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Concordant and Discordant EGFR Mutations in Patients With Multifocal Adenocarcinomas: Implications for EGFR-Targeted Therapy

      Abstract

      Purpose

      Adenocarcinoma remains the most common subtype of lung cancer in the United States. Most patients present with tumors that are invasive and often metastatic, but in some patients, multiple precursor in situ or minimally invasive adenocarcinoma tumors develop that can be synchronous and metachronous. These precursor lesions harbor the same spectrum of genetic mutations found in purely invasive adenocarcinomas, such as EGFR, KRAS, and p53 mutations. It is less clear, however, whether separate lesions in patients who present with multifocal disease share common underlying genetic driver mutations.

      Methods

      Here we review the relevant literature on molecular driver alterations in adenocarcinoma precursor lesions. We then report 4 patients with multifocal EGFR mutant adenocarcinomas in whom we performed molecular testing on 2 separate lesions.

      Findings

      In 2 of these patients, the mutations are concordant, and in 2 patients, the mutations are discordant. A review of the literature demonstrates increasing evidence that lesions with discordant mutations may confer a more favorable prognosis because they are unlikely to represent metastases.

      Implications

      Our findings suggest that the emergence of the dominant EGFR driver alteration is often independent between lesions in patients with multifocal adenocarcinomas, and thus the same targeted therapy may not be effective for all lesions. However, genetic testing of multiple lesions can help to distinguish separate primary tumors from metastatic disease.

      Key words

      Introduction

      Precursors to invasive lung adenocarcinoma have been well recognized and described. Previously known as bronchioloalveolar carcinoma (BAC),
      • Barkley J.E.
      • Green M.R.
      Bronchioloalveolar carcinoma.
      this term has now been replaced with newer designations. The 2011 revised classification of pulmonary adenocarcinoma, introduced by joint multidisciplinary specialists from the International Association for the Study of Lung Cancer, the American Thoracic Society (ATS), and the European Respiratory Society, now includes a spectrum of precursor lesions from lowest to highest risk: atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), and minimally invasive adenocarcinoma (MIA).
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      International association for the Study of lung Cancer/American Thoracic Society/European Respiratory Society International Multidisciplinary Classification of Lung Adenocarcinoma.
      The tighter delineation of lesions has proven clinical significance, as several studies have shown the newer classification to have better prognostic value.
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      Micropapillary and solid subtypes of invasive lung adenocarcinoma: clinical predictors of histopathology and outcome.
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      Prognostic significance of the IASLC/ATS/ERS classification in Chinese patients-A single institution retrospective study of 292 lung adenocarcinoma.
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      Prognostic value of the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society lung adenocarcinoma classification on death and recurrence in completely resected stage I lung adenocarcinoma.
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      Does lung adenocarcinoma subtype predict patient survival? A clinicopathologic study based on the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary lung adenocarcinoma classification.
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      Prognostic value of the IASLC/ATS/ERS classification in stage I lung adenocarcinoma patients--based on a hospital study in China.
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      Tissue-sparing application of the newly proposed IASLC/ATS/ERS classification of adenocarcinoma of the lung shows practical diagnostic and prognostic impact.
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      Tumor invasiveness as defined by the newly proposed IASLC/ATS/ERS classification has prognostic significance for pathologic stage IA lung adenocarcinoma and can be predicted by radiologic parameters.
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      The novel histologic International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification system of lung adenocarcinoma is a stage-independent predictor of survival.
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      Why do pathological stage IA lung adenocarcinomas vary from prognosis?: a clinicopathologic study of 176 patients with pathological stage IA lung adenocarcinoma based on the IASLC/ATS/ERS classification.
      The relationship between precursor lesions and invasive adenocarcinoma can be further strengthened by the finding that individual precursor lesions often harbor the same genetic mutations found in adenocarcinomas, such as EGFR,
      • Yoshida Y.
      • et al.
      Mutations of the epidermal growth factor receptor gene in atypical adenomatous hyperplasia and bronchioloalveolar carcinoma of the lung.
      • Yatabe Y.
      • et al.
      EGFR mutation is specific for terminal respiratory unit type adenocarcinoma.
      KRAS,
      • Yoshida Y.
      • et al.
      Mutations of the epidermal growth factor receptor gene in atypical adenomatous hyperplasia and bronchioloalveolar carcinoma of the lung.
      • Westra W.H.
      • et al.
      K-ras oncogene activation in atypical alveolar hyperplasias of the human lung.
      and p53 mutations.
      • Slebos R.J.
      • et al.
      p53 alterations in atypical alveolar hyperplasia of the human lung.
      Multiple foci of AAH, AIS, or MIA develop in some patients and can appear synchronously or metachronously.
      • Ebright M.I.
      • et al.
      Clinical pattern and pathologic stage but not histologic features predict outcome for bronchioloalveolar carcinoma.
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      Evolving concepts in the pathology and computed tomography imaging of lung adenocarcinoma and bronchioloalveolar carcinoma.
      • Furak J.
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      Bronchioloalveolar lung cancer: occurrence, surgical treatment and survival.
      They typically present as multiple ground glass opacities, with or without small solid foci, on computed tomography (CT) scans. These lesions can progress slowly to standard invasive carcinoma and may eventually metastasize. Therefore, current management typically includes careful CT surveillance and surgical resection and/or radiotherapy to areas that grow beyond a certain size (some propose 2 cm if the lesion remains pure ground glass) or that develop a solid component of more than a few millimeters. Prognosis can be excellent, in contrast to the typical poor prognosis for patients with metastatic lung cancer.
      • Gu B.
      • et al.
      A dominant adenocarcinoma with multifocal ground glass lesions does not behave as advanced disease.
      In patients with multifocal adenocarcinomas, the separate adenocarcinomas may share a common genetic ancestry or arise divergently over time. Here we first report 4 patients with multifocal adenocarcinoma in whom we tested EGFR mutation status for more than 1 lesion. We then review our current understanding of the underlying molecular pathologies of multifocal adenocarcinoma lesions and the potential for targeted therapies in this setting.

