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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0b013e3181d57f6e

Pathologic Diagnosis of Advanced Lung Cancer Based on Small Biopsies and Cytology: A Paradigm Shift

Travis, William D. MD*; Rekhtman, Natasha MD, PhD*; Riley, Gregory J. MD†; Geisinger, Kim R. MD‡; Asamura, Hisao MD§; Brambilla, Elisabeth MD¶; Garg, Kavita MD#; Hirsch, Fred R. MD**; Noguchi, Masayuki MD††; Powell, Charles A. MD‡‡; Rusch, Valerie W. MD§§; Scagliotti, Giorgio MD∥∥; Yatabe, Yasushi MD, PhD¶¶

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*Department of Pathology, †Thoracic Oncology Service, Memorial Sloan Kettering Cancer Center, New York, New York; ‡Department of Pathology, Wake Forest University, Winston-Salem, North Carolina; §Thoracic Surgery, National Cancer Center, Tokyo, Japan; ¶Service de Pathologie Cellulaire, Centre Hospitalier Universitaire de Grenoble, France; #Department of Radiology, University of Colorado, Aurora, Colorado; **University of Colorado Cancer Center, Aurora, Colorado; ††Department of Pathology, Tsukuba University, Tsukuba, Japan; ‡‡Pulmonary Medicine, Columbia University, New York, New York; §§Thoracic Surgery Service, Memorial Sloan Kettering Cancer Center, New York, New York; ∥∥Thoracic Oncology Unit, University of Torino, Italy; and ¶¶Department of Pathology and Molecular Diagnostics, Aichi Cancer Center, Nagoya, Japan.

Disclosure: The authors declare no conflicts of interest.

Address for correspondence: William D. Travis, MD, Department of Pathology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. E-mail:

With some exceptions, the field of lung cancer disease has been relatively static during the past several decades with few major practice-changing advances. In this issue of the journal 2, articles address the diagnosis of non-small cell lung cancer (NSCLC) based on small biopsies and/or cytology, an area of lung cancer diagnosis in which a paradigm shift has occurred for both pathologists and clinicians.1,2 This topic is important because the majority patients with lung cancer present with unresectable disease, and the diagnosis is established based on such small specimens. Moreover, with increasing use of minimally invasive biopsy methods, pathologists are being asked to do more with less tissue.

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The World Health Organization classifications of lung tumors through the 1999 edition3–5 did not address lung cancer diagnosis based on small biopsies and cytology, because these were recommendations for the histologic classifications of resection specimens. In the 2004 World Health Organization classification, cytology was addressed for the first time, but classification in small biopsies was not addressed.6 Currently, no internationally recognized standard of criteria or terminology for the diagnosis of lung cancer in small biopsies is available.

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Historically, the most important decision for pathologists in small biopsies and cytology has been the crucial distinction between small cell lung cancer and NSCLC, because it defines patients with completely different clinical tumor behavior and management. Within NSCLC, important clinical reasons to separate squamous cell carcinoma from adenocarcinoma and other histologic types have not existed.

In a literature review spanning 25 years of clinical studies evaluating chemotherapy in advanced NSCLC, there was some weak association between histology and therapeutic outcomes,7 but none of these studies reported a formal test of treatment by histology interaction. Therefore, in older studies, there was no clear evidence that histology had a prognostic (independent of treatment) or a predictive role (associated with the effectiveness of a specific treatment) for patients with NSCLC.

Recently, all this has been changed. Three clinical observations in patients with advanced lung cancer have provided a reason for pathologists to change their practice and make a better attempt to distinguish adenocarcinoma from squamous cell carcinoma. First, patients with advanced lung cancer treated with bevacizumab are at increased risk for life-threatening hemorrhage if they have squamous cell carcinoma.8 Second, patients with adenocarcinoma or NSCLC-not otherwise specified (NSCLC-NOS; reported as large cell carcinoma in some of these studies) respond significantly better to pemetrexed than those with squamous cell carcinoma.9–11 Third, EGFR mutation is strongly associated with adenocarcinoma histology, and patients with advanced NSCLC and EGFR mutation have a better outcome and response to tyrosine kinase inhibitors' as a first-line therapy, whereas patients without EGFR mutations seem to have a better outcome with chemotherapy.12 Importantly, in all these studies, the histologic classification was solely based on light microscopy with or without mucin stains. No immunohistochemistry or other special techniques were used to classify the tumors further.

