The importance of EGFR mutation testing in squamous cell carcinoma or non-small cell carcinoma favor squamous cell carcinoma diagnosed from small lung biopsies

Lung cancer is the most common cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC), accounting for approximately 80–85% of all the cases, is further divided into major histological subtypes including adenocarcinoma (ADC), squamous cell carcinoma (SqCC), large cell carcinoma (LCC), and other rare tumors including adenosquamous carcinoma (ADSC) [1]. ADSC is a rare and mixed-type NSCLC, counting for only 0.4 to 4% of all the cases. According to the World Health Organization (WHO) Classification of Lung Tumors, ADSC is defined as “a carcinoma showing components of both SqCC and ADC, with each comprising at least 10% of the tumor” [2]. ADSC has a more aggressive behavior and worse prognosis than ADC and SqCC [3, 4]. The treatment of early-stage ADSC mainly includes surgery, supplemented by chemotherapy, and other comprehensive treatment, but the curative effect is not satisfied [5]. The poor prognosis of ADSC has raised the demand for more effective treatment approaches such as targeted therapy. Recently, the development of targeted therapy toward oncogenic “driver mutations” has revolutionized the clinical management of patients with NSCLC including ADSC [6, 7].

Epidermal growth factor receptor (EGFR) mutation is the most well-characterized driver mutation in NSCLC, and is more prevalent in Asian population, never smokers, and patients with ADC subtype [8]. More than 60% of Asian ADC patients were found to have EGFR mutations [9]. EGFR tyrosine kinase inhibitors (EGFR-TKIs), giving higher response rates, longer progression-free survival and better quality of life compared to conventional chemotherapy, are now recommended as the first-line treatment in patients with advanced EGFR activating mutation-positive NSCLC [10‐13]. Currently, studies demonstrated that EGFR mutations occur at a frequency of 15 to 44% in Asian ADSC and are mainly in female and never smokers; patients with ADSC showed a similar benefit from EGFR-TKI treatment compared to those with ADC, revealing that EGFR-TKI may be a therapeutic option for ADSC patients [14‐18]. In view of this, the current guidelines recommend that EGFR mutation testing should be performed in NSCLC patients with ADC or mixed-type tumors containing an adenocarcinomatous component, for example, ADSC, and is not necessary for patients with tumors lack of any adenocarcinomatous component, such as pure SqCC or pure LCC [19, 20].

In clinical practice, accurate diagnosis of ADSC can only be made in resection specimens, because a minimum of 10% of each ADC or SqCC component is required. However, approximately 70% of lung cancer patients are diagnosed at advanced stages that are not amenable to surgical resection and small biopsies are the major tissue resources for pathological diagnosis and subtyping [21, 22]. Diagnosis of ADSC before surgical resection is not possible, since tumors present in small biopsy specimens may come from either ADC or SqCC component only. In the majority of cases, ADSC in small biopsy specimens is misdiagnosed as SqCC or poorly differentiated SqCC referring to non-small cell carcinoma (NSCC) favor SqCC due to biased sampling of the lesion, which may cause the neglect of EGFR mutation testing in these cases. A previous report by Rekhtman et al. also suggested that EGFR or KRAS mutations do not occur in pure pulmonary SqCC, and the occasional detection of these mutations may come from the challenge of ADSC diagnosis in small biopsies [23]. Furthermore, two NSCLC cases, possibly ADSC, presented by Saini et al. were initially diagnosed as SqCC in small biopsies, positive for EGFR mutations and benefitted with oral EGFR-TKI treatment [24]. The lack of EGFR mutation testing in patients who are diagnosed as SqCC from small biopsies may influence the treatment strategies for patients, especially in Asian population which has a high frequency of EGFR mutations. For this reason, we conducted an Asian retrospective study investigating the frequency and clinicopathological association of EGFR mutations in patients diagnosed as SqCC in small lung biopsies to evaluate the necessity and clinical impact of EGFR mutation testing on possibly under-sampled ADSC.

Recent advances in targeted therapy have dramatically changed the clinical management of lung cancer patients and the molecular testing for driver mutations is now routinely used to predict patients’ treatment response. The current guideline of CAP/IASLC/AMP suggests that testing for EGFR mutations and anaplastic lymphoma kinase (ALK) translocations should be recommended for adenocarcinomas and mixed lung cancers with an adenocarcinomatous component, regardless of histologic grade, and are not recommended in lung cancers that lack any adenocarcinoma component, such as “pure” SqCC, “pure” SCLC, or large cell carcinomas lacking any IHC evidence of adenocarcinoma differentiation [20]. In the era of precision medicine, accurate pathologic classification of NSCLC is mandatory for tailoring patients to specific molecular diagnostic testing. However, most of advanced lung cancers are diagnosed via tumor biopsies, which increases the difficulty in making a correct diagnosis, particularly in tumors without clear differentiation or in mixed-type tumors, such as ADSC.

