Case Report

Dramatic Response to Crizotinib in ROS1 Fluorescent In Situ Hybridization- and Immunohistochemistry-Positive Lung Adenocarcinoma: A Case Series Rita Chiari,1 Fiamma Buttitta,2 Daniela Iacono,1 Chiara Bennati,1 Giulio Metro,1 Alessia Di Lorito,2 Manuela Iezzi,2 Marcello Tiseo,3 Francesca Mazzoni,4 Federico Cappuzzo,5 Antonio Marchetti,2 Lucio Crinò1 Clinical Practice Points
Lung adenocarcinoma harboring the c-ros oncogene

1 (ROS1) translocations represents a newly discovered molecular subset of nonesmall cell lung cancer (NSCLC). Just as with anaplastic lymphoma receptor tyrosine kinase (ALK)-rearranged NSCLC, early phase clinical trials have shown a high response rate to crizotinib in these patients.
Fluorescent in situ hybridization (FISH) is considered the reference standard for detecting ROS1

translocations and ALK rearrangements. Several antiROS1 antibodies are in use, and the first results have suggested that immunohistochemistry (IHC) could be a suitable tool for identifying ROS1 rearrangement.
The results from this small series suggest that IHC is a valuable tool for identifying ROS1 rearrangement and have confirmed that this subset of patients can benefit from crizotinib-based treatment.

Clinical Lung Cancer, Vol. 15, No. 6, 470-4 ª 2014 Elsevier Inc. All rights reserved. Keywords: Crizotinib, FISH, IHC, NSCLC, ROS1

Introduction C-ros oncogene 1 (ROS1) is a newly identified receptor tyrosine kinase of the insulin receptor family. Rikova et al1 identified 2 ROS1 fusion variants as potential driver mutations in a nonesmall cell lung cancer (NSCLC) cell line (HCC78; SLC34A2-ROS1) and an NSCLC patient sample (CD74-ROS1). More recently, 5 additional fusion partners (TPM3, SDC4, EZR, LRIG3, FIG) to ROS1 have been identified, representing 12 ROS1 fusion variants in NSCLC.2 R.C. and F.B. contributed equally to this work. 1 Department of Medical Oncology, Santa Maria della Misericordia Hospital of Perugia, Perugia, Italy 2 Center of Predictive Molecular Medicine, CeSI, University-Foundation, Chieti, Italy 3 Department of Medical Oncology, University Hospital of Parma, Parma, Italy 4 Department of Medical Oncology, Careggi Hospital of Firenze, Firenze, Italy 5 Department of Medical Oncology, Istituto Toscano Tumori of Livorno, Livorno, Italy

Submitted: Apr 14, 2014; Revised: Jun 9, 2014; Accepted: Jun 17, 2014; Epub: Jun 24, 2014 Address for correspondence: Rita Chiari, PhD, Department of Medical Oncology, Ospedale Santa Maria della Misericordia, Via G. Dottori 1, Perugia 06132 Italy E-mail contact: [email protected], [email protected]

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Bergethon et al3 identified ROS1 rearrangements in 18 (1.7%) of 1073 patients with NSCLC using fluorescent in situ hybridization (FISH) and suggested that ROS1 rearrangements define a unique molecular subset of lung cancer with distinct clinical characteristics. These characteristics included younger patient age (median age, approximately 50 years) and never having smoked, clinical features that are also associated with both epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements.3 Because a high homology exists between the kinase domains of ROS1 and ALK, ALK inhibitors were tested and shown to be inhibitory in ROS1-positive cell lines and tumors.4 ROS1-rearranged tumors based on a positive break apart FISH assay were added as an additional eligibility criterion for molecularly enriched cohorts of PROFILE (Phase 1 open-label, single-arm, dose-escalation study with an expansion cohort evaluating the safety, pharmacokinetic and pharmacodynamic of crizotinib [PF-02341066], administered orally to patients with advanced cancer.) 1001 since May 2010. Ou et al5 reported the results of this phase I trial in 36 patients, showing a overall response rate (ORR) of 61%, suggesting the high clinical efficacy of crizotinib in this subset.

1525-7304/$ - see frontmatter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cllc.2014.06.004

Table 1 Methods for Detection of ROS1 Rearrangements FISH Pros

Cons

IHC

Used in clinical trials User friendly Validated kit with Simultaneous standard procedures evaluation of available morphology Can detect unusual Cost effective variants Technically challenging Lack of kit with Costly standard procedures Not used in clinical trials

RT-PCR High sensitivity High specificity

High quality samples needed Based on complex multiplex PCR

Abbreviations: FISH ¼ fluorescent in situ hybridization; IHC ¼ immunohistochemistry; RT-PCR ¼ reverse transcriptase polymerase chain reaction.

