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effects. A CT scan after two cycles of therapy showed an increase of the lung lesion, complete obstruction of the bronchus, consensual parenchymal atelectasis, and tumor infiltration of the right pulmonary artery, the superior vena cava, the azygos vein, and a large osteolytic area in the manubrium. Because of worsening dyspnea and severe sternal and lumbar pain, the patient was hospitalized and gefitinib was discontinued. An unblocking procedure with multiple biopsies was performed on the superior right bronchus. Morphologic and immunohistochemical features of poorly differentiated SqCC were preserved, and molecular evaluation confirmed the original exon 19 microdeletion plus a resistant T790M mutation in exon 20. Considering the rapid disease progression that the patient experienced while receiving tyrosine kinase inhibitor (TKI) therapy, we retrospectively reassessed the pretreatment specimen for a primary resistant mutation. A T790M mutation in exon 20 was identified. No mutations were found in normal tissue, thereby excluding germline alterations. The Eastern Cooperative Oncology Group performance status (PS) of the patient dramatically worsened (PS of 3), and palliative radiation therapy on the sternal lesionwasperformedwhilecontinuingzoledronicacid.InFebruary2012, the patient experienced a rapid worsening of dyspnea (grade 4) and PS and began receiving best supportive care. Two months later the patient died.

EGFR-Driven Behavior and Intrapatient T790M Mutation Heterogeneity of Non–Small-Cell Carcinoma With Squamous Histology Case Report 1 In October 2011, a lesion measuring 8 ⫻ 5 cm was found in the right upper lung lobe of a 48-year-old woman with no history of smoking. Computed tomography (CT) scanning, bone scintigraphy, and magnetic resonance imaging highlighted pleura, hilar, mediastinal, and bilateral axillary lymph node involvement along with a right femur lesion. Bronchoscopy revealed obstruction of the right upper lobe, and multiple biopsies positive for p63, p40, and cytokeratin 5/6 and negative for cytokeratin 7 and thyroid transcription factor 1 favored a diagnosis of poorly differentiated squamous cell carcinoma (SqCC) of the lung. Considering the patient’s sex and never-smoker status, EGFR assessment was requested, and an exon 19 microdeletion (L747-P753⬎S; 2240-2257del18) was identified. First-line treatment with gefitinib (250 mg per day) with zoledronic acid (4 mg per month) and palliative radiation therapy was preferred over conventional histotype-guided therapy. The patient had no remarkable adverse

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Case Report 2 In December 2010, a 70-year-old woman with no history of smoking was referred to our institution with a diagnosis of SqCC of the left upper lobe of the lung, stage IV, with spinal metastases (L3). The patient had good PS and grade 1 dyspnea with severe back pain. In a different institution she had undergone a first-line platinum-based regimen that was discontinued after two cycles because of pulmonary thromboembolism and progressive disease in the left upper lobe with two novel liver metastases (2.6 cm and 3.9 cm in diameter) in segments IV and VI (Figs 1A and 1B, respectively, show CT scans of the pretreatment lung and segment VI liver lesions [gold arrows]). Palliative radiation therapy for the spinal metastases and zoledronic acid (4 mg per month) were prescribed. Histology was reassessed with appropriate immunohistochemical markers and showed positivity for cytokeratin 5/6, p63, and p40, and negativity for cytokeratin 7 and thyroid transcription factor 1 (Figs 2A and 2B show hematoxylin and eosin [H & E] staining and p40 immunoreactivity of the pretreatment biopsy from the primary tumor, respectively). EGFR analysis revealed an exon 21 L858R mutation with a preponderant mutant signal, indicative of mutant allele–specific imbalance, along with high amplification (Figs 3A and 3B, EGFR amplification detected by fluorescent in situ hybridization and L858R T⬎G mutation in the pretreatment primary tumor). In January 2011, gefitinib (250 mg per day) was prescribed as second-line treatment. During the first 2 months, the patient experienced diarrhea as the only adverse event, with good PS and bone pain control. CT scan re-evaluation after two cycles of TKI therapy showed stable disease in the lung and partial response of both liver metastases (Figs 1C and 1D, CT scan of the lung lesion and responsive segment VI

