The importance of a satisfactory biopsy for the diagnosis of lung cancer in the era of personalized treatment

Advances in molecular biology are improving the understanding of lung cancer and changing the approach to treatment. A satisfactory biopsy that allows for histologic characterization and mutation analysis is becoming increasingly important. Most patients with lung cancer are diagnosed at an advanced stage, and diagnosis is often based on a small biopsy or cytology specimen. Here, we review the techniques available for making a diagnosis of lung cancer, including bronchoscopy, ultrasound-guided bronchoscopy, mediastinoscopy, transthoracic needle aspiration, thoracentesis, and medical thoracoscopy. We also discuss the indications, complications, and tissue yields of those techniques, especially as they pertain to testing for molecular markers.


INTRODUCTION
Discoveries in molecular biology are changing the approach to the treatment of patients with non-smallcell lung cancer (nsclc).In recent years, the histologic characterization of lung cancer has markedly advanced, moving beyond the simple distinction of small-cell or non-small-cell disease.
The observation that certain histologic subtypes respond differently to particular chemotherapeutic agents and the increasing use of targeted therapies have created a need for precise histologic characterization of biopsy specimens.For example, several trials have shown that response rate and survival with pemetrexed are significantly better in patients with non-squamous histology [1][2][3] .Trials using tyrosine kinase inhibitors have observed that greater benefit from treatment with those agents is seen in patients with nsclc tumours harbouring EGFR mutations than in patients with wild-type tumours 4 .Ongoing clinical trials are examining the use of Alk inhibitors in patients with nsclc characterized by EML4-ALK gene translocations 5 .Those mutations are found almost exclusively in adenocarcinomas.
Lung cancer remains the leading cause of cancer death in North America.In Canada in 2010, an estimated 25,300 Canadians were diagnosed with lung cancer, and 20,600 died of the disease 6 .At diagnosis, 75% of patients have either locally advanced or metastatic disease 7 .The goal in this latter group of patients is to establish the diagnosis and, ideally, to confirm the disease stage with the least invasive technique possible.As a result, biopsy specimens have become increasingly smaller.Of nsclc patients receiving chemotherapy for advanced disease, 80% will have only a small biopsy specimen or cytology samples available for diagnosis 8 .
In this review, we discuss the minimally invasive and invasive techniques available for the diagnosis and staging of lung cancer, with their success rates and complications.We also discuss the size of the tissue samples obtained by the various techniques, as that size pertains to maximizing the histologic characterization of lung cancer in an era of personalized medicine.Lastly, we recount our own experience in obtaining adequate tissue samples in our Rapid Investigation Clinic at McGill University.

Fiberoptic Bronchoscopy
The primary diagnostic tool in lung cancer is the bronchoscope.Flexible bronchoscopy, usually performed under local anesthesia and with minimal sedation, provides a thorough examination of all segmental bronchi within minutes.Complications are rare.Surveys of more than 75,000 procedures reveal mortality rates between 0.01% and 0.5%, and major complication rates between 0.08% and 5% 9 .
Endobronchial tumour may manifest as submucosal infiltration or an exophytic mass (Figure 1).Forceps biopsies of endobronchial tumour and mucosal abnormalities are performed under direct vision.The diagnostic yield increases when computed tomography (ct) images are available for review before the bronchoscopy, because the bronchoscopist is then better able to localize the bronchial segment containing tumour 11 .
If the lesion in the bronchus is seen, the diagnostic yield for endobronchial biopsy is 70%-90% 12 .Historically, 4 specimens have been shown to be adequate for optimal diagnostic yield in central lesions 13,14 .When the tumour is located on the lateral wall of the airway, biopsy specimens are difficult to obtain using standard forceps.To optimize specimens, use of a spear forceps that has a small needle between the biopsy jaws to anchor into the airway wall is recommended.The optimal sequence of techniques for sampling endobronchial disease has been examined by Chaudhary and colleagues.Their study showed that more malignant cells are obtained when bronchial wash is performed after bronchial brushing and bronchial biopsy 15 .Most bronchoscopists perform bronchial brushing, biopsy, and washing in that order.
Biopsy specimens are, in general, smallaveraging about 300 malignant cells in aggregate biopsies.The amount of tumour contained in the specimens is relatively low.Coghlin et al. 16 found that the mean percentage area of tumour in an endobronchial biopsy sample is 33%.In addition, not every biopsy contains tumour.In fact, fewer than half the cases (48%) in the study contained tumour in all biopsy specimens.Although 4 specimens may be enough to make a diagnosis of lung cancer, they may not provide enough tissue to perform a more detailed molecular analysis.Consideration should be given to obtaining up to 6 specimens.
Transbronchial needle aspiration (tbna) guided by ct images has an established role in the sampling of central adenopathy and masses.However, the technique is underused because of limited training of physicians, fear of complications, and need for onsite evaluation by a cytopathologist to optimize the yield 17 .Where the technology is available, "blind" tbna is increasingly likely to be replaced by tbna performed under real-time endobronchial ultrasonography (ebus) guidance.
In the case of peripheral nodules, where the endobronchial exam is normal, the diagnostic yield falls to 40% 18,19 .When positive, the diagnoses in these cases are usually cytologic, relying on bronchoalveolar lavage or bronchial brushings to make the diagnosis.Ultrasonography techniques using a radial ebus probe allow for transbronchial biopsy specimens of peripheral nodules to be obtained; this technique is further discussed in the next subsection.

