Most oncologists are familiar with the clinical significance

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1 Brief Report RET-Rearranged Lung Adenocarcinomas with Lymphangitic Spread, Psammoma Bodies, and Clinical Responses to Cabozantinib Sanjay Mukhopadhyay, MD,* Nathan A. Pennell, MD, Siraj M. Ali, MD, PhD, Jeffrey S. Ross, MD, Patrick C. Ma, MD, MSc, and Vamsidhar Velcheti, MD, Abstract: Oncogenic rearrangements of the RET gene have recently been described in 1% to 2% of lung adenocarcinomas. We report five cases of RET-rearranged lung adenocarcinoma with an unusual constellation of clinical and histologic features that has not previously been described in tumors with this genomic alteration. The age at diagnosis of the five patients (4F, 1M) ranged from 44 to 77 years. All were never-smokers. Radiologically, four tumors showed lymphangitic spread within the lungs at presentation; three of these had multiple bilateral lung nodules. Histology showed psammoma bodies within the tumor in four of five cases. Molecular testing for activating EGFR mutations by standard genotyping and ALK expression by immunohistochemistry was negative in all cases. Additional molecular analysis was prompted by the clinical profile in that all five patients were never-smokers with metastatic, relapsed, and/or refractory disease; and also by unusual histologic findings in two cases. Comprehensive genomic profiling performed by means of a clinical grade cancer gene panel next-generation sequencing assay demonstrated a KIF5B-RET fusion in three; and fluorescence in-situ hybridization documented a RET rearrangement in two. Three of the patients were treated with the RET inhibitor cabozantinib. By Response Evaluation Criteria In Tumors (RECIST) criteria, two had a confirmed partial response (at 6 weeks and 4 weeks) and one had stable disease. Our findings suggest that the combination of lymphangitic spread and psammoma bodies may be characteristic of a subset of advanced stage RET-rearranged lung adenocarcinomas. These findings should prompt additional molecular testing for RET translocations, particularly in never-smokers with EGFR- and ALK-negative lung adenocarcinoma. Key Words: RET, KIF5B-RET, Lung, Adenocarcinoma, Nextgeneration sequencing. (J Thorac Oncol. 2014;9: ) *Departments of Pathology, Tumor Oncology, and Translational Hematology and Oncology Research, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH; Foundation Medicine, Cambridge, MA; and Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, NY. Disclosure: The authors declare no conflict of interest. Address for correspondence: Vamsidhar Velcheti, MD, Department of Tumor Oncology, Cleveland Clinic, 9500 Euclid Avenue/L25, Cleveland, OH velchev@ccf.org DOI: /JTO ISSN: /14/ Most oncologists are familiar with the clinical significance of EGFR mutations and ALK rearrangements in lung adenocarcinomas. However, the clinicopathologic profile and therapeutic significance of other, less common novel rearrangements, such as those involving the ROS1, RET, and NTRK1 genes, are not well understood. In 2012, several groups reported fusion of the KIF5B (kinesin family member 5B) gene to the RET (rearranged during transfection) gene in adenocarcinomas of the lung. 1 4 These and subsequent reports have demonstrated that the KIF5B-RET fusion as well as other RET fusions such as the TRIM33- RET, NCOA4-RET, and CCDC6-RET fusions are present in 1% to 2% of pulmonary adenocarcinomas Although in vitro preclinical responses to the RET-targeting inhibitors vandetanib, sunitinib, and sorafenib have been reported in RET-driven lung cancer models, 2 4 there are only two reports of clinical responses to RET inhibitors in RET fusion-positive lung adenocarcinomas. 