liver metastasis [gold arrows] after two cycles of TKI treatment). TKI treatment continued for two additional cycles, when CT scan reevaluation detected pulmonary and hepatic progression (Figs 1E and 1F, CT scan showing progressive disease of the lung and segment VI liver metastasis [gold arrows] after four cycles of TKI). Gefitinib was discontinued. Given that the patient maintained a good PS, pathologic and molecular reassessment of the lung and liver lesions was suggested. After written informed consent was obtained from the patient, a bronchoscopic rebiopsy of the lung tumor and percutaneous ultrasonography-guided biopsy of the liver segment VI metastasis were performed. On both pulmonary and liver biopsies, SqCC diagnosis was histologically and immunohistochemically confirmed (Figs 2C and 2D, H & E staining and p40 immunoreactivity of the lung after four cycles of TKI; Figs 2E and 2F, H & E staining and p40 immunoreactivity of liver segment VI after four cycles of TKI). Molecular biology was reassessed, including EGFR T790M evaluation, on both pre- and post-TKI specimens (after four cycles of TKI). The L858R mutation and EGFR amplification were still present in both specimens (Figs 3D and 3E, EGFR amplification and L858R mutation in the post-TKI lung specimen; Figs 3G and 3H, EGFR amplification and L858R mutation in the post-TKI liver specimen), but, surprisingly, a resistant T790M C⬎T mutation was found only in the liver metastasis and not in the lung rebiopsy (Figs 3C and 3F show exon 20 wild-type status of the pre-TKI and post-TKI lung biopsies; Fig 3I shows the T790M mutation in the liver rebiopsy). The mutant signal was more prominent also for the T790M allele. The lung rebiopsy was retested by Scorpion Amplification Refractory Mutation System polymerase chain reaction (Therascreen-EGFR test; Qiagen, Valencia, CA), which

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confirmed the L858R but not the T790M mutation, further supporting a differing T790M status for the two lesions (data not shown). C-Met fluorescent in situ hybridization analysis and a search for KRAS or BRAF mutations as alternative resistance mechanisms were negative. In July 2011, the patient’s Eastern Cooperative Oncology Group PS significantly deteriorated, and 1 month later, the patient died. Discussion Herein we describe two patients, both women and nonsmokers, with SqCC of the lung that displayed the characteristic EGFR-driven behavior of a lung adenocarcinoma. Activating EGFR mutations have been reported in small subsets (1% to 15%) of nonadenocarcinoma non–small-cell lung cancers, but controversy exists as to whether a so-called pure SqCC histotype may harbor them.1-3 Rekhtman et al4,5 have suggested, after comparing small biopsies with surgical specimens from the same patients, that EGFR mutations are not in bona fide SqCCs but in poorly differentiated adenocarcinomas that morphologically mimic SqCC; the authors thus concluded that, when EGFR mutations are found, a so-called pure SqCC should be excluded. However, both Rekhtman et al and we have proposed that, www.jco.org

with a specific panel of antibodies, an effective distinction between adenocarcinoma and SqCC can be confidently achieved in small biopsies. In our report, both primary and metastatic specimens from the same patients were morphologically and immunophenotypically shown to be pure SqCC. Our first patient, despite an activating mutation, experienced rapid disease progression while receiving TKI therapy. Retrospective evaluation found that a T790M-resistant mutation had been present from the start. Both mutations were maintained after TKI therapy. In the second patient, the T790M mutation emerged during TKI therapy in the responsive liver metastasis and not in the stable primary tumor, showing a different molecular status in behaviorally distinct tumor foci even in the same individual and confirming that T790M-associated resistance is likely to arise where pharmacologic pressure is more effective. The prominent signals of both sensitive and resistant mutations suggested that they were in cis on the same allele and, consequently, on the same mutant protein. On the basis of recent evidence regarding the synergistic oncogenic effect of the double mutation when in cis, the rapid relapse observed in the liver injury is biologically justified.6 C-Met amplification, epithelial-mesenchymal transition, and KRAS and BRAF mutations were ruled out as © 2014 by American Society of Clinical Oncology