Our study focuses on investigating the impact of EGFR mutation testing on ADSC in small biopsy diagnostics, especially in Asian patients that have high incidence of EGFR mutations. The misdiagnosis of ADSC remains a problem in up to 20% of small biopsy cases, even using immunohistochemistry [23, 27]. A recent study reported by Zhang et al. showed that 38.8% (26/67) of post-operatively pathologically proven ADSC patients were pre-operatively misdiagnosed as SqCC and 29.8% (20/67) were misdiagnosed as poorly differentiated SqCC via biopsy or cytology specimens [28]. In this study, we found EGFR mutations are detected in 8.8% of small biopsy-diagnosed SqCC or NSCC favor SqCC, and significantly associated with never smokers and a diagnosis of NSCC favor SqCC. It has been reported that the incidence of EGFR mutations in Asian SqCC is less than 3% [29]. The frequency of EGFR mutations in our cases is higher than that reported in SqCC, suggesting that some of these small biopsy cases may not be single-histology SqCC. Indeed, three of 4 (75%) EGFR mutation-positive cases in our study are subsequently diagnosed as ADSC, suggesting that most of EGFR-mutated SqCC diagnosed from small biopsies may result from undersampling of ADSC.

The limitation of this study is that this is a retrospective study and patients who are diagnosed as SqCC in small biopsies would not receive EGFR-TKI treatment according to current guidelines, therefore, no treatment results are available for analysis. Moreover, due to the rarity of ADSC and its unfeasibility in small biopsy diagnosis, data on the efficacy of EGFR-TKI in ADSC patients is limited. Although there were only a limited number of cases published, some studies demonstrated that using EGFR-TKIs is an effective treatment for patients with EGFR-mutated ADSC (Table 3) [17, 28, 30]. Song et al. showed that the progression-free survival of ADSC patients with EGFR-TKIs treatment was 4.3 months which is comparable to 4.2 months of ADC. They also revealed higher efficacy of EGFR-TKIs in ADSC patients with EGFR mutations compared to those without EGFR mutations. In Fan et al. and Zhang et al. studies, EGFR-TKIs were only applied to EGFR activating mutation-positive patients, and the average of disease control rate and overall survival were 75.4% and 41 months. Zhang et al. also showed that ADSC patients harboring EGFR-sensitizing mutations who were treated with EGFR-TKIs had a significantly better prognosis than those receiving chemotherapy or chemoradiotherapy alone [28]. However, these studies only included patients with clinical stage I to IIIA, and a prospective study toward efficacy of EGFR-TKIs in advanced stage ADSC patients needs to be further conducted in the future.

Our multivariate analyses showed that never-smokers and NSCC favor SqCC, but not female gender, are independently associated with EGFR mutations. Lung cancer in never smokers has significant gender and geographic variations, occurring more frequently in female and in Asia. One possible explanation is that the majority of female NSCLC patients in Asian populations have no smoking history. Studies showed that the proportion of never smokers in female lung cancer cases are 83% in South Asia, while only 15% in the United States [31]. Moreover, lung cancer in Asian never smokers are often diagnosed as ADC that can be classified into different molecular subtypes based on oncogenic driver mutations. On the contrary, SqCC is more significantly associated with smokers than other subtypes of NSCLC [32]. From a molecular point of view, several studies showed that EGFR mutations are more prevalent in tumors of never smokers, while TP53 and KRAS mutations are more frequently found in smokers [31, 33‐35]. Taken together, in our study cohort, the clinicopathological characteristics of EGFR mutation-positive cases are similar to those of ADC, implying that some of these cases might be EGFR-mutated ADSC. Furthermore, our findings that EGFR mutations significantly associate with a diagnosis of NSCC favor SqCC, which is defined as poorly differentiated NSCLC without definite SqCC morphology features, also suggest these cases may possibly be ADSC.

EGFR-TKI is a therapeutic option of EGFR-mutated ADSC which is often misdiagnosed as SqCC in small lung biopsies, resulting in absence of EGFR mutation testing. Our findings demonstrated the importance of EGFR mutation testing in small biopsy-diagnosed lung SqCC, especially in never smokers and patients whose diagnoses need to be assisted by immunohistochemistry.