      Case Series

      Based on the previously cited reports of interlesional heterogeneity, we were interested in examining available tumor molecular data from our patients with multifocal minimally invasive adenocarcinoma (mMIA). From 2009 to 2015, we identified 4 patients who had resection or biopsy of more than 1 adenocarcinoma lesion in the setting of additional ground glass opacities, with results of molecular testing available for multiple separate lesions. We report their clinical characteristics and molecular testing results. This retrospective chart review project was performed using an institutional review board–approved protocol. The clinical characteristics of these patients and representative images are reported in the following and shown in Figure 1 and Table I.
      Figure 1
      Figure 1Imaging of Lesions Studied in Case Series. Representative CT imaging of analyzed lesions in four patients with mMIA. (labeled with large light green arrow when there is more than one lesion present). RUL: right upper lobe; RML: right middle lobe; LUL: left upper lobe; LLL: left lower lobe.
      Table ISummary of target lesions in patients.
      PatientSexAge, ySmoking StatusLesion LocationLesion size (greatest dimension)Lesion Radiological AppearanceLesion HistologyMutation(s)
      1F56NeverRUL5 cmIrregular mixed nodular and ground glass massAdenocarcinomaEGFR exon 19 L747_T751 deletion
      RML1.8 cmOpacityInvasive adenocarcinoma, acinar predominantEGFR exon 21 L858R
      2M79Distant 30 packs/yearLLL2.0 cmPredominantly solid lobulated lesion containing cystic air lucenciesModerately differentiated adenocarcinoma, lepidic predominantEGFR L861Q
      RUL1 cmSpiculated noduleWell-differentiated adenocarcinomaEGFR exon 19 L747_T751 deletion, p53 H178D
      3F60NeverRUL3.9Spiculated RUL apical massAcinar adenocarcinoma with papillary and lepidic patternsEGFR L858R, PIK3CA E545K, FBXL7 H98Y
      LUL1 cmSpiculated noduleInvasive adenocarcinoma, acinar predominantEGFR L858R, PIK3CA E545K, FBXL7 H98Y
      4F70NeverRML1.8 cmNoduleWell-differentiated invasive adenocarcinoma, mixed lepidic and acinar patternsEGFR exon 21 L858R
      LUL2.1 cm and 1.3 cmInfiltratesWell-differentiated adenocarcinoma, lepidic predominantEGFR exon 21 L858R
      F = female; LLL = left lower lobe; LUL = left upper lobe; M = male; RML = right middle lobe; RUL = right upper lobe.