11,12 We describe the clinical, radiologic, and histologic findings in five patients with RET-rearranged lung adenocarcinoma presenting with metastatic disease to draw attention to an unusual constellation of clinical and histologic findings in these tumors, and to add further evidence of clinical responses of these tumors to RET-targeting inhibitor therapy. PATIENTS AND METHODS We identified five patients with RET-translocated lung adenocarcinoma who were treated at the Cleveland Clinic. Routine molecular testing (mutant-allele-specific polymerase chain reaction for EGFR mutations and immunohistochemistry using the ultrasensitive D5F3 antibody for ALK expression) was negative in all cases. A clinical grade CLIA-certified targeted next-generation sequencing ()- based assay was performed using the FoundationOne platform (Foundation Medicine, Cambridge, MA) which involves comprehensive genomic profiling that identifies all four classes of genomic alterations within 236 genes, and rearrangements of selected introns in 19 genes frequently rearranged in cancer, including RET. 13 Fluorescence in-situ hybridization (FISH) was performed using a break-apart probe for the RET gene Journal of Thoracic Oncology Volume 9, Number 11, November 2014

2 Journal of Thoracic Oncology Volume 9, Number 11, November 2014 RET-Rearranged Lung Adenocarcinomas RESULTS Clinical Findings The clinical findings are summarized in Table 1. Case 1. A 77-year-old woman presented with cough and weight loss for over a year and was found to have innumerable bilateral lung nodules in a lymphangitic distribution (Fig. 1A). A wedge biopsy of lung showed adenocarcinoma with extensive lymphangitic spread, psammoma bodies, and a mucinous cribriform pattern. Since these histologic findings have been reported in ROS1-translocated adenocarcinomas 14 additional molecular testing () was performed, leading to the unexpected finding of a KIF5B-RET fusion. The patient had a poor performance status (Eastern Cooperative Oncology Group 3) and multiple brain metastases at presentation. She received whole brain irradiation and subsequently was started on first line treatment with cabozantinib at a daily dose of 140 mg. Her symptoms improved with treatment and she had a radiologically confirmed partial response by RECIST criteria (Fig. 1B pretreatment, Fig. 1C posttreatment). However, after 4 weeks of therapy she developed significant grade 3 stomatitis requiring dose reduction to 100 mg. However, her stomatitis continued to worsen despite dose reduction and aggressive supportive oral care. She was readmitted for severe oral pain and decreased oral intake and cabozantinib was discontinued. Given her poor performance status she was transitioned to hospice care and subsequently expired. Case 2. A 67-year-old man presented with weight loss, cough, and shortness of breath. Chest computed tomography (CT) showed a right middle lobe lung mass and also lymphangitic spread of tumor in the right upper lobe (Fig. 1D). Metastatic tumor lesions were found in the scapula, vertebral body, iliac crest, and liver. He progressed after receiving first line systemic treatment with carboplatin paclitaxel. Review of the lung biopsy at our institution showed signet-ring cells and psammoma bodies, prompting the decision to perform additional molecular testing. A RET translocation was demonstrated by FISH and corroborated by a CCDC6-RET fusion on. Cabozantinib therapy was initiated at 140 mg per day. The patient had significant improvement in dyspnea and exercise tolerance accompanied by a radiographic partial response by RECIST criteria after 4 weeks of therapy (Fig. 1D pretreatment, Fig. 1E posttreatment). He continued to have partial response on most recent scans at 8 weeks and the response is ongoing. Case 3. A 57-year-old woman presented with weight loss and flank pain. She was found to have innumerable bilateral tiny pulmonary nodules in a lymphangitic distribution and metastases to the mediastinal lymph nodes and liver. After progressing on initial therapy with carboplatin pemetrexed and subsequently second line docetaxel, she was tested for additional molecular abnormalities and a KIF5B-RET fusion was found by. Cabozantinib, 140 mg daily, was started. A positron emission tomography (PET) scan 2 months posttherapy showed slight shrinkage of tumor. Although the response was classified as stable disease by RECIST measures, many of the lesions (e.g., the liver metastases) became PET-negative posttreatment and she felt much better symptomatically. The dose was reduced to 60 mg 3 months later due to severe stomatitis. Overall, she was on cabozantinib for a total of 4 months. Therapy was stopped after the 4-month restaging scan showed progression, and she died one month after stopping the drug (3 years after the initial diagnosis). Case 