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One major underlying problem in addressing more precise classification of small biopsies and cytology specimens is the inherent histologic heterogeneity that exists in a subset of NSCLC.2 Roggli et al.13 addressed this in a comprehensive study of 100 lung cancer cases in which 39 adenocarcinomas showed a squamous component in two cases (5%) and in 27 squamous cell carcinomas (excluding the cases with small cell carcinoma), adenocarcinoma was found in four cases (15%). Another difficulty is in tumors in which a solid component is sampled in small biopsy or cytology specimens, and the morphologic features of squamous or adenocarcinoma differentiation are focal or not distinguishable. This has been the subject of numerous articles that demonstrate the error rate in classification of small biopsies when compared with final histologic classification of the surgical specimen.14 In one such study, 10% of squamous cell carcinomas, 14% of adenocarcinomas, and 50% of large cell carcinomas were misclassified on bronchial biopsies.14

Despite the issues of morphologic heterogeneity and overlapping features in tumors with solid histology, recent molecular studies reveal that there are clear biologic differences between lung adenocarcinoma and squamous cell carcinoma. In particular, gene-expression-profiling studies show distinct molecular clusters for adenocarcinoma and squamous cell carcinoma.15–17 In addition, EGFR and KRAS mutations are largely confined to adenocarcinoma but are rare in squamous cell carcinoma.18,19

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Based on the recent clinical observations, there is a growing literature regarding the pathologic characteristics using special techniques to distinguish adenocarcinoma from squamous cell carcinoma. The current articles by Loo et al. and Nicholson et al. are the latest additions to this literature. It seems from the studies by Loo et al.2 and Nicholson et al.1 that thyroid transcription factor-1 (TTF-1) and mucin versus p63 stains are the best stains for detecting adenocarcinoma versus squamous cell carcinoma, respectively. However, none of the antibodies used in immunohistochemistry are 100% specific and sensitive for this differential diagnosis. For example, in the study by Nicholson et al., TTF-1 was positive only in 13 of the 16 cases (81%), finally classified as adenocarcinoma including 4 of the 7 (63%) with adenocarcinoma morphology and 9 of the 9 (100%) initially diagnosed as NSCLC-NOS. One case classified as squamous cell carcinoma also showed occasional staining for mucin. p63 is consistently expressed in squamous cell carcinoma, but it can also be seen in a subset of adenocarcinomas. As pointed out in both the articles, high-molecular-weight cytokeratin antibody 34βE12 (CK903) is highly nonspecific for distinguishing adenocarcinoma and squamous cell carcinoma. Despite the limitations of the individual markers, evidence is emerging, as demonstrated in these two studies, that high accuracy can be achieved using a panel approach. A number of other antibodies have been reported recently including desmocollin-3 and napsin.20–22 More prospective investigation is needed to determine whether these novel markers provide any added value to TTF-1, p63, and mucin stains to establish the optimal marker panel for the greatest diagnostic accuracy. Other molecular techniques, such as micro-RNA, have been reported to distinguish squamous cell carcinoma and adenocarcinoma.23 However, whether they provide any advantage to light microscopy and immunohistochemistry remains to be determined. Ultimately, a simple panel of stains should be used that does not exhaust tissue, which may be needed for predictive and prognostic molecular studies. The combination of these novel techniques with traditional morphology will hopefully lead to a more biologically precise classification of NSCLC in morphologically equivocal cases. Such an approach has been quite successful in other diseases, especially in the diagnosis and classification of lymphomas24 and sarcomas.25 The prognostic and predictive impact of this approach for lung cancer will depend on analysis in prospective clinical studies.

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Both articles recognize that light microscopy is sufficient to classify lung cancer in a subset of small biopsies or cytology specimens, but there remain cases that cannot be further classified for which they propose to use the term NSCLC-NOS. Using special stains, the authors identified characteristics that allowed them to classify most of these tumors further. Nicholson et al. showed that although 53% of the cases (17 of 32) were classified as NSCLC-NOS after the initial light microscopic review, after attempts to perform special stains 6 of the 32 (19%) remained unclassified. NSCLC-NOS is a more appropriate term for these unclassified cases than large cell carcinoma, as has been used in some, in the absence of standardized criteria and terminology for small biopsies or cytology.10 In some cases in which biopsies show NSCLC-NOS, but tumor tissue is too sparse for immunohistochemistry or mucin stains for more precise classification or for molecular studies, if clinically indicated, additional biopsies may be warranted. Large cell carcinoma is a term that should be reserved for classification of undifferentiated NSCLC in resected specimens.

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During the past 2 years, the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society have been developing a new International Multidisciplinary Lung Adenocarcinoma Classification. The articles by Loo et al. and Nicholson et al. are projects that were, in part, generated in response to questions raised during discussions for this classification. This classification document will be published soon in the Journal of Thoracic Oncology.

In this new classification, an algorithm will be presented with recommended terminology, special stains, and approaches to tissue management.26 Implementation of these recommendations will require fundamental changes in the approach of obtaining small tissue and cytology samples by clinicians, radiologists, and surgeons. It will also require changes in pathologists' approach to diagnosis including specimen processing and evaluation, as well as diagnostic terminology.

The growing evidence for the utility of ancillary studies underscores the importance of obtaining sufficient tissue, whenever clinically feasible.26 Although minimal material may be sufficient to distinguish NSCLC from small cell lung cancer, additional tissue could be needed to perform immunostains for subtyping of NSCLC. Together with predictive marker testing (often molecular tests), these new paradigms emphasize the need for close communication between pathologists and clinical colleagues to ensure tissue adequacy and optimal triage for all appropriate diagnostic studies.