EBUS Using a Guide Sheath
Endobronchial ultrasonography using a guide sheath and electromagnetic navigation can increase the diagnostic yield of flexible bronchoscopy, especially in the setting of peripheral lung nodules.An increase to 58%-80% from 36% in lesions less than 2 cm in size is possible 20 .The technique allows for direct visualization of the lesion before biopsy (Figure 2).Advantages of ebus with a guide sheath are the abilities to analyze the internal structure of lesions, to repeat the access to the bronchial lesion for sampling, and to protect against bleeding from the biopsy site into the proximal bronchus 21 .
Biopsies can be performed using fluoroscopy.In the case of electromagnetic navigation, biopsies are directed in real time to the site of the lesion using a three-dimensional reconstruction of the airways from ct scans, a steerable probe with position sensor, and an electromagnetic board 22 .

EBUS Transbronchial Needle Aspiration
Accurate staging is an essential step in the management of lung cancer patients.The involvement of mediastinal lymph nodes is one of the most adverse prognostic factors in nsclc because metastases to mediastinal nodes suggest inoperability in most cases 23 .Mediastinal staging requires invasive procedures to achieve the necessary tissue confirmation, and mediastinoscopy remains the standard of practice.Transbronchial needle aspiration under ebus guidance is a novel technique that can achieve minimally invasive staging of the mediastinum.The technique is performed using a dedicated flexible bronchoscope with an integrated ultrasound transducer (Figure 3).It allows for sampling of the mediastinal and hilar lymph nodes under direct vision and is performed as an outpatient procedure in the endoscopy suite, using local anesthesia and moderate sedation.The upper and lower paratracheal, prevascular, and retrotracheal, and the subcarinal and hilar lymph node stations can all be sampled using this technique 24 (Figures 4 and 5).
Studies performed to date indicate that ebus-tbna is a safe and accurate method for the mediastinal staging of lung cancer.A randomized trial of conventional compared with ebus-guided tbna demonstrated significantly better yields for ebus-tbna in all stations except for the subcarinal 25 .In 105 patients with suspected lung cancer and enlarged mediastinal nodes, ebus-tbna had a sensitivity of 95% and a specificity of 100%.Findings from ebus-tbna had a considerable impact on patient management, avoiding 29 mediastinoscopies, 4 video-assisted thoracoscopic surgeries, 8 thoracotomies, and 9 ct-guided lung biopsies 26 .In the largest study to date, Herth et al. 27 performed ebus-tbna in 502 patients with suspected lung cancer and enlarged mediastinal nodes, comparing the biopsy results with operative findings.The reported sensitivity was 94% and the specificity, 100%.No complications occurred.A meta-analysis of ebus-tbna for mediastinal staging in patients with lung cancer reported a pooled specificity of 1.00 (95% confidence interval: 0.96 to 1.00) and a pooled sensitivity of 0.88 (95% confidence interval: 0.79 to 0.94) 28 .
Endobronchial ultrasound is expected to have an increasingly important role in the diagnosis and staging of lung cancer 29 .Current guidelines on lung cancer staging recommend that a negative ebus-tbna result should be confirmed by mediastinoscopy 30 .Recent studies have directly compared the diagnostic accuracy of ebus-tbna with mediastinoscopy, reporting comparable results for mediastinal staging 31 .
It is important to recognize that there is a learning curve to ebus-tbna, with guidelines recommending initial training consisting of 40-50 supervised procedures 32 .However, once optimal diagnostic results are achieved on ebus-tbna, confirmation of results may not be necessary.Also, ebus-tbna has been successfully used for restaging of the mediastinum, where repeat mediastinoscopy is typically difficult because of fibrotic changes from the earlier procedure 33 .One caveat is that the papers published to date come from a small group of authors with extensive expertise, and further studies by different operators are needed.
Tissue samples from ebus-tbna are acquired using a dedicated 22-gauge needle, or the more recently introduced 21-gauge needle (Figure 6).Where available, rapid on-site evaluation by a cytopathologist of aspirated samples is invaluable.In the absence of rapid on-site evaluation, Lee and colleagues 34 demonstrated that maximum diagnostic values are achieved with 3 aspirations per lymph node.The needle aspirates are typically processed using a combination of slides and cell block preparations.
Optimal methodology for specimen handling is an important part of improving the yield of ebustbna, particularly as molecular analysis becomes increasingly important.Nakajima and Yasufuku 35 recently reported on their technique to improve the success rate in obtaining histologic cores.The socalled tissue coagulum-clot technique for cell block preparation has been compared with the conventional saline-rinse cell block method, and the former technique was found to significantly increase the cellular yield of cell block preparations 36 .Several authors have reported on the successful use of ebus-tbna specimens for molecular analysis for EGFR, ALK, and KRAS mutations.Nakajima and colleagues reported the successful detection of EGFR mutations using dna extracted from paraffin-embedded ebus-tbna samples 37 .Other groups have reported that molecular analysis for EGFR with or without KRAS could be performed in 72%-77% of patients with lung adenocarcinoma who had undergone ebus-tbna 38,39 .The results of mutation analysis on cytology aspirates and histologic samples obtained by surgical staging have been compared and found to be reliable 40 .
To summarize: For lung cancer, ebus-tbna is an innovative procedure whose main role is in diagnosing and staging patients.Specimens from this technique allow for conventional cytology and molecular mutation analysis.Optimal methodology for specimen handling is key to maximizing the yield of lymph node cytology.