4. A 48-year-old woman with a 2-year history of intermittent dry cough presented with a skin lump under her right clavicle suspicious for a subcutaneous cyst. Excisional biopsy of the lesion unexpectedly revealed TTF-1-positive metastatic adenocarcinoma, consistent with a lung primary. Chest CT showed a left lower lobe infrahilar irregular-shaped infiltrative 3.5-cm lung mass with evidence of possible lymphangitic spread. She received three cycles of induction chemotherapy (cisplatin pemetrexed) with partial tumor response, followed by tumor resection with bilobectomy (with mediastinal positive margins), and then three cycles of adjuvant cisplatin pemetrexed and subsequent chest radiation therapy. Her tumor was tested by FISH for RET rearrangement and was positive. However, she was later found to have central nervous system (CNS) failure with multiple new brain metastases treated with whole brain radiation and gamma knife radiosurgery. Since she did not have clinically significant extra-cns disease at the time, she was not started on RET-targeted therapy. Case 5. A 47-year-old woman noticed a newly enlarged supraclavicular lymph node; she was otherwise asymptomatic. Imaging showed a lung mass with mediastinal, supraclavicular, and abdominal lymphadenopathy. No signs of lymphangitic spread were noted. A biopsy of an abdominal lymph node showed metastatic adenocarcinoma. She initially received erlotinib which was discontinued after testing for EGFR was reported as negative. This was followed by six cycles of cisplatin paclitaxel bevacizumab followed by radiation to the chest, with an excellent response. Subsequently, a small PET-positive subdiaphragmatic periaortic lymph node was detected and biopsied, revealing adenocarcinoma. Pemetrexed was administered for 23 cycles and then stopped due to cumulative toxicity. After a treatment break for one year, she developed recurrence in the mediastinum, prompting comprehensive molecular testing by which revealed a KIF5B- RET fusion. Although pemetrexed was restarted, the plan is to use RET inhibitor therapy in the event of progression. Histologic Findings The histologic findings are summarized in Table 2 and illustrated in Figure 2. All tumors were adenocarcinomas, with a solid pattern being predominant in most. Four tumors showed prominent high-grade nuclei. In four of five cases, the tumor was associated with concentrically lamellated psammoma bodies, which were present only focally in each case. The peak count of psammoma bodies ranged from 3 to 12 per 40 high power field. In all cases, psammoma bodies were associated with solid-pattern tumor rather than with papillary or micropapillary foci. A lymphangitic (intralymphatic) distribution of tumor along the pleura, bronchovascular 1715

3 Mukhopadhyay et al. Journal of Thoracic Oncology Volume 9, Number 11, November 2014 TABLE 1. Clinical Features of Five Patients with RET-Rearranged Lung Adenocarcinoma Case Age/Sex Ethnicity Smoking Status Radiology at ation Stage at ation EGFR ALK RET Treatment Outcome 1 77/F Caucasian 2 67/M Caucasian 3 57/F Asian 4 48/F Asian 5 47/F Caucasian Bilateral nodules, lymphangitic distribution Right middle lobe mass, with lymphangitic reticulonodular opacities in right upper lobe Bilateral nodules, lymphangitic distribution 5-cm left infrahilar mass, increased peribronchial markings c/w lymphangitic spread, bilateral tiny lung nodules Lung mass, mediastinal, supraclavicular and abdominal lymphadenopathy IV (brain metastases) Negative Negative KIF5B-RET by IV (bone and liver metastases) Negative Negative Positive for RET rearrangement by FISH (10q11.2 breakapart probe); CCDC6-RET by IV (liver metastases) Negative Negative KIF5B-RET by IV (oligometastasis to skin) IV (metastasis to supraclavicular lymph node) Negative Negative Positive for RET rearrangement by FISH (10q11.2 breakapart probe) Negative Negative KIF5B-RET by Cabozantinib with partial response after 4 weeks of therapy; severe dose-limiting stomatitis Carboplatin Taxol, progressed; started on cabozantinib with partial response after 4 weeks Cabozantinib with partial response; severe dose-limiting stomatitis Induction chemotherapy (three cycles cisplatin pemetrexed) with partial response, followed by surgery, and then sequential adjuvant