Newly Diagnosed Lung Cancer: Which Molecular Tests Are Needed for Optimal Treatment Decision Making?

June 15, 2018

In this article, an approach to the diagnostic evaluation of patients with newly diagnosed advanced NSCLC is suggested, in an attempt to identify the best treatment options.

The management of advanced non–small-cell lung cancer (NSCLC) has changed dramatically over the past few years due to our expanded knowledge of the molecular basis of lung cancer (driver mutations and immune targets) and drugs that affect these pathways, namely targeted agents and checkpoint inhibitors. Consequently, it is important to test patients with newly diagnosed advanced NSCLC for potentially significant molecular abnormalities prior to the initiation of treatment. This requires close coordination between the surgical pathologist, the molecular pathologist, and the medical oncologist to ensure that the biopsy specimen is used judiciously to get all necessary information. In this article, I suggest an approach to the diagnostic evaluation of patients with newly diagnosed advanced NSCLC, in an attempt to identify the best treatment options for each.


Lung cancer is the leading cause of cancer-related deaths not only in the United States, but worldwide.[1] Historically, lung cancer has been classified, based on histology, as either non–small-cell lung cancer (NSCLC) or small-cell lung cancer. Adenocarcinoma (38.5%) is the most common histologic subtype of NSCLC.[2] Approximately 57% of patients with NSCLC present with metastatic disease at diagnosis,[1] and systemic therapy forms the backbone of treatment for these patients.

Role of Histology

Once metastatic disease has been confirmed either by a tissue biopsy or by imaging studies, it is important to identify the correct histology. A diagnosis of simply “NSCLC” is no longer considered adequate, since management is dependent on the specific histologic subtype. For example, pemetrexed and bevacizumab are not indicated for patients with squamous cell carcinoma,[3,4] while necitumumab appears to be useful in this subtype.[5] At the same time, it is important to preserve enough tissue for molecular analysis. Given these conflicting tensions in pathology tissue utilization, current guidelines recommend using only one immunohistochemistry stain each for adenocarcinoma (thyroid transcription factor 1 [TTF-1] or napsin A) and squamous cell carcinoma (p40 or p63), rather than a large panel.[6,7]

Role of Molecular Pathology

While chemotherapy has long been the mainstay of treatment for patients with metastatic NSCLC, response rates to conventional cytotoxic chemotherapy range from approximately 20% with platinum-based doublets, to approximately 35% with the addition of bevacizumab to platinum doublet therapy.[4] Over the past decade, an increased understanding of the pathogenesis of lung cancer, especially lung adenocarcinoma, has led to the emergence of molecularly targeted agents that have revolutionized the management of the disease. The most common abnormalities seen in lung adenocarcinoma are KRAS mutations, but unfortunately, attempts to identify therapeutic agents targeting the relevant pathways have not been successful.

Therapies that target EGFR, ALK, and ROS1 rearrangements have increased progression-free survival (PFS) in patients with these mutations, with much less toxicity, compared with cytotoxic chemotherapy.[8,9] Of the targetable abnormalities, activating mutations in the EGFR gene are the most common, followed by ALK translocations. Additionally, a study from the Lung Cancer Mutation Consortium evaluated 1,007 metastatic lung adenocarcinoma patients and identified less common abnormalities (Table 1).[10]

There have been a number of clinical trials comparing specific epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibitors vs chemotherapy in the first-line setting in patients whose tumors harbor the respective mutations (Table 2).[11-20] While none of the studies have shown an overall survival benefit with targeted agents, they all uniformly show an increase in response rates, as well as improved PFS, compared with cytotoxic chemotherapy. Moreover, the molecularly targeted agents are better tolerated. There have been no trials comparing chemotherapy with targeted agents for patients with less common mutations like ROS1 and BRAF. However, single-arm studies in these two settings have demonstrated response rates and PFS similar to those seen in the randomized trials mentioned previously. In a phase I study of ROS1-rearranged NSCLC, treatment with crizotinib showed an objective response rate of 72% (95% CI, 58%–84%), and produced a median PFS of 19.2 months (95% CI, 14.4 months–not reached).[21]

Similarly, the BRF113928 study treated 93 patients with BRAF V600E mutation–positive metastatic NSCLC using a combination of dabrafenib and trametinib. Of these patients, 36 had received no prior systemic therapy for metastatic NSCLC, while 57 had progressed following at least one platinum-based regimen. The previously treated group had an overall response rate of 63% (95% CI, 49%–76%), with a median duration of response of 12.6 months (95% CI, 5.8 months–not estimable). The treatment-naive group had an overall response rate of 61% (95% CI, 44%–77%); the median duration of response was not reached.[22] Given these data, molecularly targeted agents are the recommended treatment option for patients with metastatic lung cancer whose tumors harbor these driver mutations.