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alternative resistance mechanisms in the T790M-negative lung rebiopsy, suggesting that alternative oncogene aberrations such as HER2/ Neu amplification, hepatocyte growth factor release by the tumor microenvironment, or other unidentified pathways contributed to the TKI resistance that was observed in the primary lesion. Several studies have highlighted successful TKI treatment of EGFR-mutated squamous/adenosquamous lung cancers, although with different rates,7-11 but evidence of EGFR T790M intrapatient heterogeneity has never been reported, to our knowledge. Tumor heterogeneity, which is responsible for tumor clone selection during therapy, is still a largely unexplored issue that, with the advent of novel highly sensitive analytic assays such as sequence-specific polymerase chain reaction or next-generation sequencing, is being rapidly elucidated. Our findings imply that multifocal tumors, whether synchronous or metachronous, should be differentially sampled and separately examined for genetic alterations whenever possible, as already occurs for other tumor types such as breast and colon cancer. Moreover, the knowledge of possible heterogeneity should be taken into account for better planning of timing and strategy of potential multimodal therapies and for the validation of novel TKIs developed against EGFR in patients with acquired resistance. In the first patient described here, the resistant mutation was present at diagnosis but not initially examined. This suggests that if one looks at EGFR, screening should be performed for all critical mutations; particularly in the case of this patient, alternative therapy could have been offered first and gefitinib kept for subsequent treatment. The costbenefit ratio for routine baseline EGFR assessment might favor this possibility, if, as suggested by American Society of Clinical Oncology guidelines, women never-smokers with SqCC are considered. The presence of EGFR mutations in squamous cell–featuring carcinomas is rare, but current views suggest that predictive factors such as smoking status and sex can play a key role in the diagnostic algorithm.12 In our limited experience of 25 patients with SqCC tested for EGFR mutations, of which 22 were former smokers or never-smokers, we found four patients with EGFR mutations, all of whom were women and never-smokers. In conclusion, to provide the best therapy approach for this patient population, regardless of histology, EGFR assessment should be considered, especially when dealing with small biopsies and never-smokers.

Paolo Graziano, Filippo de Marinis, Bruno Gori, Rita Gasbarra, Rita Migliorino, and Stefano De Santis San Camillo-Forlanini Hospitals, Rome, Italy

Giuseppe Pelosi Fondazione Istituto di Ricovero e Cura a Carattere Scientifico, Istituto dei Tumori; and Universita` degli Studi, Milan, Italy

Alvaro Leone San Camillo-Forlanini Hospitals, Rome, Italy

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The author(s) indicated no potential conflicts of interest. REFERENCES 1. Miyamae Y, Shimizu K, Hirato J, et al: Significance of epidermal growth factor receptor gene mutations in squamous cell lung carcinoma. Oncol Rep 25:921-928, 2011 2. Perez-Moreno P, Brambilla E, Thomas R, et al: Squamous cell carcinoma of the lung: Molecular subtypes and therapeutic opportunities. Clin Cancer Res 18:2443-2451, 2012 3. Baik CS, Pritchard CC, Eaton KD, et al: EGFR mutations in squamous cell lung cancer in never-smokers. J Thorac Oncol 8:e6-e7, 2013 4. Pelosi G, Fabbri A, Bianchi F, et al: ⌬Np63 (p40) and thyroid transcription factor-1 immunoreactivity on small biopsies or cellblocks for typing non-small cell lung cancer: A novel two-hit, sparing-material approach. J Thorac Oncol 7:281290, 2012 5. Rekhtman N, Paik PK, Arcila ME, et al: Clarifying the spectrum of driver oncogene mutations in biomarker-verified squamous carcinoma of the lung: Lack of EGFR/KRAS and presence of PIK3CA/AKT1 mutations. Clin Cancer Res 18:1167-1176, 2012 6. Godin-Heymann N, Bryant I, Rivera MN, et al: Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation. Cancer Res 67:7319-7326, 2007 7. Tanaka K, Hata A, Kida Y, et al: Gefitinib for a poor performance status patient with squamous cell carcinoma of the lung harboring EGFR mutation. Intern Med 51:659-661, 2012 8. Iwanaga K, Sueoka-Aragane N, Nakamura T, et al: The long-term survival of a patient with adenosquamous lung carcinoma harboring EGFR-activating mutations who was treated with gefitinib. Intern Med 51:2771-2774, 2012 9. Paik PK, Varghese AM, Sima CS, et al: Response to erlotinib in patients with EGFR mutant advanced non-small cell lung cancers with a squamous or squamous-like component. Mol Cancer Ther 11:2535-2540, 2012 10. Tseng JS, Yang TY, Chen KC, et al: Retrospective study of erlotinib in patients with advanced squamous lung cancer. Lung Cancer 77:128-133, 2012 11. Hata A, Katakami N, Yoshioka H, et al: How sensitive are epidermal growth factor receptor-tyrosine kinase inhibitors for squamous cell carcinoma of the lung harboring EGFR gene-sensitive mutations? J Thorac Oncol 8:89-95, 2013 12. de Marinis F, Rossi A, Di Maio M, et al: Treatment of advanced non-smallcell lung cancer: Italian Association of Thoracic Oncology (AIOT) clinical practice guidelines. Lung Cancer 73:1-10, 2011

DOI: 10.1200/JCO.2013.49.5697; published online ahead of print at www.jco.org on April 21, 2014 ■ ■ ■

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EGFR-Driven Behavior and Intrapatient T790M Mutation Heterogeneity of Non-Small-Cell Carcinoma With Squamous Histology.

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