Currently, crizotinib has been approved by the Food and Drug Administration and European Medicines Agency for the treatment of ALK-positive NSCLCs, defined by FISH, according to the important clinical effect shown in a phase III trial (PROFILE 1007). This second-line trial comparing crizotinib with either docetaxel or pemetrexed included 347 patients with ALK-rearranged stage IIIB-IV NSCLC who had already undergone chemotherapy. The trial results showed that crizotinib prolonged progression-free survival to a median of 7.7 months compared with 3.0 months for those patients who had received chemotherapy (hazard ratio [HR], 0.49; 95% CI, 0.37-0.64; P < .0001). The ORR was also significantly greater in the patients treated with crizotinib (65% vs. 20%; P < .0001). An interim analysis of overall survival showed no significant improvement with crizotinib compared with chemotherapy (HR, 1.02; P ¼ .54).6 To our knowledge, few clinical findings from patients with crizotinib-treated ROS1-rearranged NSCLC with adequate followup data have been reported to date. Different methodologic approaches, including FISH, reverse transcriptase polymerase chain reaction (RT-PCR), and IHC, are currently available for the analysis of ROS1 rearrangement. FISH has been considered the reference standard test for ROS1 rearrangement; however, it is expensive and requires experience. RT-PCR has mainly been used for research purposes, because it is reliable only when the mRNA quality is adequate. IHC is easy to perform and allows the simultaneous evaluation of morphology and protein expression. The pros and cons of the different

diagnostic tests are summarized in Table 1. In addition to ALK rearrangements, several anti-ROS1 antibodies are in use. The first results have suggested that IHC could be a valuable tool for identifying ROS1 mutation, because it is easy to interpret and costeffective to apply as a primary screening test.2 We report a case series of stage IV ROS1-positive lung adenocarcinomas showing concordance between FISH and IHC and a dramatic response to crizotinib.

Case Report We identified 8 patients with stage IV lung adenocarcinoma positive for ROS1 rearrangement using both FISH and IHC. These 8 patients had been treated at Santa Maria della Misericordia Hospital of Perugia (n ¼ 5), the University Hospital of Parma (n ¼ 1), Istituto Toscano Tumori of Livorno (n ¼ 1), and the Careggi Hospital of Firenze (n ¼ 1). The genetic tests for ROS1 rearrangement by FISH were performed at the Center of Predictive Molecular Medicine, CeSI, University-Foundation, Chieti (n ¼ 6) and at the Division of Medical Oncology of the University of Colorado (n ¼ 2). IHC testing with anti-ROS1 (clone D4D6, Cell Signaling Technology, Danvers, MA) monoclonal antibody was performed at the Center of Predictive Molecular Medicine, CeSI, University-Foundation, Chieti. The median patient age at diagnosis was 44.7 years (range, 37-71 years). All 8 patients were white women; 6 were never smokers and 2 were light former smokers (4 and 5 pack-years, respectively) (Table 2). Genetic testing of tumor samples showed no EGFR mutation or ALK rearrangement. All patients received platinum-based first-line chemotherapy and crizotinib as the second or subsequent line of treatment. We observed 1 complete response and 7 partial responses to crizotinib (ORR, 100%). Two patients had died 3 and 10 months after starting crizotinib treatment, and 6 patients were alive. The median duration of treatment with crizotinib was 9.0 months (range, 5.0-18.0 months). As an example, we present the radiologic, clinical, and ROS-1 IHC and FISH data for 2 representative patients.

Patient 1 A 51-year-old woman who had never smoked had been diagnosed with stage IV lung adenocarcinoma in January 2012 (cT1aN3M1b TNM, 7th edition, bone and nodal metastases). She

Table 2 Demographic and Clinical Characteristics of Patients Pt. No. 1 2 3 4 5 6 7 8

Agea (years)

Sex

Histologic Type

Tobacco Use

Ethnicity

First-Line Chemotherapy

47 58 71 45 51 42 44 37

Female Female Female Female Female Female Female Female

Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma

4 pack-y NS NS NS NS NS NS 5 pack-y

White White White White White White White White

Cisplatin þ pemetrexed Carboplatin þ paclitaxel Cisplatin þ pemetrexed Cisplatin þ pemetrexed Cisplatin þ pemetrexed Cisplatin þ pemetrexed Cisplatin þ pemetrexed Carboplatin þ pemetrexed

Abbreviations: NS ¼ not significant; Pt. No. ¼ patient number. a Median age was 44.7 years.

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Response to Crizotinib in ROS1 FISH- and IHC-positive Lung Cancer received 6 cycles of platinum-based chemotherapy, obtaining a partial response. The radiologic findings were stable until September 2012 when a positron emission tomography-computed tomography (PET-CT) scan showed disease progression at known sites and new liver and adrenal gland lesions. The presence of ROS-1 rearrangement was tested using FISH (Figure 1A,B) and IHC, with a positive immunoreaction in > 80% of the tumor cells (see Figure 3A). She began receiving crizotinib at the standard dose of 250 mg twice daily and experienced clinical improvement within 1 week. One month later, a PET-CT scan showed a nearly complete response that was confirmed after 2 months (Figure 1C,D). The patient died after 5 months of treatment with crizotinib of brain and liver progression (Figure 2F).