Therefore, it is important to test for molecular abnormalities prior to initiation of therapy for metastatic lung adenocarcinoma. This is especially important in patients who have never smoked or have a light/remote history of smoking, since the more common abnormalities (ie, EGFR activating mutations and ALK translocations) are seen in never/light smokers. Hence, I perform molecular testing in never/light smokers regardless of the histologic subtype, even if it means performing another biopsy to obtain sufficient tissue.


More recently, there has been a greater understanding of the role of the immune system in the regulation of cancer growth. In multiple malignancies, including lung cancer, checkpoint inhibition has been utilized to enhance and restore the capability of the immune system to recognize and eliminate cancer cells by overcoming the mechanisms used by malignancies to evade the immune response. Immunotherapy with programmed death 1 (PD-1) inhibitors is now part of the armamentarium for the treatment of advanced NSCLC, with at least some patients benefiting from frontline therapy.

Pembrolizumab is an immunoglobulin G4 monoclonal antibody that attaches to PD-1 receptors expressed on T cells. This disrupts binding to programmed death ligand 1 (PD-L1), and restores cytotoxic T-cell effector function.[23] Pembrolizumab was compared with chemotherapy in the phase III KEYNOTE-024 study of 305 patients with treatment-naive metastatic NSCLC and PD-L1 expression ≥ 50% (EGFR mutation– and ALK translocation–negative).[24] The median PFS was higher with pembrolizumab (10.3 vs 6 months; hazard ratio for disease progression or death, 0.50; 95% CI, 0.37–0.68; P < .001). The trial was stopped prematurely, and the US Food and Drug Administration (FDA) approved the use of pembrolizumab for patients meeting the criteria used in the trial.

The recently published results of the KEYNOTE-189 study demonstrated that the addition of pembrolizumab to platinum/pemetrexed was associated with improved overall survival, regardless of PD-L1 expression.[25] While it is tempting to omit PD-L1 testing and use the triplet chemoimmunotherapy combination for all patients, the role of cytotoxic chemotherapy is unclear in patients with advanced lung adenocarcinoma who have high PD-L1 expression.

PD-L1 expression on tumors is detected by immunohistochemistry. There are currently four different antibody assays on two different platforms registered with the FDA to test for PD-L1 expression on lung cancer specimens: the Dako 28-8, Dako 22C3, Ventana SP142, and Ventana SP263. A recent study compared these four assays and found a high concordance between the 22C3 and 28-8 antibodies.[26] When the four assay results were dichotomized according to clinically relevant cutoffs, the pairwise comparisons showed poor to moderate concordance (κ = 0.2–0.58), and the positive percent agreement was between 15% and 90%. Since the KEYNOTE-024 study used the 22C3 antibody on the Dako platform, my practice is to use this assay while deciding whether or not to treat with pembrolizumab.

Interestingly, initial studies demonstrated that PD-L1 expression on lung adenocarcinomas was significantly associated with the presence of EGFR mutations.[27] Moreover, EGFR mutation–positive patients with PD-L1 expression were more likely to respond to gefitinib or erlotinib than PD-L1–negative patients. However, clinical evidence suggests that patients whose tumors harbor EGFR mutations or ALK translocations do not respond to PD-1 inhibitors regardless of the level of PD-L1 expression on the tumors.[28] Given the similarities between these and other molecular drivers in terms of response to targeted agents, it is reasonable to extrapolate these results to patients whose tumors harbor ROS1 and BRAF, even though these tumors have not been specifically tested for response to immunotherapy.

In my practice, I do not test for PD-L1 expression in patients with adenocarcinoma whose tumors harbor a driver mutation. My approach to pretreatment analysis of the tumor specimen is illustrated in the Figure. This approach is also endorsed by the current National Comprehensive Cancer Network guidelines, which recommend routine molecular testing for all patients with advanced disease.[29] Though they recommend that testing be conducted as part of broad molecular profiling, this is not always feasible; in these cases, patients should be tested for EGFR mutations, ALK translocations and, if possible, ROS1 translocations and BRAF mutations at the very least. Similarly, for squamous cell carcinoma, the guidelines recommend testing for these markers in never-smokers, patients with small biopsy specimens, and those with mixed histology.


