Although the sophistication of patient selection for treatment has increased, the size of lung cancer samples to obtain that information has reduced. The challenge for the pathologist and lung cancer multidisciplinary team is to optimize diagnostic specimens and staging while supplying sufficient information to guide oncological therapy. Because at least 75% of patients have inoperable disease, the information to guide treatment algorithms must be obtained from small histology or cytology specimens.
EBUS-TBNA is an important investigation for the diagnosis of mediastinal and hilar lymphadenopathy in patients with lung cancer. It has been recommended as an initial investigation by the National Institute of Health and Clinical Excellence in patients with enlarged mediastinal lymph nodes because it may provide an inoperable disease stage and a pathological diagnosis in a single investigation (2
). This large, multicenter, pragmatic implementation study demonstrates that routine samples from EBUS-TBNA provide sufficient information to allow subtyping in 77% and EGFR mutation testing in 90% of patients with NSCLC.
The proportion of patients with NSCLC for whom the final diagnosis using EBUS-TBNA specimens was NSCLC NOS was 23%. This is consistent with data from alternative biopsy techniques. An analysis of the California Cancer Registry of 175,298 patients diagnosed with lung cancer between 1989 and 2006 demonstrated a NSCLC-NOS rate of 22.1% (13
). The rate of NSCLC-NOS was higher in the patients who had a cytological diagnosis alone (37%). The UK National Lung Cancer Audit published data on 26,731 patients diagnosed with NSCLC in England and Wales in 2010 (14
). These patients underwent diagnosis and staging of lung cancer in a real-world setting, and the audit demonstrated an overall NSCLC-NOS rate of 24.4%. This highlights that EBUS-TBNA may be as good as other sample acquisition techniques for subtyping and that there will always be a proportion of patients with NSCLC in whom further subtyping is not possible due to lack of differentiation. EBUS-TBNA is also able to sample central parenchymal lung lesions that would otherwise not be accessible without a more invasive approach (15
). Therefore, increased application of EBUS-TBNA may improve the rate of histological confirmation in patients with NSCLC, which stands at a mean of 72% in the UK National Lung Cancer Audit (14
Previous studies have shown that samples from cytology are valid when compared with subsequent larger samples. Indeed, the morphologic features that distinguish squamous cell carcinoma (predominantly keratinized cytoplasm and intercellular bridges) from adenocarcinoma (mucin vacuoles and gland formation) span less than the 250-μm inner diameter of a 25-gauge fine needle (16
). In a recent retrospective study of 48 patients (17
), cell block samples from EBUS-TBNA were compared with histological specimens obtained by alternative procedures such as bronchoscopy and CT-guided biopsy. All subtypes diagnosed by EBUS-TBNA were validated by histological samples. When immunohistochemistry was performed on cell blocks, there were six cases diagnosed as NSCLC-NOS on EBUS-TBNA samples, which were diagnosed with a specific cell type on alternative histological samples (three adenocarcinomas, two squamous cell carcinomas, and one large cell undifferentiated carcinoma). A further study of 101 individuals demonstrated a 93% concordance between small biopsy and cytology specimens (18
). As in this study, a lack of supporting immunohistochemistry contributed to unclassified cytology cases. In another report, 158 (85%) cases of NSCLC were typed by cytology and 28 (15%) were classified as NSCLC-NOS (19
). Using histological specimens from the same patients, 183 (98%) cases were subtyped by histology, and only 3 (2%) cases were classified as NSCLC-NOS. There was 88% concordance between cytological and histological typing. The available data therefore confirm that cytological specimens are reliable for subtyping, with no false-positive results from cytological subtyping observed, and that use of immunohistochemistry can reduce the NSCLC-NOS rate.
Immunohistochemistry profiles do not feature in the diagnostic criteria for squamous cell or adenocarcinoma in the current WHO classification of NSCLC, which is based on resected surgical specimens (20
). However, when morphological criteria are unable to distinguish subtypes in smaller samples, a panel of antibodies, including TTF-1, p63, and CK5/6, as well as a mucin stain has been recommended to minimize the proportion of NSCLC tumors that remain unclassified and to make the key distinction between squamous and nonsquamous subtypes (21
). The current study shows that samples obtained by EBUS-TBNA are suitable for this approach from any accessible lymph node station and even when sampling lymph nodes less than 1 cm in size.
