxylin-eosin, original magnifications ×40 [A] and ×400 [B])

Close modal

As a result, there was a significant association between the existence of BAC component or MPP and EGFR mutations (P = .01 and P = .04, respectively). In addition, both BAC component and MPP were significantly associated with the hobnail cell type (P < .001 and P = .01, respectively), as compared with the combined group of columnar and polygonal cell types. However, there was no association between BAC components and MPP (P = .75).36 

The MPP is a distinct pathologic subtype first reported in lung cancers by Amin et al.39 Among early stage lung adenocarcinomas, MPP-positive cancers show a significantly poorer prognosis than those that are MPP negative.38 We speculated that the distinct MPP feature reflects a step of tumor progression from well-differentiated papillary adenocarcinoma of the hobnail cell type to a less differentiated state, unrelated to smoking. From their pathologic presentation and relatively unfavorable outcome, it is suggested that cancers with MPP should be classified as moderately differentiated rather than well differentiated. This pattern is often observed in nonsmokers and correlates with a high degree of tumor aggression. We also have demonstrated that metastasis to lymph nodes, pleural invasion, intrapulmonary metastases, and nonsmoking status are significantly more frequent in MPP-positive cases with a significantly poorer survival.38 In our study, the presence of MPP components significantly correlated with EGFR mutations. Kim et al40 referred to an association between the presence of MPP and tumor sensitivity to an EGFR TK inhibitor, although their analysis was limited to 36 relapsed lung adenocarcinomas. A notable characteristic of MPP is its frequent presence at the periphery of cancers and its predominance in metastatic foci.39,41 These clinicopathologic observations, accompanied by our findings of a high mutational frequency for EGFR, may explain the dramatic responses to gefitinib in lung adenocarcinomas with diffuse micronodular intrapulmonary metastasis.42 

Both BAC components and MPP are prevalent among nonsmokers.38,43 Considering the etiologic relevance and its correlation with EGFR mutations, we speculate that lung cancers with these features belong to the same lineage characterized by thyroid transcription factor–135 and the hobnail cell type. Also, the results imply that lesions featuring MPP may be at a slightly more advanced stage than those with BAC components, because the MPP is an adverse prognostic marker for pathologic stage I disease.38 Lung cancers in nonsmokers are considered to be less genetically complex than those in smokers44,45 and, therefore, they may have distinct characteristics depending on simple signaling pathways, such as EGFR/Akt, for maintenance and survival.2 Consequently, patients with tumors harboring these pathologic features could be good candidates and benefit from EGFR TK inhibitors.

There is no doubt that the EGFR TK inhibitors and EGFR monoclonal antibodies offer innovative molecular-targeted drugs, effective for some NSCLCs. However, the possibility of acute lung damage and interstitial pneumonia as negative side effects must be borne in mind. Therefore, the fact that in patients who are young, female, never-smokers, and of East Asian ethnicity, one subtype of NSCLC positively responds to EGFR TK inhibitors— because of the presence of EGFR mutations—is of great importance.

Pathologically, the hobnail cell type, MPP, and BAC components of lung adenocarcinomas are associated with a high incidence of EGFR mutations. Adenocarcinomas with these features form a distinct subtype, a fact suggesting that a genetic background confers susceptibility to EGFR TK inhibitors. The immunohistochemical analysis has a potential vulnerability because different antibodies might yield different results. Hence, these histologic features of lung adenocarcinomas with EGFR mutations, which can be detected by hematoxylin-eosin staining, are meaningful. These findings could provide a clue for selection of patients who might benefit from such treatment, as well as insights into biologic mechanisms of phenotype-genotype correlations.

Parts of this study were supported financially by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of the Japan Society for the Promotion of Science and by grants from the Ministry of Health, Labour and Welfare, the Smoking Research Foundation, the National Institute of Biomedical Innovation, and the Vehicle Racing Commemorative Foundation, as well as a Grant-in-Aid for Young Scientists (B).

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The authors have no relevant financial interest in the products or companies described in this article.

Author notes

Reprints: Osamu Matsubara, MD, PhD, Department of Basic Pathology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan ([email protected])