Mediastinoscopy
Cervical mediastinoscopy is used predominantly in the staging of lung cancer and is considered the  standard of practice in staging the mediastinum.However, ebus, although still imperfect, is increasingly being used in mediastinal staging.In other words, ebus is useful to rule nodal metastases in, but not to rule them out.
Cervical mediastinoscopy is performed in an operating room under general anesthesia.A small incision is made at the base of the neck and the mediastinoscope is introduced.The sensitivity of mediastinoscopy for detecting cancer in mediastinal lymph nodes varies between 80% and 95% 33,41 .False-negative rates of mediastinoscopy vary between 5% and 9% and are attributable to an inability to access the paraesophageal, inferior pulmonary ligament, and aortopulmonary nodes.The tissue samples obtained are on the order of millimetres to centimeters in size depending on the size of the node.Repeat mediastinoscopy cannot easily be performed in the same person because of adhesions and fibrotic changes from the earlier procedure 42 .Complications are on the order of 2%-5% and include hoarseness, infection, and bleeding 43 .

Transthoracic Needle Aspiration
In 10%-20% of cases, nsclc will present as a solitary pulmonary nodule.In patients who are not candidates for surgery or who have advanced disease in which the most accessible site for biopsy is a peripheral lung nodule or mass, transthoracic needle aspiration (ttna) and biopsy are useful diagnostic procedures.
A ttna can be performed under either fluoroscopy or ct guidance.Studies comparing the two approaches showed a higher sensitivity for ct guidance 44 .In most centres, a coaxial system, in which a large-gauge needle is inserted into the edge of the lesion and a smaller needle is passed through the larger needle to obtain the specimen, is used.This approach allows for a single pleural puncture, because the larger needle is left in place until all specimens are collected.The major complications of ttna are pneumothorax and bleeding.Contraindications to ttna are previous pneumonectomy; severe chronic obstructive lung disease, especially if large bullae are present; mechanical ventilation; and high risk for bleeding 45 .
The specimens obtained are cutting-needle core biopsies and needle aspirates.Core-needle biopsy specimens usually contain enough cellular material for molecular analysis: approximately 500 cells per core biopsy.Needle aspirates obtained using a 21-gauge needle yield approximately 100 cells 46 .The pooled sensitivity of ttna for a diagnosis of peripheral bronchogenic cancer is 90% (95% confidence interval: 0.88 to 0.91) 47 .Discordance between ttna lung cancer cell type and surgical pathology is between 6% and 39% 47 .The false-negative rate with ttna is relatively high (range: 20%-30%), and so it is not considered a useful diagnostic test to rule out lung cancer 19 .If a ttna is negative, further diagnostic testing is recommended.