chemotherapy and chest radiation. CNS metastases at recurrence 14 months after surgery were treated with whole brain radiation and gamma knife radiosurgery Erlotinib, cisplatin paclitaxel bevacizumab, followed by chest radiation. At recurrence on pemetrexed, stopped for one year and now restarted. Plan to use RET inhibitor if she progresses Died of disease 5 months from diagnosis Alive 5 months from diagnosis; Ongoing response to cabozantinib. Died of disease 3 years after first biopsy Alive with disease to date 20 months after lobectomy; recurrent CNS metastases Alive with disease 3+ years after biopsy CNS, central nervous system; FISH, fluorescence in-situ hybridization;, next-generation sequencing. 1716

4 Journal of Thoracic Oncology Volume 9, Number 11, November 2014 RET-Rearranged Lung Adenocarcinomas FIGURE 1. Radiologic features and responses to RET inhibitor (cabozantinib) therapy in RET-rearranged lung adenocarcinomas. A C, case 1. A, Bilateral reticulonodular opacities with solid and cavitary nodules in a lymphangitic distribution. PET-CT at presentation (B) and 1 month after cabozantinib therapy (C), demonstrating decrease in size and FDG avidity of nodules. D E, case 2. CT scan at presentation (D) and 1 month after cabozantinib therapy (E), showing a decrease in size of the confluent right infrahilar mass and adenopathy involving the central right middle lobe, development of focal cavitation, and improvement in patency of the right middle lobe bronchus. TABLE 2. Case Histologic Features of Five RET-Rearranged Lung Adenocarcinomas Specimen Type 1 Wedge biopsy of lung 2 Bronchoscopic lung biopsy Mediastinal lymph node biopsies Lobectomy and partial lingulectomy Supraclavicular lymph node biopsy Nuclear Grade Predominant Psammoma Pattern Bodies Intermediate Acinar bundles, and interlobular septa was confirmed histologically in case 1. Mucin was present in three cases and was prominent in two. One of these showed a mucinous cribriform pattern while the other was composed entirely of signet-ring cells. Large, irregular, and intracytoplasmic vacuoles were Mucin Prominent, mucinous cribriform pattern Prominent, signet ring cells Intranuclear Cytoplasmic Inclusions Immunophenotype TTF-1-pos, PAX-8-neg TTF-1-pos Focal Prominent TTF-1-pos, PAX-8-neg TTF-1-pos, PAX-8-neg TTF-1-pos noted within the tumor cells in two cases. All five tumors were TTF-1-positive, consistent with lung origin; in three of these, PAX-8 was negative, excluding the most common carcinomas traditionally associated with psammoma bodies (thyroid and ovary). 1717

5 Journal of Thoracic Oncology Volume 9, Number 11, November 2014 Mukhopadhyay et al. FIGURE 2. Histologic features of lung adenocarcinomas with RET rearrangements. A, Case 1. Psammoma bodies within solidpattern tumor. B, Case 3. Tumor cells and psammoma bodies within a lymphatic. C, Case 4. -grade nuclei, intranuclear cytoplasmic inclusion (top right), and a psammoma body (top center). D, Case 1. Mucinous cribriform pattern. E, Case 2. Signet-ring cells. Psammoma bodies were also present (not shown). F, Case 4. Large intracytoplasmic vacuoles. DISCUSSION This small series of stage IV RET-translocated adenocarcinomas of the lung highlights an unusual constellation of clinical and histologic findings remarkably similar to those reported in adenocarcinomas bearing ROS1 and ALK translocations, including never-smoker status, female predominance, psammoma bodies, and some forms of mucinous morphology such as the mucinous cribriform pattern and signet-ring cells.11,14,15 The reported prevalence of psammoma bodies in adenocarcinoma of the lung varies by mutation status. Reported rates include 0.5% (ROS1-negative), 0.6% to 3% (ALK-negative), 3.4% (KRAS-positive), 11% (EGFR-positive), 11.8% (triple-negative), 17% to 56.4% (ALK-positive), and 33% to 66% (ROS1-positive) Interestingly, none of the prior studies of RET-translocated lung adenocarcinoma have reported psammoma bodies. It is unclear whether psammoma bodies were absent in their cases or whether their presence was not reported. Similarly, lymphangitic spread has not been previously reported in RET-translocated adenocarcinomas Although one could make the argument that all neversmokers with metastatic disease, adenocarcinoma histology and