      Patients With Discordant EGFR Mutations

      Patient 1 is a 56-year-old Asian woman, a never smoker, who had first received a diagnosis of lung adenocarcinoma after presenting with atypical chest pain. A CT scan performed initially showed a 5-cm right upper lobe (RUL) nodule; then a subsequent positron emission tomography (PET)–CT scan showed 3 focal lesions: in the RUL, right middle lobe (RML), and left upper lobe (LUL). Biopsy of the RUL nodule showed adenocarcinoma with EGFR exon 19 deletion mutation (L747_T751) by outside EGFR testing, and the LUL nodule showed atypical cells. A subsequent workup, including magnetic resonance imaging of the brain and mediastinoscopy, was negative for metastases. The patient underwent an RML wedge resection 2 months later that revealed adenocarcinoma, 1.8-cm in the largest diameter, with an EGFR exon 21 (L858R) mutation. She was treated with erlotinib for 4 months with stable disease as her best response, with a plan to proceed with surgical resection of the RUL and RML and stereotactic ablative radiotherapy (SABR) to LUL nodule; however, repeat PET/CT then showed new mediastinal lymph node involvement. The patient was then started on systemic “neoadjuvant” chemotherapy with carboplatin and pemetrexed, followed by definitive surgery with a right thoracotomy and right upper and middle bilobectomy with mediastinal lymph node dissection.
      Patient 2 is a 79-year-old Asian man with a distant 30-packs per year smoking history. He received an incidental diagnosis of multiple lung nodules, all ~1 to 2 cm in diameter, that appeared mostly to have mixed solid and ground glass components. Biopsy of the left lower lung nodule showed adenocarcinoma with an EGFR exon 21 (L861Q) mutation by multiplex polymerase chain reaction (PCR) followed by single nucleotide mutation detection, and a subsequent workup showed no metastases. He underwent left lower lobectomy that revealed T1N0M0 moderately differentiated adenocarcinoma, predominantly lepidic subtype. At delayed follow-up 3 years later, a repeat CT scan showed that 1 RUL nodule had been slowly growing, as well as multiple other sites that were likely low-grade adenocarcinoma. Biopsy of the RUL nodule showed well-differentiated adenocarcinoma, with EGFR exon 19 (L747_T751) deletion mutation and TP53 (H178D) mutation by next-generation sequencing (NGS). The patient declined surgery in favor of SABR to the RUL nodule with favorable response.

      Patients With Concordant EGFR Mutations

      Patient 3 is a 60-year-old Asian woman, a never smoker, who received a diagnosis of right pheochromocytoma when she presented with headaches, palpitations, flushing, and sweating. After surgical resection, she continued to experience occasional tachycardia. A PET-CT scan was ordered and showed a 3.9 cm hypermetabolic, spiculated RUL apical mass, as well as a 1-cm nodular opacity in the LUL. Patient eventually underwent right video-assisted thoracoscopic right upper lobectomy revealing a T3 N0 adenocarcinoma. The final pathology showed acinar adenocarcinoma with papillary and lepidic patterns, and molecular testing by NGS showed the following mutations: EGFR L858R, PIK3CA E545K, and FBXL7 H98Y. Six months after surgery, the patient’s repeat CT scan showed enlargement of the previously present small anterior LUL nodule and confluence/growth of a 10-mm spiculated multilobulated nodule in the posterior LUL. She then underwent LUL wedge excision 9 months after the initial lobectomy, and pathology showed invasive adenocarcinoma, 0.7-cm in diameter, with the same 3 mutations noted at the previous surgical resection by NGS. The patient was to undergo SABR for her other LUL lesion; however, biopsy-proven chest wall metastases then developed and she was started on systemic erlotinib.
      Patient 4 is a 70-year-old Asian woman, a never smoker, who had a diagnosis of multiple lung lesions when a cough developed. A CT scan showed an ill-defined 1.8-cm nodule in the RUL, and 2 lesions in the LUL, 2.1 cm and 1.3 cm infiltrates. PET-CT and magnetic resonance imaging of the brain showed no metastatic disease, although PET-CT noted additional opacities in the RLL. A CT-guided biopsy of the right nodule revealed adenocarcinoma. She underwent surgical resection with an RML lobectomy as the tumor was palpated to be in the middle lobe during surgery, and pathology showed well-differentiated invasive adenocarcinoma, with mixed lepidic and acinar patterns, 1.3 cm, with an EGFR L858R mutation by NGS. The patient was under regular surveillance until 2 years later when a CT scan showed that the lesions in the LUL were slowly progressing. She then underwent left video-assisted thoracoscopic and lingual-sparing LUL lobectomy. Pathology showed a 1.6-cm and a 1.2-cm well-differentiated adenocarcinomas, lepidic predominant, the larger of which was positive for EGFR L858R mutation by NGS. A year later, a surveillance CT scan showed a slight gradual increase in nodularity along the R fissure, for which the patient underwent SABR. She continues to do well and remains under active surveillance.

