Originally discovered in lymphomas,1 the ALK fusion oncogene with gain-of-function cytoplasmic tyrosine kinase activity has since been identified as an oncogenic driver in 3% to 10% of patients with non–small cell lung cancer (NSCLC).2,3 FDA approval of crizotinib, a small molecule ALK tyrosine kinase inhibitor (TKI), only 3 years after the discovery of the ALK fusion oncogene represents one of the most rapid bench-to-bedside translational advances in the history of targeted cancer therapy.4 Among the many fusion partners that have been reported, EML4 is the most common gene partner of ALK through a paracentric inversion of chromosome 2 inv(2) (p21;p23). We previously reported that in a cohort of 200 NSCLC specimens, the EML4-ALK–positive transcripts included 109 variant 1 (V1; 54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and 3 V5a (1.5%) variants.5 Most (n=188; 94.0%) EML4-ALK–positive NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK variants and individual tumors, with V3 having the lowest ALK expression level by reverse transcription polymerase chain reaction (RT-PCR) assay.5,6 Although EML4-ALK V1 has been shown to be most common in Caucasians, V3 has been shown to be most common in the Chinese population.7 Recent studies have shown that EML4-ALK V1 and V3 may have different sensitivity to ALK inhibitors and different resistant mutations.8,9 Little is known regarding whether rare variants other than EML4-ALK V1, V3, or different fusion partners affect the clinical benefit of ALK inhibitors.
Histologic transformation from lung adenocarcinoma (LUAD) to small cell lung carcinoma (SCLC) or large cell neuroendocrine tumor has been associated with resistance to EGFR10–12 and ALK TKIs.13 Although histologic transformation from LUAD to lung squamous cell carcinoma (LUSC) has been reported as a mechanism of resistance to EGFR inhibition,14,15 it has not been reported for ALK inhibition. This report describes the clinical course of a female former light smoker with metastatic LUAD whose tumor underwent histologic transformation from a well-differentiated LUAD to a well-differentiated LUSC in the same location in the left mainstem bronchus while maintaining the ALK fusion as the driver oncogene without any known resistance mutation detected by clinical next-generation sequencing (NGS) assays.
Materials and Methods
The patient provided consent to participate in an Institutional Review Board–approved study (University of California, Davis, #226210), as well as consent for publication. The clinical history, radiographic images, and histologic and immunohistochemistry stains were performed as standard of care clinical tests at our institution. Fluorescence in situ hybridization (FISH) assay was performed using the Vysis ALK Break Apart FISH Probe Kit (Abbott Laboratories) at a commercial CLIA-certified laboratory. A board-certified radiologist (E.H.M) and pathologists (J.P.G., Y.Z.) selected the images. Plasma cell-free DNA (cfDNA) and tumor specimens were subjected to the hybridization capture–based NGS (FoundationACT for blood and FoundationOne for tissue) assays performed at a CLIA-certified, College of American Pathologists (CAP)–accredited, New York State–approved laboratory (Foundation Medicine, Inc).16,17
In January 2013, a 60-year-old Hispanic female former light smoker (4 pack-year history who quit 33 years prior to diagnosis) was diagnosed with LUAD with a lingular primary, at least 9 subcentimeter metastases to the brain, and with additional lesions in the liver, spleen, and bones. The patient underwent bronchoscopy, which revealed diffuse submucosal hemorrhage in the left mainstem bronchus and a tumor obstructing approximately 30% to 50% of the left upper lobe which corresponded to the chest CT findings (Figure 1A). The diagnostic tumor specimen tested negative for EGFR L858R, exon 19 deletion, and T790M mutations by RT-PCR, but positive for ALK gene rearrangement by FISH, without any information on the fusion partner. Figure 2 and supplemental eTable 1 (available with this article at JNCCN.org) summarize the major diagnostic and treatment course of this patient over almost 4 years (in order): 30 Gy of palliative whole-brain radiation therapy (WBRT) in 10 daily fractions; crizotinib for 13 months; 30 Gy of palliative external-beam RT (EBRT) to T11–L1 in 10 daily fractions; ceritinib for 10 months; alectinib for 7 months; 30 Gy of palliative EBRT to T4–T9 in 10 fractions. The patient also received zoledronic acid first and then denosumab for approximately 2 years to prevent fracture, spinal cord compression, or the need for RT.