Pleural Fluid Analysis and Medical Thoracoscopy
The choice of technique in the diagnosis of lung cancer depends on the presumed stage of the disease.In patients with suspected lung cancer presenting with an accessible pleural effusion, thoracentesis is recommended to distinguish between a malignant and a paramalignant effusion 19 .The yield of pleural cytology is 60%-80% under optimal conditions and repeat sampling 48,49 .The use of cytology specimens for molecular testing is under evaluation.The 2010 European consensus for EGFR testing in nsclc cautioned that further research was required before testing of cytology specimens could be recommended.Cell blocks of pleural fluid have been used and have a low insufficiency rate of 3.7% (1 in 27 specimens) for EGFR and KRAS mutation testing 50 .
When 2 cytology specimens are nondiagnostic, or when the material is insufficient for histologic classification, thoracoscopy is recommended.Closed pleural biopsy has a low yield (17% in one series in patients with nondiagnostic cytology) 51 .Biopsy performed under direct vision is preferred, because pleural nodules are often scattered on the parietal pleural surface (Figure 7).
Thoracoscopic examination of the pleural cavity can be performed using medical thoracoscopy.Unlike surgical thoracoscopy, medical thoracoscopy can be performed in a dedicated sterile endoscopy suite under local anesthesia and conscious sedation.A pneumothorax is artificially induced and a rigid thoracoscope is introduced into the pleural cavity.This procedure has two clinical uses: the diagnosis of pleural effusions, and the application of pleurodesis should an effusion be identified as malignant.Obtained under direct vision, the diagnostic yield of medical thoracoscopy for malignancy is 93%-97% 52 .At the McGill University Health Centre, a "fasttrack" diagnostic evaluation clinic was established with the goal of accelerating the outpatient diagnostic assessment of patients with suspected lung cancer.After prompt imaging, preference is given to the invasive diagnostic technique-bronchoscopy, ebus, ttna, mediastinoscopy, or open-lung biopsy-that is felt to have the best yield-risk ratio based on ct findings.Procedures that allow for simultaneous diagnosis and staging are favoured.
Of the 36 patients who initially had a bronchoscopy that confirmed the diagnosis of nsclc, 18 required further tissue sampling for staging.We reviewed the 18 patients who had no other invasive tissue sampling after bronchoscopy.In 17 of the 18, the pathology specimens consisted of endobronchial specimens varying in size from 1 cm to 0.1 cm.In 1 of the 18, only a bronchial wash was positive by cytology.
Overall, tissue sampling for molecular testing was adequate in 116 of 124 patients.Pathology confirmation of malignancy was not achieved in 4 patients.The specimens that could not be analyzed because of inadequate cellularity included 1 bronchial wash, 1 radial ebus wash, 1 bone biopsy, and 1 pleural fluid sample.

OPTIMIZING SPECIMENS FOR MOLECULAR ANALYSIS
Most routinely available pathology specimens can be used for biomarker analysis, including formalinfixed, paraffin-embedded tissue and cell block preparations.The minimum number of malignant tumour cells required for biomarker testing is not well established.In general, larger samples with at least 200-400 available malignant cells are preferable 54 .
To avoid overconsumption of tumour for additional immunohistochemistry or biomarker analysis if the clinical and radiographic context is not appropriate, it is important that the pathologist be part of the multidisciplinary team.
Isolation of tumour cells often requires macroand microdissection.Optimizing testing on extracted dna means minimizing dna from non-neoplastic cells in the sample 8 .

SUMMARY AND RECOMMENDATIONS
Advances in molecular biology are improving the understanding of lung cancer and changing the approach to treatment.The importance of a satisfactory biopsy that allows for histologic characterization and mutation analysis cannot be overemphasized.In this article, we reviewed the techniques available for diagnosing lung cancer and the tissue yields associated with each technique.In general, the most easily accessible site should be chosen for biopsy, and a sufficient sample should be obtained.Biopsy specimens are preferable to cytology specimens, but there is increasing evidence to suggest that, when handled judiciously, cytology specimens can be used for molecular analysis.To optimize the analysis of limited tissue available for biomarker testing, it is important that the pathologist be involved in the multidisciplinary team.It is equally important that the respirologist, interventional radiologist, and thoracic surgeon understand the importance of obtaining a satisfactory amount of material, because the specimens obtained ultimately affect the patient's management and prognosis.

figure 2
figure2 Clockwise from top left: biopsy forceps; stopper that marks the biopsy forceps at the correct length; ultrasonography probe; ultrasonography probe and sheath secured with stopper.