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Of these two studies, the article by Nicholson et al. presents the more integrated approach to address this problem. With their approach, they include biopsy histology, cytology, immunohistochemistry, and EGFR mutation testing.1 Cytology is a powerful modality that, in some cases, may allow for a more specific classification that is not possible based on histology or immunohistochemistry. This article also points out the benefits of making cell blocks from cytology specimens, to allow for immunohistochemistry or molecular studies.1 Cell blocks were made from pleural effusions and transbronchial needle aspirations including procedures that were blinded or guided by esophageal endoscopic ultrasound or endobronchial ultrasound.1

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With the more important role that is being assigned to newer diagnostic tests, a critical need exists for clinical trials that stratify data according to various diagnostic methods (i.e., light microscopic with or without mucin or immunohistochemical stains), to validate which approach provides the best discrimination for predicting toxicity or efficacy of chemotherapeutic regimens and other treatment modalities. At a minimum, clinical trials should report diagnostic criteria and methods used to classify tumors from enrolled patients. Finally, any studies that incorporate new techniques should compare them with currently accepted ones (i.e., TTF-1 and p63) to determine whether they provide any added value.

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The authors acknowledge all panel members of the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Adenocarcinoma Classification for their help in developing some of the concepts in this editorial.

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1. Nicholson AG, Gonzalez D, Shah P, et al. Refining the diagnosis and EGFR status of non-small cell carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6 and P63 and EGFR mutation analysis. J Thorac Oncol (in press).

2. Loo PS, Thomas SC, Nicolson MC, et al. Subtyping of undifferentiated non-small cell carcinomas in bronchial biopsy specimens. J Thorac Oncol (in press).

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4. World Health Organization. Histological Typing of Lung Tumors. Geneva: World Health Organization, 1981.

5. Travis WD, Colby TV, Corrin B, et al. Histological Typing of Lung and Pleural Tumors. Berlin: Springer, 1999.

6. Travis WD, Brambilla E, Muller-Hermelink HK, et al. Pathology and Genetics: Tumours of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC Press, 2004.

7. Hirsch FR, Spreafico A, Novello S, et al. The prognostic and predictive role of histology in advanced non-small cell lung cancer: a literature review. J Thorac Oncol 2008;3:1468–1481.

8. Johnson DH, Fehrenbacher L, Novotny WF, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 2004;22:2184–2191.

9. Scagliotti G, Hanna N, Fossella F, et al. The differential efficacy of pemetrexed according to NSCLC histology: a review of two Phase III studies. Oncologist 2009;14:253–263.

10. Scagliotti GV, Parikh P, von Pawel J, et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 2008;26:3543–3551.

11. Ciuleanu T, Brodowicz T, Zielinski C, et al. Maintenance pemetrexed plus best supportive care versus placebo plus best supportive care for non-small-cell lung cancer: a randomised, double-blind, phase 3 study. Lancet 2009;374:1432–1440.

12. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947–957.

13. Roggli VL, Vollmer RT, Greenberg SD, et al. Lung cancer heterogeneity: a blinded and randomized study of 100 consecutive cases. Hum Pathol 1985;16:569–579.

14. Cataluna JJ, Perpina M, Greses JV, et al. Cell type accuracy of bronchial biopsy specimens in primary lung cancer. Chest 1996;109:1199–1203.

15. Fujii T, Dracheva T, Player A, et al. A preliminary transcriptome map of non-small cell lung cancer. Cancer Res 2002;62:3340–3346.

16. Bhattacharjee A, Richards WG, Staunton J, et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci USA 2001;98:13790–13795.

17. Takeuchi T, Tomida S, Yatabe Y, et al. Expression profile-defined classification of lung adenocarcinoma shows close relationship with underlying major genetic changes and clinicopathologic behaviors. J Clin Oncol 2006;24:1679–1688.

18. Kosaka T, Yatabe Y, Endoh H, et al. Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res 2004;64:8919–8923.

19. Tam IY, Chung LP, Suen WS, et al. Distinct epidermal growth factor receptor and KRAS mutation patterns in non-small cell lung cancer patients with different tobacco exposure and clinicopathologic features. Clin Cancer Res 2006;12:1647–1653.

20. Ring BZ, Seitz RS, Beck RA, et al. A novel five-antibody immunohistochemical test for subclassification of lung carcinoma. Mod Pathol 2009;22:1032–1043.

21. Monica V, Ceppi P, Righi L, et al. Desmocollin-3: a new marker of squamous differentiation in undifferentiated large-cell carcinoma of the lung. Mod Pathol 2009;22:709–717.

22. Bishop JA, Sharma R, Illei PB. Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol 2010;41: 20–25.

23. Lebanony D, Benjamin H, Gilad S, et al. Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma. J Clin Oncol 2009;27:2030–2037.

24. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th Ed. Lyon: International Agency of Research on Cancer, 2008.

25. Fletcher CD, Unni KK, Mertens F. Pathology and Genetics: Tumours of Soft Tissue and Bone. Lyon: International Agency for Research on Cancer Press, 2002.

26. Travis WD, Brambilla E, Noguchi M, et al. The new IASLC/ATS/ERS international multidisciplinary lung adenocarcinoma classification. J Thorac Oncol (in press).

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© 2010International Association for the Study of Lung Cancer


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