In summary, there have been a number of changes in the management of advanced NSCLC over the past few years. It is important to provide optimal personalized treatment to every patient with advanced disease. It is in the patients’ best interest that we obtain all molecular information prior to initiation of treatment, which has necessitated a closer interaction between the medical oncologist, the surgical pathologist, and the molecular pathologist.

Financial Disclosure:The author has no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.


1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68:7-30.

2. Dela Cruz CS, Tanoue LT, Matthay RA. Lung cancer: epidemiology, etiology, and prevention. Clin Chest Med. 2011;32:605-44.

3. 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;26:3543-51.

4. Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med. 2006;355:2542-50.

5. Reck M, Thomas M, Kropf-Sanchen C, et al. Necitumumab plus gemcitabine and cisplatin as first-line therapy in patients with stage IV EGFR-expressing squamous non-small-cell lung cancer: German subgroup data from an open-label, randomized controlled phase 3 study (SQUIRE). Oncol Res Treat. 2016;39:539-47.

6. Lindeman NI, Cagle PT, Aisner DL, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors. J Thorac Oncol. 2018;13:323-58.

7. Travis WD, Brambilla E, Nicholson AG, et al. The 2015 World Health Organization classification of lung tumors: impact of genetic, clinical and radiologic advances since the 2004 classification. J Thorac Oncol. 2015;10:1243-60.

8. Lee CK, Brown C, Gralla RJ, et al. Impact of EGFR inhibitor in non-small cell lung cancer on progression-free and overall survival: a meta-analysis. J Natl Cancer Inst. 2013;105:595-605.

9. Shaw AT, Kim DW, Nakagawa K, et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med. 2013;368:2385-94.

10. Kris MG, Johnson BE, Berry LD, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA. 2014;311:1998-2006.

11. Fukuoka M, Wu YL, Thongprasert S, et al. Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS). J Clin Oncol. 2011;29:2866-74.

12. Maemondo M, Inoue A, Kobayashi K, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010;362:2380-8.

13. Mitsudomi T, Morita S, Yatabe Y, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010;11:121-8.

14. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361:947-57.

15. Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012;13:239-46.

16. Wu YL, Zhou C, Hu CP, et al. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial. Lancet Oncol. 2014;15:213-22.

17. Zhou C, Wu YL, Chen G, et al. Final overall survival results from a randomised, phase III study of erlotinib versus chemotherapy as first-line treatment of EGFR mutation-positive advanced non-small-cell lung cancer (OPTIMAL, CTONG-0802). Ann Oncol. 2015;26:1877-83.

18. Sequist LV, Yang JC, Yamamoto N, et al. Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol. 2013;31:3327-34.

19. Solomon BJ, Mok T, Kim DW, et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med. 2014;371:2167-77.

20. Soria JC, Tan DSW, Chiari R, et al. First-line ceritinib versus platinum-based chemotherapy in advanced ALK-rearranged non-small-cell lung cancer (ASCEND-4): a randomised, open-label, phase 3 study. Lancet. 2017;389:917-29.

21. Shaw AT, Ou SH, Bang YJ, et al. Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J Med. 2014;371:1963-71.

22. Odogwu L, Mathieu L, Blumenthal G, et al. FDA approval summary: dabrafenib and trametinib for the treatment of metastatic non-small cell lung cancers harboring BRAF V600E mutations. Oncologist. 2018 Feb 7. [Epub ahead of print]

23. Disis ML. Mechanism of action of immunotherapy. Semin Oncol. 2014;41(suppl 5):S3-S13.

24. Reck M, Rodriguez-Abreu D, Robinson AG, et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med. 2016;375:1823-33.

25. Gandhi L, Rodríguez-Abreu D, Gadgeel S, et al. Pembrolizumab plus chemotherapy in metastatic non-small-cell lung cancer. N Engl J Med. 2018 Apr 16. [Epub ahead of print]

26. Hendry S, Byrne DJ, Wright GM, et al. Comparison of four PD-L1 immunohistochemical assays in lung cancer. J Thorac Oncol. 2018;13:367-76.

27. D’Incecco A, Andreozzi M, Ludovini V, et al. PD-1 and PD-L1 expression in molecularly selected non-small-cell lung cancer patients. Br J Cancer. 2015;112:95-102.

28. Gainor JF, Shaw AT, Sequist LV, et al. EGFR mutations and ALK rearrangements are associated with low response rates to PD-1 pathway blockade in non-small cell lung cancer: a retrospective analysis. Clin Cancer Res. 2016;22:4585-93.

29. National Comprehensive Cancer Network. Non-small cell lung cancer guidelines, version 3.2018.