Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma

RESEARCH ARTICLE Intratumor Heterogeneity of ALKRearrangements and Homogeneity of EGFRMutations in Mixed Lung Adenocarcinoma Federica Zito Marino1, Giuseppina Liguori1, Gabriella Aquino1, Elvira La Mantia1, Silvano Bosari2, Stefano Ferrero3, Lorenzo Rosso4, Gabriella Gaudioso2, Nicla De Rosa5, Marianna Scrima6, Nicola Martucci7, Antonello La Rocca7, Nicola Normanno8, Alessandro Morabito9, Gaetano Rocco7, Gerardo Botti1, Renato Franco1* 1 Pathology Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy, 2 Department of Pathophysiology and Organ Transplantation, University of Milan Medical School, Milan, Italy; Division of Pathology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico Milan, Italy, 3 Department of Biomedical, Surgical and Dental Sciences, University of Milan Medical School, Milan, Italy; Division of Pathology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico Milan, Italy, 4 Thoracic Surgery and Lung Transplantation Unit, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico Milan, Italy, 5 Department of Oncology and Anatomic Pathology, Hospital of National Relevance (AORN) Vincenzo Monaldi, Naples, Italy, 6 Biogem scarl, Institute for Genetic Research “Gaetano Salvatore”, Ariano Irpino (Avellino), Italy, 7 Division of Thoracic Surgery, Department of Thoracic Surgical and Medical Oncology, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy, 8 Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy, 9 Medical Oncology Unit, Department of Thoracic Surgical and Medical Oncology, Istituto Nazionale Tumori “Fondazione G. Pascale”IRCCS, Naples, Italy OPEN ACCESS Citation: Marino FZ, Liguori G, Aquino G, La Mantia E, Bosari S, Ferrero S, et al. (2015) Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma. PLoS ONE 10(9): e0139264. doi:10.1371/journal.pone.0139264 Editor: Gianpaolo Papaccio, Second University of Naples, ITALY Received: July 23, 2015 Accepted: September 10, 2015 Published: September 30, 2015 Copyright: © 2015 Marino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * Abstract Background Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be ‘major’, characterized by a single tumor showing two different histologic types, and ‘minor’, due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas. Methods 590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR. PLOS ONE | DOI:10.1371/journal.pone.0139264 September 30, 2015 1 / 15 ALK-Rearrangements in Mixed Lung Adenocarcinoma Results 10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components. Conclusion Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes. Introduction Lung cancer represents a very heterogeneous tumor not only for its clinical and radiologic presentation but also for its histological and molecular features. Particularly, major histologic heterogeneity, characterized by a single tumor showing at least two different histologic types, and minor histologic heterogeneity with a single tumor showing just one histologic type but at least two different growth patterns, are generally recorded [1]. Adenocarcinoma (ADC) is the most frequently diagnosed histological type of primary lung cancer accounting for almost half of all lung cancers [2]. Histologic intratumoral heterogeneity in ADCs is expressed both in terms of a frequent minor heterogeneity due to the occurrence of several growth patterns, such as lepidic, acinar, papillary, micropapillary and solid adenocarcinoma in mixed adenocarcinomas (mADCs), and an unusual major heterogenity as in the adenosquamous lung carcinomas (AdSqLCs). According to the 2004 World Health Organization (WHO) classification, more than 80% of lung adenocarcinomas falls into the mixed subtype [3]. In recent years, major revisions have been made to the classification and grading of ADCs, leading to an improvement of the diagnosis of these tumors with a subsequent prognostic stratification. Thus, the 2011 IASLC/ATS/ERS (International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society) classification of lung adenocacinomas has introduced some significant revisions to the 2004 WHO ADCs classification [4]. In particular, a 5% increment in the comprehensive histologic subtyping of the different patterns of mADCs is recommended, in order to determine the predominant pattern. Many series have shown the clinicopathological relevance and the prognostic role of the 2011 IASLC/ ATS/ERS classification being the predominant pattern of adenocarcinoma related to different prognosis [5]. Moreover, Sica et al. proposed a grading system based on the histologic subtyping of mADCs, providing a grading score based on the two main growth patterns [6]. This pattern-based scoring system has proved to improve both mADCs subclassification and better prognostic stratification [7]. In the past decade, the advancement on the treatment targeting mutated Epidermal Growth Factor Receptor (EGFR) and Anaplastic Lymphoma Kinase (ALK) have changed considerably the possibility of therapy for Non Small Cell Lung Cancer (NSCLC) patients. EGFR gene most common and clinically relevant mutations, accounting for approximately 80–90 (...truncated)