      Review of Current Literature

      Molecular Tumor Biology

      The understanding of how recurrent oncogenic mutations “drive” invasive lung adenocarcinoma has advanced over the years, with the research effort focused on development and testing of potential targeted therapies. However, a deeper understanding of molecular alterations in minimally invasive precursor lesions and their treatment is still lacking. Another challenge is that many of the studies were done using the previous designation of BAC rather than the newer term AIS or MIA. For clarity in this review, the term BAC will still be used for studies performed using this previous designation.
      Whether multifocal adenocarcinomas share a common genetic origin or arise independently remains controversial. Barsky et al
      • Barsky S.H.
      • et al.
      The multifocality of bronchioloalveolar lung carcinoma: evidence and implications of a multiclonal origin.
      showed BAC clonal nonidentity or multiclonality in 3 separate cases by using PCR amplification of a 511–base pair region located within the first intron of the human hypoxanthine phosphoribosyltransferase gene. On the other hand, Holst et al
      • Holst V.A.
      • et al.
      Bronchioloalveolar adenocarcinoma of lung: monoclonal origin for multifocal disease.
      studied multifocal BAC in 28 patients using a topographic genotyping approach for the presence of KRAS exon 1 mutations and p53 loss of heterozygosity (LOH) and suggested a monoclonal origin with spread via the intra-alveolar route, intrapulmonary lymphatics, or aerosolization, leading to implantation at different sites. In a study by Zhong et al
      • Zhong W.Z.
      • et al.
      Genetic evolution of epidermal growth factor receptor in adenocarcinoma with a bronchioloalveolar carcinoma component.
      looking at multifocal disease using EGFR analysis, the results suggest that both of these models can exist. In a more recent study by Murphy et al,
      • Murphy S.J.
      • et al.
      Identification of independent primary tumors and intrapulmonary metastases using DNA rearrangements in non-small-cell lung cancer.
      the authors addressed this question using next-generation sequencing performed using an Illumina mate-pair library protocol. A total of 41 tumor samples were sequenced, with a range of 3 to 276 break points per tumor identified. Lung tumors predicted to be independent primary tumors based on different histologic subtypes did not share any genomic rearrangements. In patients with primary lung tumors and paired distant metastases, shared rearrangements were identified in all tumor pairs, emphasizing the patient specificity of identified breakpoints. Concordance between histology and genomic data occurred in the majority of samples. Discrepant tumor samples were resolved by genome sequencing. This study suggests the importance of detailed molecular testing in distinguishing independent primary tumors from intrapulmonary metastases. However, a remaining question is how frequently do separate primary lesions share common underlying genetic driver mutations, especially in patients with little smoking history. In another similar study by Wu et al,
      • Wu C.
      • et al.
      High Discrepancy of Driver Mutations in Patients with NSCLC and Synchronous Multiple Lung Ground-Glass Nodules.
      the authors analyzed tumors from 35 patients with multiple lesions resected, including confirmed nonsmall cell lung cancer (NSCLC) and at least 1 ground glass nodule (GGN), were analyzed for mutations in EGFR, KRAS, HER2, BRAF, and PIK3CA together with fusions in ALK, ROS1, and RET. A total of 72 lesions (60 were GGNs) were analyzed. Of these, 33 tumor lesions (45.8%) were found to harbor EGFR mutations: 13 tumors with exon 19 deletion, 18 with L858R on exon 21, and 2 with both exon 19 del and L858R mutation. There were 5 tumors (6.9%) harboring EML4-ALK fusion, 4 HER2 mutations (5.6%), 3 KRAS mutations (4.2%), 1 ROS1 fusion, and 1 BRAF mutation. Only 6 of 30 patients harbored identical mutations. The discordance rate of driver mutations was 80% (24 of 30) in those patients harboring at least 1 of the detected driver mutations. The authors concluded that there is a high discrepancy of driver mutations among NSCLC patients with GGNs and a favorable prognosis after multiple lesion resection.

      Loss of Heterozygosity

      LOH is an indirect index of genetic alterations in tumors. A number of markers have also been implicated in the stepwise progression from precursor lesions to fully invasive adenocarcinomas. LOH of 3p and 9p might be an early event of carcinogenesis, as in a study by Yamasaki et al
      • Yamasaki M.
      • et al.
      Correlation between genetic alterations and histopathological subtypes in bronchiolo-alveolar carcinoma and atypical adenomatous hyperplasia of the lung.
      in which the authors compared 2 types of BAC with AAH. Takamochi et al
      • Takamochi K.
      • et al.
      Loss of heterozygosity on chromosomes 9q and 16p in atypical adenomatous hyperplasia concomitant with adenocarcinoma of the lung.
      analyzed AAH and concomitant adenocarcinoma in 11 patients, and the results suggested a causal relationship of LOH on 9q and 16p in a fraction of AAH lesions and adenocarcinomas of the lung. In another study by Sasatomi et al,
      • Sasatomi E.
      • et al.
      Genetic profile of cumulative mutational damage associated with early pulmonary adenocarcinoma: bronchioloalveolar carcinoma vs. stage I invasive adenocarcinoma.
      the authors found the most frequently affected chromosome regions in BAC were 8q and 17p. LOH of 1p, 3p, 7q, and 18q as well as fractional allele loss were more frequent in stage 1 adenocarcinomas than BAC. Although these genomic alterations are present in many precursor lesions, it is challenging to use LOH analysis to identify shared ancestry between independent lesions.

      KRAS, EGFR, and p53 Mutations

      KRAS mutations are the primary driver genetic alterations in as many as 25% of NSCLC adenocarcinomas. It has been hypothesized that these lung adenocarcinomas develop through a series of genetic mutations that sequentially accumulate, leading eventually to the phenotype of invasive adenocarcinoma. This model was based on several lines of evidence. One mouse model study using a conditionally activated allele of oncogenic KRAS showed progression from AAH to conditions similar to BAC to adenocarcinoma.
      • Jackson E.L.
      • et al.
      Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.
      In a separate study, regional pulmonary stem cells were identified, termed bronchioalveolar stem cells, that are capable of giving rise to bronchiolar Clara cells and alveolar cells of the distal lung. Bronchioalveolar stem cells expanded in response to oncogenic KRAS in culture and in precursors of lung tumors in vivo and thus may be the putative cells of origin for this subtype of lung cancer.
      • Kim C.F.
      • et al.
      Identification of bronchioalveolar stem cells in normal lung and lung cancer.
      EGFR mutations are also frequently found in both precursor lesions and invasive adenocarcinomas, with the reported frequency ranging from 11% to 58% depending on the patient population.
      • Yoshida Y.
      • et al.
      Mutations of the epidermal growth factor receptor gene in atypical adenomatous hyperplasia and bronchioloalveolar carcinoma of the lung.
      • Haneda H.
      • et al.
      A correlation between EGFR gene mutation status and bronchioloalveolar carcinoma features in Japanese patients with adenocarcinoma.
      • Marchetti A.
      • et al.
      EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment.
      EGFR and KRAS mutations are mutually exclusive in BAC lesions.
      • Marchetti A.
      • et al.
      EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment.
      Another distinction between these 2 mutations is that KRAS mutations are more frequently found in mucinous disease, whereas EGFR mutations are more frequently found in nonmucinous disease, with the latter more responsive to EGFR-targeted therapies even without EGFR mutation testing.
      • Marchetti A.
      • et al.
      EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment.
      • Garfield D.H.
      • et al.
      West, Bronchioloalveolar carcinoma: the case for two diseases.
      • Sakuma Y.
      • et al.
      Distinctive evaluation of nonmucinous and mucinous subtypes of bronchioloalveolar carcinomas in EGFR and K-ras gene-mutation analyses for Japanese lung adenocarcinomas: confirmation of the correlations with histologic subtypes and gene mutations.
      EGFR mutations and HER2 overexpression (defined by positive immunohistochemistry staining) often coexist in BAC lesions, but the role of HER2 as a codriver or passenger genomic alteration remains unclear.
      • Erman M.
      • et al.
      Epidermal growth factor receptor, HER-2/neu and related pathways in lung adenocarcinomas with bronchioloalveolar features.
      Mutations in p53 are commonly found in invasive adenocarcinomas, with incidence of 50% or higher depending on testing methodology.
      • Mitsudomi T.
      • et al.
      Prognostic significance of p53 alterations in patients with non-small cell lung cancer: a meta-analysis.
      However, the incidence of p53 mutation is much lower in BAC lesions, reported to be 0 to 11%,
      • Ahrendt S.A.
      • et al.
      p53 mutations and survival in stage I non-small-cell lung cancer: results of a prospective study.
      • Koga T.
      • et al.
      Clinicopathological and molecular evidence indicating the independence of bronchioloalveolar components from other subtypes of human peripheral lung adenocarcinoma.
      leading to speculation that p53 mutation may be a later event in the development of invasive adenocarcinoma from precursor lesions.
      • Slebos R.J.
      • et al.
      p53 alterations in atypical alveolar hyperplasia of the human lung.

      Mutational Progression Within Precursor Lesions

      A recent study by Izumchenko et al
      • Izumchenko E.
      • et al.
      Targeted sequencing reveals clonal genetic changes in the progression of early lung neoplasms and paired circulating DNA.
      examining genetic drivers in each of the precursor categories of adenocarcinoma (using the 2011 classification) ties some of the knowledge about individual gene alterations together. The authors studied the full spectrum of early histologic progression using next-generation sequencing of coding regions from 125 well-characterized cancer-driving genes. They found that in AAH lesions, the most frequently mutated genes were not the classic drivers, but rather genes involved in DNA repair and chromatin remodeling, suggesting that these lesions are predisposed to clonal expansion and acquisition of secondary genetic mutations. In AIS tumors, the mutational landscape varied considerably, but began to include more driver mutations such as EGFR and KRAS. In MIA tumors, even more driver oncogenes were identified, but mostly in the denser or invasive zones of tumors. Genes such as KRAS, EGFR, and TP53 are highly connected nodes in the mutational landscape, consistent with their potential as drivers of glandular tumorigenesis, and likely have a role in aggressive transformation of small premalignant lesions. In addition, there appears to be a high degree of intertumoral and even intratumoral genetic heterogeneity, even in a single patient.

      Targeted Therapy in Multifocal Precursor Disease

      As multifocal minimally invasive adenocarcinoma became recognized as a distinct clinical entity, it was recognized that this histology predicted a better response to gefitinib, an EGFR tyrosine kinase inhibitor. This is likely due to the high prevalence of EGFR mutations in these lesions.
      • Fukuoka M.
      • et al.
      Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected].
      • Kris M.G.
      • et al.
      Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial.
      • Lynch T.J.
      • et al.
      Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.
      • Miller V.A.
      • et al.
      Bronchioloalveolar pathologic subtype and smoking history predict sensitivity to gefitinib in advanced non-small-cell lung cancer.
      Several studies examining the effects of EGFR-targeted therapy have since been conducted.
      In a 2004 study by Kris et al
      • Kris M.G.
      • et al.
      Cigarette smoking history predicts sensitivity to erlotinib: Results of a phase II trial in patients with bronchioloalveolar carcinoma (BAC).
      69 patients with stage IIIB/IV inoperable NSCLC were found to have pure or mixed BAC and treated with erlotinib. Partial responses to erlotinib occurred in 15 of 59 patients evaluated (25% [95% CI, 15%–38%]). Responses occurred in 1 of 14 patients (7%) with “pure” BAC and 13 of 44 (30%) with adenocarcinoma with BAC features. Never smoking and a lower number of cigarettes smoked predict sensitivity to erlotinib in patients with BAC.
      In the Phase II S0126 study by West et al,
      • West H.L.
      • et al.
      Gefitinib therapy in advanced bronchioloalveolar carcinoma: Southwest Oncology Group Study S0126.
      a total of 136 chemotherapy-naïve and chemotherapy-pretreated patients with advanced BAC were treated with gefitinib 500 mg/d until progression or prohibitive toxicity. The Response Evaluation Criteria in Solid Tumors response rate was 17%, with 6% complete responses (CRs) in 69 previously untreated patients with measurable disease and 9% with no CRs in 22 pretreated patients. Exploratory subset analyses revealed improved survival among women (P = 0.031), patients in whom a rash developed (P = 0.003), never smokers (P = 0.061), and patients with a performance status of 0 or 1 (P = 0.015). Further analysis by Hirsch et al
      • Hirsch F.R.
      • et al.
      Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study.
      showed that increased EGFR gene copy number detected by fluorescence in situ hybridization is associated with improved survival after gefitinib therapy in patients with advanced BAC: 12 of 19 EGFR/fluorescence in situ hybridization–positive patients (63%) demonstrated disease control versus 14 of 36 patients (39%) in the fluorescence in situ hybridization–negative group (P = 0.087). In separate analysis, Franklin et al
      • Franklin W.A.
      • et al.
      Association between activation of ErbB pathway genes and survival following gefitinib treatment in advanced BAC (SWOG 0126).
      showed low phosphorylated mitogen-activated kinase and combined low ErbB2 and phosphorylated mitogen-activated kinase predict increased survival with gefitinib therapy in patients with advanced BAC and that dual inhibition of ErbB1 and ErbB2 may lead to improved therapeutic efficacy.
      In another Phase II study by Miller et al,
      • Miller V.A.
      • et al.
      Molecular characteristics of bronchioloalveolar carcinoma and adenocarcinoma, bronchioloalveolar carcinoma subtype, predict response to erlotinib.
      101 patients were treated with BAC (n = 12) or adenocarcinoma, BAC subtype (n = 89) with erlotinib 150 mg/d. The overall response rate was 22% (95% CI, 14%–31%). In patients treated with pure BAC, the response rate and median survival were 20% and 4 months, respectively, compared with 23% and 19 months, respectively, in those with adenocarcinoma, BAC subtype. Patients with EGFR mutations had an 83% response rate (15 of 18; 95% CI, 65%–94%) and 23-month median overall survival (OS).
      The Phase II IFCT-0401 trial enrolled 88 chemotherapy-naïve patients with BAC who were treated with 250 mg/d gefitinib. Disease control was achieved in 25 patients (29.4%); 11 patients (12.9%) had a partial response, and 14 (16.4%) had stable disease. Median progression-free survival was 2.9 months (95% CI, 2.3–3.2), and median OS was 13.2 months (95% CI, 10.2–17.3). Never smokers, patients with low respiratory symptoms score, occurrence of cutaneous rash, and nonmucinous subtype had an increased probability of disease control.
      • Cadranel J.
      • et al.
      IFCT-0401 Trial: a phase II study of gefitinib administered as first-line treatment in advanced adenocarcinoma with bronchioloalveolar carcinoma subtype.
      Ramalingam et al
      • Ramalingam SS1
      • Lee J.W.
      • Belani C.P.
      • et al.
      Cetuximab for the treatment of advanced bronchioloalveolar carcinoma (BAC): an Eastern Cooperative Oncology Group phase II study (ECOG 1504).
      conducted a Phase II study using cetuximab in which patients with advance-stage pure BAC or adenocarcinoma with BAC features, >2 previous chemotherapy regimens and no previous EGFR therapy, and Eastern Cooperative Oncology Group performance status of 0 to 2 were eligible. Approximately 50% of patients received >2 cycles of therapy (>8 weeks). The confirmed response rate was 7%, and stable disease was observed in 35%. The median survival and progression-free survival were 13 and 3.3 months, respectively.
      Overall, response rates to EGFR inhibitor therapy (7%–25%) in these studies are substantially lower than in patients with widely metastatic EGFR in whom response rates of 60% or better are anticipated. This suggests that intralesional and interlesional heterogeneity may limit the effectiveness of a single targeted therapy in this patient population.

      Prognostic Value of EGFR and KRAS in Early-stage NSCLC

      The prognostic value of molecular testing of a single site of disease remains controversial and may depend on the mutation itself. In a systematic review and meta-analysis that included 16 studies (N = 3337) to determine the prognostic value of EGFR mutations in resected NSCLC by Zhang et al,
      • Zhang Z.
      • et al.
      Prognostic value of epidermal growth factor receptor mutations in resected non-small cell lung cancer: a systematic review with meta-analysis.
      the combined hazard ratio evaluating EGFR mutations on disease-free survival (DFS) was 0.96 (95% CI, 0.79–1.16; P = 0.65). The combined hazard ratio evaluating EGFR mutations on OS was 0.86 (95% CI, 0.72–1.04; P = 0.12). The subgroup analysis based on univariate and multivariate analyses in DFS and OS showed no statistically significant difference. There was also no difference in DFS and OS of stage I NSCLC patients. In a separate study examining the prognostic value of KRAS mutation status in resected stage I lung adenocarcinoma, Izar et al
      • Izar B.
      • et al.
      The prognostic impact of KRAS, its codon and amino acid specific mutations, on survival in resected stage I lung adenocarcinoma.
      examined mutation status in a total of 312 patients who had complete resection of stage I lung adenocarcinoma without any adjuvant therapy, using a multiplex PCR-based assay; 127 patients harbored KRAS mutations (KRAS-MUT) and 185 had KRAS-wild type (KRAS-WT) tumors. Compared with KRAS-WT, KRAS-MUT was associated with significantly shorter OS and DFS. When stratifying KRAS-WT patients based on EGFR status, KRAS-MUT patients had worse OS and DFS than patients with EGFR-MUT and EGFR-WT/KRAS-WT (WT/WT). Multivariate analysis identified KRAS mutation as an independent predictor of worse OS (P = 0.001) and DFS (P < 0.0001). The authors concluded that KRAS appears to be an independent prognostic marker in resected stage I lung adenocarcinoma.

      Conclusions

      Multifocal adenocarcinoma remains a unique disease entity with a distinct presentation from typical NSCLC. It is important, but not always easy, to differentiate between multiple primary tumors versus metastatic intrapulmonary disease. Controversy also still exists as to how these lesions develop, whether through clonal expansion and metastasis or multiclonal origin. From our limited case series and review of the literature, we believe that molecular testing of >multiple lesions helps to distinguish these possibilities for staging and treatment purposes, with some tumors sharing a common genetic driver and some tumors appearing to have arisen independently. In our experience, molecular testing may not only provide treatment options but also potentially be of prognostic value because multifocal lesions that share the same mutations are more likely to behave like metastatic disease over time. Deeper NGS-based molecular analysis of these tumors may identify additional somatic tumor gene alterations helping resolve whether they are concordant or discordant. Targeted therapy based on the molecular status of a dominant lesion is an appropriate first step in therapy, as is often the practice for metastatic disease; however, the same targeted therapy may not be effective for all lesions. Further evaluation of underlying mutations in these synchronous and metachronous lesions can shed light on these pathogenic processes and potentially identify therapies to halt the process of tumorigenesis within these lesions before they progress.
      • Aberle D.R.
      • et al.
      Reduced lung-cancer mortality with low-dose computed tomographic screening.

      AUTHOR CONTRIBUTIONS

      J.C. Chuang were responsible for study design, patient identification, creation of the figures, literature search, and writing of the first draft.
      J.B. Shrager were responsible for study design, patient identification, editing of manuscript. H.A.Wakelee were responsible for study design, editing of manuscript.
      J.W. Neal were responsible for study design, patient identification, editing of manuscript.
      Grant support: none reported.

      Conflicts of Interest

      The authors report no funding from industry that influenced this work. There was no industry sponsorship of this manuscript.

      Acknowledgments

      No one other than the authors listed helped write or revise this manuscript.

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