The EGFR-tyrosine kinase inhibitors erlotinib and gefitinib have become established as first-line treatments for patients with advanced lung cancer that harbor an EGFR mutation. Current European Society of Medical Oncology guidelines recommend that all never- or former light smokers (<15 pack years) or patients with nonsquamous histology should be tested for EGFR mutation status regardless of performance status (22
). Cytological samples in alcohol-based fixatives may preserve nucleic acids better than formalin (23
), and molecular profiling of cytology samples has been shown to be reliable when compared with histological samples from the same patient (24
). In this study, EGFR mutation testing was requested in 119 patients, and the test was possible and deemed reliable in 107 (90%) cases. In the remaining cases, there was insufficient tumor sample to perform the investigation. Previous studies have assessed the utility of EBUS-TBNA samples for EGFR testing with variable results. In one study, EGFR mutation testing was possible in 27 out of 35 patients (77%) undergoing EUS-FNA or EBUS-TBNA (25
). Another study of 36 patients in Spain undergoing EBUS-TBNA suggested EGFR mutation analysis was feasible in 26 (72%) cases (26
). Billah demonstrated that 96% of specimens from EBUS-TBNA in a cancer center were able to undergo EGFR mutation testing (27
). Similarly high rates of reliable EGFR mutation testing of EBUS-TBNA samples have been observed by Nakajima and colleagues (28
). A recent study, in which cell blocks were prepared from 128 lung cancer cytology specimens, demonstrated that molecular analysis was possible in 98% of specimens (30
It is widely accepted that NSCLC may contain areas of mixed adenocarcinoma, large cell and squamous cell carcinoma. Up to 25% of small cell carcinomas are thought to contain areas of NSCLC differentiation (31
). This pathological heterogeneity implies that smaller cytological samples may not be representative of the entire lesion. Another potential area of controversy in NSCLC is that of genetic tumor heterogeneity. Conflicting evidence exists. Three studies comparing EGFR mutation status in primary tumor and local lymph node metastases demonstrated significant discrepancies between the sites (32
). However, a recent study showed that when highly sensitive techniques for mutation detection were used, no discordant mutation patterns were detected among 77 paired primary and metastatic tumors (35
). These authors suggested that weak EGFR mutation signals in an area without EGFR amplification may not reach the threshold of detection because of the mixture with normal cells resulting in pseudoheterogeneity. The authors concluded that true genetic heterogeneity is rare (35
). This latter view would support EGFR mutation status being assessed in the most accessible tissue only rather than multiple sites being sampled.
This study confirms the high yield from EBUS-TBNA of detecting malignancy in intrathoracic lymph nodes in a real world setting. A sensitivity of 88% in 774 patients was observed, which is similar to a sensitivity of 93% observed in a meta-analysis of 1,299 patients (36
). This study contains the first reported death attributed to EBUS-TBNA. The patient was likely immunosuppressed due to widely metastatic malignancy and succumbed to sepsis within 72 hours of the procedure. Sepsis may be attributed to the process of introducing pharyngeal microorganisms into the lower respiratory tract. The large number of patients included in this study renders subgroup analyses powerful. EBUS-TBNA of lymph nodes less than 1 cm has a significantly lower sensitivity than when the procedure is performed in nodes larger than 1 cm. This may be due to the increased technical difficulty of sampling smaller lymph nodes. However, when small lymph nodes were sampled successfully (regardless of lymph node location), the samples were suitable for NSCLC subtyping and EGFR mutation analysis.
There are several limitations of this study. Pathological samples in this study did not undergo central review; however, this reflects the pragmatic nature of the study and results in strong external validity. The centers included in the study carry out a high volume of EBUS-TBNA procedures with experienced operators and pathologists. Despite the observational design of this study, data were collected prospectively in each center. A final issue is that not all negative EBUS-TBNA cases underwent mediastinoscopy. However, all patients underwent at least 6 months of clinical follow-up to allow a clinical diagnosis to be made.
Recent guidance has suggested a novel algorithm for the diagnosis of adenocarcinoma in small biopsies and cytological samples (37
). In patients with positive cytology and classic morphology for adenocarcinoma or squamous cell carcinoma, no further markers are required, and those with adenocarcinoma can be submitted directly for EGFR mutation testing. Samples that are classified as NSCLC-NOS on morphology are recommended to undergo a panel of immunohistochemistry that includes one squamous cell carcinoma marker and one adenocarcinoma marker with or without mucin staining. If the NSCLC tumor remains unclassified, then molecular analysis is recommended. This multicenter study clearly demonstrates that samples from EBUS-TBNA obtained in routine practice are suitable for entry into this new diagnostic algorithm and provides further impetus for the use of EBUS-TBNA as an initial diagnostic procedure in patients with suspected lung cancer.