Patient 2 A 46-year-old woman who had smoked (4 pack-years) had undergone right superior lobectomy and systematic lymphadenectomy

in June 2011 for stage IA, grade 3 lung adenocarcinoma (pT1aN0M0, TMN 7th edition). The radiologic findings were negative until June 2012, when a CT scan showed mediastinal nodal enlargement (Figure 2A,B). ROS-1 rearrangement was determined using FISH (Figure 2A,B) and IHC (Figure 3B). After 3 cycles of platinum plus pemetrexed chemotherapy, she experienced nodal disease progression and the appearance of pericardial effusion. On August 2012, the patient began crizotinib therapy. She had obtained a nearly complete response 1 month later that was confirmed after 2 months (Figure 2C,D) and was still ongoing after 18 months of treatment with no evidence of disease progression (Figure 2F).

Discussion We observed a dramatic and long lasting response to crizotinib treatment in all patients with NSCLC harboring a ROS1 rearrangement. Our data have confirmed the real clinical impact of

Figure 1 Patient 1. Diagnosis of Anaplastic Lymphoma Receptor Tyrosine Kinase (ALK)-Negative and c-Ros Oncogene 1 (ROS1)-Positive Adenocarcinoma Using Abbot Molecular ALK/ROS1 Break Apart Probe, and Response to ROS1 Inhibition. (A) Green Probes are Hybridized to 50 Region of ALK and Orange Probes to 30 Region. White Arrows Denote Fused Signals (Nonrearranged ALK Allele), and Green and Orange Arrows Denote 50 and 30 Regions of ALK, Respectively, That have been Separated by Rearrangement. (B) Gold Probes are Hybridized to 50 Region of ROS1 and Aqua Probes to 30 Region. White Arrows Denote Fused Signals (Nonrearranged ROS1 Allele), and Aqua and Gold Arrows Denote 30 and 50 Regions of ROS1, Respectively, That have been Separated by Rearrangement. Results of Positron Emission Tomography Computed Tomography Scans (C) At Baseline, (D) After 1 Month, and (E) After 3 Cycles of Therapy. (F) Liver Progression Seen After 5 Months of Treatment With Crizotinib

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Rita Chiari et al Figure 2 Patient 2. Diagnosis of Anaplastic Lymphoma Receptor Tyrosine Kinase (ALK)-Negative and c-Ros Oncogene 1 (ROS1)-Positive Adenocarcinoma Using Abbot Molecular ALK/ROS1 Break Apart Probe, and Response to ROS1 Inhibition. (A) Green Probes are Hybridized to 50 Region of ALK and Orange Probes to 30 Region. White Arrows Denote Fused Signals (Nonrearranged ALK Allele), and Orange Arrows Denote 30 Regions of ALK That have been Separated by Rearrangement (No Single Green Signals Were Seen). (B) Gold Probes are Hybridized to 50 Region of ROS1 and Aqua Probes to 30 Region. White Arrows Denote Fused Signals (Nonrearranged ROS1 Allele), and Aqua and Gold Arrows Denote 30 and 50 Regions of ROS1, Respectively, That have been Separated by Rearrangement. Results of Positron Emission Tomography Computed Tomography Scans (C) At Baseline, (D) After 1 Month, (E) After 3 Months of Therapy, and (F) After 15 Months of Treatment

Figure 3 (A,B) Immunohistochemical Staining of Formalin-Fixed, Paraffin-Embedded Tumor Sections From 2 Patients Using Anti-c-Ros Oncogene 1 (ROS1) (Clone D4D6) Monoclonal Antibody. Intense, Positive ROS1 Cytoplasmic and Submembrane Immunoreaction in > 80% of Tumor Cells Evident. Normal Stromal Cells Were Negative. Original Magnification 3 200

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Response to Crizotinib in ROS1 FISH- and IHC-positive Lung Cancer Figure 4 Algorithm for c-Ros Oncogene 1 (ROS1) Testing

protein (prescreening), with FISH being required only if the IHC findings were equivocal (Figure 4). Considering the pro and cons of FISH analysis reported in Table 1, this algorithm could help save time, money, and tissue for additional diagnostic tests. Because of the poorness of data in ROS1-positive patients, in Italy, crizotinib is available for this subset of patients only as offlabel use, depending on the insurer regulatory authorities and local ethic committees. In the present case series, we wished to focus the attention on such patients who could benefit from treatment with crizotinib and other ROS1 inhibitors. Although the detection of ROS1 mutation is a rare event, occurring in less than about 2% of cases, considering the annual incidence of NSCLC, targeting ROS1 and approving a clinically effective treatment could be beneficial for thousands of patients each year.

Disclosure The authors have stated that they have no conflicts of interest.

References Abbreviations: FISH ¼ Fluorescent In Situ Hybridization; IHC ¼ Immunohistochemistry.

crizotinib in this subset of patients. In this Italian series, all the patients with ROS1 rearrangement were women. The number of cases investigated using IHC and FISH in our study was very limited. However, our experience suggests that IHC could be a suitable and useful tool for detecting ROS1 mutations. If our data are confirmed in future studies, an algorithm for ROS1 testing could be hypothesized, based on the detection of the ROS1

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