In July 2016, the patient presented with chest and back pain and dyspnea. Chest CT showed disease progression with confluent soft tissue attenuation and adenopathy within the subcarinal space that invaded and occluded the left mainstem bronchus and extended into the left upper and lower lobes with postobstructive consolidation (Figure 1B), small left pleural effusion, right-sided mediastinal adenopathy, and new pleural nodularity along the medial aspect of the right hemithorax. A bronchoscopy on August 2, 2016, revealed that the left mainstem bronchus was completely obliterated by friable tissue. Cryoablation was performed with restoration of approximately 50% patency. Pathology showed invasive well-differentiated LUSC (Figure 1D) that was TTF-1–negative (Figure 1F) and P40–positive (data not shown) compared with the original LUAD (Figure 1C) that was TTF-1–positive (Figure 1E). Tumor genomic profiling assay of this tumor and plasma cfDNA revealed a rare driver oncogene, EML4-ALK V5 (supplemental eTable 2). Other notable genomic alterations in the tumor specimen included PTEN loss of exon 1, CDKN2A and CDKN2B loss, and FAS loss. The tumor had high tumor mutational burden (TMB) of 15.17 mutations per megabase (Muts/Mb), and positive PD-L1 expression with a tumor proportion score of 3% by PD-L1 IHC 22C3 pharmDx assay (Agilent).
Due to her poor performance status, the patient was deemed not a candidate for chemotherapy. Instead, she received 30 Gy of EBRT to the left hilum in 10 daily fractions, and showed symptomatic improvement. Shortly thereafter, brain MRI revealed diffuse progression of intracranial metastases. After extensive discussion, an additional course of WBRT to 25 Gy in 10 fractions was performed. She received pembrolizumab for 2 cycles without any significant clinical improvement, and died after enrolling in home hospice care several weeks later.
NGS for Detecting Rare Variants of EML4-ALK Fusion Oncogene in Tissue and Blood
Histology assessment of lung cancer at diagnosis is important for selecting chemotherapy and molecularly targeted therapy.18 Currently, testing for ALK rearrangements is recommended for patients diagnosed with adenocarcinoma and mixed lung cancers with an adenocarcinoma component, regardless of clinical characteristics.19 At initial diagnosis in early 2013, our patient was found to have ALK-rearranged LUAD by the ALK break-apart FISH assay. However, FISH is less sensitive or unable to detect the fusion variants and partners compared with NGS.20
Despite exceptional tumor response rates and improved survivals, almost all patients with ALK-rearranged NSCLC eventually develop acquired resistance to the ALK TKIs.21 Both ALK-dependent and -independent mechanisms can contribute to the development of resistance mechanisms to ALK inhibitors, which include the presence of a second-site mutation in the tyrosine kinase domain of ALK gene in 20% to 30% of cases, ALK amplification, MET proto-oncogene amplification, activation of bypass pathways, and, rarely, histologic transformation.22 In the present case, we found histologic transformation from well-differentiated LUAD to well-differentiated keratinizing LUSC in the same location of the left main bronchus site in August 2016. We could not determine the ALK fusion partner and variant of the original tumor, because there was insufficient tumor specimen remaining from the initial diagnosis. Detection of the oncogenic EML4-ALK V5 without any known molecular resistance mutations in both the tumor and blood after disease had progressed on 3 ALK inhibitors suggests histologic transformation might be a resistance mechanism to the ALK inhibitors.
Potential Mechanisms and Clinical Implication of Histologic Transformation
Histologic transformation from LUAD to SCLC has been reported in EGFR-mutant and ALK-rearranged LUAD.23–25 Recent reports suggest the transformation occurs from early clonal evolution and coexpression of molecular markers (such as loss of RB and P53) found in the transformed histology (ie, SCLC).26,27 These transformed tumors behave biologically like SCLC and have a treatment response and survival comparable to those of typical SCLC.28
We do not know the timing and the mechanism controlling the histologic transformation at the same tumor site after ALK inhibitors and RT. Similar to our reported results, FoundationOne identified several genomic alterations, including PTEN loss, CDKN2A and CDKN2B loss, FAS loss, and FBXW7 E113D – subclonal (supplemental eTable 2), that are frequently reported in LUSC but not LUAD. In addition, the tumor had a high TMB of 15.17 Muts/Mb. LUSC shows a higher TMB than LUAD (9.6 vs 6.3 Muts/Mb, respectively) in a large human genomic database.29 Unfortunately, our patient was paralyzed from spine metastasis and had significant physical deconditioning, rendering her unsuitable for chemotherapy. Although no adenocarcinoma or small cell component was identified in the resistant tumor with squamous cell transition, intertumor heterogeneity or sampling artifacts cannot be completely ruled out. Tumor genomic profiling identified the rare EML4-ALK V5 as the only genomic alteration in plasma cfDNA at the time of tumor sampling of the squamous cell transition and 4 months later, and did not reveal any other resistant mutations to ALK inhibitors or other alterations (supplemental eTable 2), suggesting that the ALK-rearranged gene was the dominant oncogenic driver. In support of this, we recently observed that mice bearing xenografts established from the tumor biopsy performed at the time of the squamous cell transformation have significant tumor shrinkage in response to a newer ALK inhibitor brigatinib (data not shown).
Multidisciplinary Management for Patients With NSCLC and ALK Fusion Oncogenes
Patients with ALK-rearranged NSCLC have been found to have a higher incidence of brain metastasis either at initial diagnosis or after control of systemic disease with crizotinib.21,30 In approximately half of the patients treated with crizotinib, brain metastases develop or progress. Our patient was diagnosed with LUAD with a lingular primary with high TMB in both central nervous system and extracranial sites. After experiencing disease progression on crizotinib, our patient was enrolled in clinical trials which enabled her to receive the second-generation ALK inhibitors ceritinib and alectinib before FDA approval. RT has been increasingly integrated into the clinical management of patients with metastatic NSCLC, both for symptom relief and for local disease control of oligometastatic progression while on otherwise successful systemic therapy, particularly in patients with oncogene-driven NSCLC on molecularly targeted therapy.31,32 Our patient received 5 courses of palliative RT during her disease course. Use of symptom-directed RT in this patient in tandem with ALK inhibitors offered disease and symptom control that may have improved her survival. Although ICIs have revolutionized the treatment of patients with advanced NSCLC, and consistent with previous reports,33,34 we did not observe any clinical response to pembrolizumab despite squamous cell transformation, positive PD-L1 immunohistochemical expression, and high TMB. Further study is needed to define the role of ICIs in ALK-rearranged NSCLC.
To our knowledge, this is the first report of histologic transformation from LUAD to LUSC at the same tumor location while maintaining the ALK-rearranged driver oncogene. Tumor genomic profiling using NGS can identify the rare EML4-ALK V5 fusion gene. Liquid biopsy and plasma cfDNA offer a quick and reliable alternative to invasive tissue biopsy to detect the rare driver oncogene and explore the resistance mechanisms. There are unmet needs to delineate the molecular mechanisms underlying historical transformation from LUAD to LUSC, to develop novel strategies to overcome the resistance to ALK TKIs with activity for both extracranial and intracranial metastases, and to develop effective cancer immunotherapy strategies for ALK-rearranged lung cancer.
Disclosures: Dr. Gregg has disclosed that he is a consultant for Foundation Medicine; receives honoraria from and serves on a scientific advisory board for AstraZeneca; and is a consultant for Bristol-Myers Squibb. Dr. Yoneda has disclosed that he is a scientific advisor for AstraZeneca and Guardant Health. Dr. Daly has disclosed that she receives research funding from EMD Serono. Dr. Li has disclosed that she is a scientific advisor for Foundation Medicine, Takeda, and PUMA, and receives grant/research support from Foundation Medicine, Pfizer, AstraZeneca, Hengrui, and Eureka. The remaining authors have disclosed that they have not received any financial consideration from any person or organization to support the preparation, analysis, results, or discussion of this article.
Hui MUppin SGStalin BJ. Histological transformation of adenocarcinoma to small cell carcinoma lung as a rare mechanism of resistance to epidermal growth factor receptor-tyrosine kinase inhibitors: report of a case with review of literature. Lung India 2018;35:160–163.
Lindeman NICagle PTAisner DL. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Thorac Oncol 2018;13:323–358.
Weickhardt AJScheier BBurke JM. Continuation of EGFR/ALK inhibition after local therapy of oligoprogressive disease in EGFR mutant (Mt) and ALK+ non-small cell lung cancer (NSCLC) [abstract]. J Clin Oncol 2012;30(Suppl):Abstract 7526.
Gan GNWeickhardt AJScheier B. Stereotactic radiation therapy can safely and durably control sites of extra-central nervous system oligoprogressive disease in anaplastic lymphoma kinase-positive lung cancer patients receiving crizotinib. Int J Radiat Oncol Biol Phys 2014;88:892–898.