negative EGFR/ALK status may benefit from additional molecular testing regardless of any associated features, comprehensive genotyping may not be feasible for all patients because of cost or limited tumor cellularity in small biopsies. Therefore, clinical, radiologic, or histologic clues that increase the yield of molecular testing for oncogenic drivers may still be of value to oncologists faced with difficult decisions regarding whom to test and what molecular abnormalities to test for. Our findings suggest that the combination of lymphangitic spread and psammoma bodies in stage IV lung adenocarcinoma should provide an impetus to perform additional molecular testing for RET translocations, particularly in never-smokers with EGFR- and ALK-negative tumors. However, we do not mean to imply that the absence of these findings should preclude comprehensive molecular testing in never-smokers with EGFR-negative, ALK-negative tumors. Our experience with cabozantinib therapy in three patients adds to the limited published clinical data

6 Journal of Thoracic Oncology Volume 9, Number 11, November 2014 RET-Rearranged Lung Adenocarcinomas suggesting that some RET-translocated lung adenocarcinomas respond to RET-inhibitor therapy. The role of RET-targeted therapy in RET-translocated lung adenocarcinomas requires further validation in clinical trial studies with novel RET inhibitors. REFERENCES 1. Ju YS, Lee WC, Shin JY, et al. A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing. Genome Res 2012;22: Kohno T, Ichikawa H, Totoki Y, et al. KIF5B-RET fusions in lung adenocarcinoma. Nat Med 2012;18: Lipson D, Capelletti M, Yelensky R, et al. Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nat Med 2012;18: Takeuchi K, Soda M, Togashi Y, et al. RET, ROS1 and ALK fusions in lung cancer. Nat Med 2012;18: Suehara Y, Arcila M, Wang L, et al. Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mrna-based screen for tyrosine kinase fusions. Clin Cancer Res 2012;18: Wang R, Hu H, Pan Y, et al. RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer. J Clin Oncol 2012;30: Yokota K, Sasaki H, Okuda K, et al. KIF5B/RET fusion gene in surgicallytreated adenocarcinoma of the lung. Oncol Rep 2012;28: Go H, Jung YJ, Kang HW, et al. Diagnostic method for the detection of KIF5B-RET transformation in lung adenocarcinoma. Lung Cancer 2013;82: Sasaki H, Shimizu S, Tani Y, et al. RET expression and detection of KIF5B/RET gene rearrangements in Japanese lung cancer. Cancer Med 2012;1: Tsuta K, Kohno T, Yoshida A, et al. RET-rearranged non-small-cell lung carcinoma: a clinicopathological and molecular analysis. Br J Cancer 2014;110: Drilon A, Wang L, Hasanovic A, et al. Response to Cabozantinib in patients with RET fusion-positive lung adenocarcinomas. Cancer Discov 2013;3: Gautschi O, Zander T, Keller FA, et al. A patient with lung adenocarcinoma and RET fusion treated with vandetanib. J Thorac Oncol 2013;8:e43 e Frampton GM, Fichtenholtz A, Otto GA, et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol 2013;31: Sholl LM, Sun H, Butaney M, et al. ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas. Am J Surg Pathol 2013;37: Yoshida A, Tsuta K, Nakamura H, et al. Comprehensive histologic analysis of ALK-rearranged lung carcinomas. Am J Surg Pathol 2011;35: Kim H, Jang SJ, Chung DH, et al. A comprehensive comparative analysis of the histomorphological features of ALK-rearranged lung adenocarcinoma based on driver oncogene mutations: frequent expression of epithelial-mesenchymal transition markers than other genotype. PLoS One 2013;8:e Nishino M, Klepeis VE, Yeap BY, et al. Histologic and cytomorphologic features of ALK-rearranged lung adenocarcinomas. Mod Pathol 2012;25: Yoshida A, Kohno T, Tsuta K, et al. ROS1-rearranged lung cancer: a clinicopathologic and molecular study of 15 surgical cases. Am J Surg Pathol 2013;37: Erratum Outcome of Multimodality Treatment for 188 Cases of Type B3 Thymoma: Erratum The affiliation for the authors of the article beginning on page 1329 of the October 2013 issue was incorrectly listed as Shanghai Thoracic Hospital. The correct affiliation is Shanghai Chest Hospital. Reference: Gao L, Wang C, Fang W, Zhang J, Lv C, Fu S. Outcome of Multimodality Treatment for 188 Cases of Type B3 Thymoma. J Thorac Oncol. 2013;8: