NEJM-Crizotinib in ROS1-rearranged Non-Small-Cell Lung Cancer

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N Engl J Med. 2014 November 20; 371(21): 1963–1971. doi:10.1056/NEJMoa1406766.
Crizotinib in ROS1-Rearranged Non–Small-Cell Lung Cancer

Alice T. Shaw, M.D., Ph.D., Sai-Hong I. Ou, M.D., Ph.D., Yung-Jue Bang, M.D., Ph.D., D.
Ross Camidge, M.D., Ph.D., Benjamin J. Solomon, M.B., B.S., Ph.D., Ravi Salgia, M.D.,
Ph.D., Gregory J. Riely, M.D., Ph.D., Marileila Varella-Garcia, Ph.D., Geoffrey I. Shapiro,
M.D., Ph.D., Daniel B. Costa, M.D., Ph.D., Robert C. Doebele, M.D., Ph.D., Long Phi Le, M.D.,
Ph.D., Zongli Zheng, Ph.D., Weiwei Tan, Ph.D., Patricia Stephenson, Sc.D., S. Martin
Shreeve, M.D., Ph.D., Lesley M. Tye, Ph.D., James G. Christensen, Ph.D., Keith D. Wilner,
Ph.D., Jeffrey W. Clark, M.D., and A. John Iafrate, M.D., Ph.D.
Massachusetts General Hospital Cancer Center (A.T.S., L.P.L., Z.Z., J.W.C., A.J.I.), Dana–
Farber Cancer Institute (G.I.S.), and Beth Israel Deaconess Medical Center (D.B.C.) — all in
Boston; University of California at Irvine, Irvine (S.-H.I.O.), and Pfizer Oncology, La Jolla (W.T.,
S.M.S., L.M.T., J.G.C., K.D.W.) — both in California; Seoul National University Hospital, Seoul,
South Korea (Y.-J.B.); University of Colorado, Aurora (D.R.C., M.V.-G., R.C.D.); Peter MacCallum
Cancer Centre, Melbourne, VIC, Australia (B.J.S.); University of Chicago, Chicago (R.S.);
Memorial Sloan Kettering Cancer Center, New York (G.J.R.); Karolinska Institutet, Stockholm
(Z.Z.); and Rho, Chapel Hill, NC (P.S.)
Abstract
BACKGROUND—Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene
receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non–small-cell lung
cancers (NSCLCs) that may be susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a
small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and
another proto-oncogene receptor tyrosine kinase, MET.
METHODS—We enrolled 50 patients with advanced NSCLC who tested positive for ROS1
rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with
crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety,
pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of
next-generation sequencing or reverse-transcriptase–polymerase-chain-reaction assays.
RESULTS—The objective response rate was 72% (95% confidence interval [CI], 58 to 84), with
3 complete responses and 33 partial responses. The median duration of response was 17.6 months
(95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4
to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that
were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No
correlation was observed between the type of ROS1 rearrangement and the clinical response to
Copyright © 2014 Massachusetts Medical Society.

Address reprint requests to Dr. Shaw at the Massachusetts General Hospital Cancer Center, Yawkey 7B, 32 Fruit St., Boston, MA
02114, or at [email protected].
Drs. Shaw and Ou contributed equally to this article.
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.
Shaw et al. Page 2
crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged
NSCLC.
CONCLUSIONS—In this study, crizotinib showed marked antitumor activity in patients with
advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of
NSCLC for which crizotinib is highly active.
The ROS1 oncogene encodes an orphan receptor tyrosine kinase related to anaplastic
lymphoma kinase (ALK), along with members of the insulin-receptor family.1 First
discovered as the oncogene product of an avian sarcoma RNA tumor virus,2–4 ROS1 (ROS1
proto-oncogene receptor tyrosine kinase) is activated by chromosomal rearrangement in a
variety of human cancers, including non–small-cell lung cancer (NSCLC),
cholangiocarcinoma, gastric cancer, ovarian cancer, and glioblastoma multiforme.5–9
Rearrangement leads to fusion of a portion of ROS1 that includes the entire tyrosine kinase
domain with 1 of 12 different partner proteins.10 The resulting ROS1 fusion kinases are
constitutively activated and drive cellular transformation. Whether the various ROS1 fusion
kinases may have different oncogenic properties is unknown.
ROS1 rearrangements occur in approximately 1% of patients with NSCLC.11 Of the

estimated 1.5 million new cases of NSCLC worldwide each year, approximately 15,000 may
be driven by oncogenic ROS1 fusions. As with ALK rearrangements, ROS1 rearrangements
are more commonly found in patients who have never smoked or have a history of light
smoking and who have histologic features of adenocarcinoma.11,12 However, at the genetic
level, ALK and ROS1 rearrangements rarely occur in the same tumor, with each defining a
unique molecular subgroup of NSCLC.11
Several lines of evidence suggest that ROS1 may represent another therapeutic target of the
ALK inhibitor crizotinib (Xalkori, Pfizer). First, the kinase domains of ALK and ROS1
share 77% amino acid identity within the ATP-binding sites. Crizotinib binds with high
affinity to both ALK and ROS1, which is consistent with this homology.13 Second, in cell-
based assays for inhibition of autophosphorylation of different kinase targets, both ALK and
ROS1 are sensitive to crizotinib, with a half-maximal inhibitory concentration of 40 to 60
nM.14 Third, in cell lines expressing ROS1 fusions, crizotinib potently inhibits ROS1
signaling and cell viability.12,15,16 Finally, case reports have described marked responses to
crizotinib in patients with ROS1-rearranged NSCLC.12,17 Here we report the efficacy and
safety of crizotinib in patients with advanced, ROS1-rearranged NSCLC.
METHODS
PATIENTS
Eligible patients had histologically confirmed, advanced NSCLC with a ROS1
rearrangement. In 49 of 50 patients (98%), we identified the ROS1 rearrangement using
break-apart fluorescence in situ hybridization (FISH).12,17 In the remaining patient, we
identified the ROS1 rearrangement using a reverse-transcriptase–polymerase-chain-reaction
(RT-PCR) assay. We used FISH results that were obtained at most of the participating sites.
All patients with positive results on FISH had more than 15% split signals. Other eligibility
criteria included an age of at least 18 years, an Eastern Cooperative Oncology Group

Shaw et al. Page 3
performance status of 0 to 2 (on a scale of 0 to 5, with 0 indicating that the patient is fully
active and able to carry on all predisease activities without restriction and 5 indicating that
the patient has died),18 adequate organ function, and measurable disease according to the
Response Evaluation Criteria in Solid Tumors (RECIST), version 1.0.19 (For details, see the
Supplementary Appendix, available with the full text of this article at NEJM.org.)
The protocol, which is available at NEJM.org, was approved by the institutional review
board or independent ethics committee at each site and complied with the International
Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical
Practice guidelines, the Declaration of Helsinki, and local laws. All patients provided written
informed consent.
STUDY DESIGN AND TREATMENT

This phase 1 study was originally designed to include an initial dose-escalation phase,
followed by an expansion phase in which the maximum dose established in the initial phase
would be evaluated in molecularly defined cohorts of patients.20 Details on the dose-
escalation phase have been reported previously,21 as have efficacy and safety data from the
ALK-positive expansion cohort.20,22 In November 2009, the study was amended to include
an expansion cohort of patients with advanced, ROS1-rearranged NSCLC. The primary end
point of this expansion study was the response rate.
Crizotinib was administered orally at the standard dose of 250 mg twice daily in continuous
28-day cycles. Treatment continued until the occurrence of RECIST-defined disease
progression or clinical deterioration, unacceptable toxic effects, withdrawal from the study,
or death. In patients with RECIST-defined progression, the study treatment could be
continued at the investigator’s discretion and with approval from the sponsor.
STUDY ASSESSMENTS
Patients underwent baseline tumor imaging, with computed tomography or magnetic
resonance imaging of the chest, abdomen, and pelvis. Brain and bone scans were obtained at
baseline if disease at these sites was suspected. Tumor assessments were performed by the
investigators every 8 weeks until RECIST-defined disease progression; starting with cycle
15, tumor assessments could be performed every 16 weeks, as determined by the
investigator. All tumor responses were confirmed at least 4 weeks after the initial response.
Adverse events were assessed from the time informed consent was obtained until at least 28
days after the last dose of crizotinib was administered. All adverse events were classified
and graded with the use of the Common Terminology Criteria for Adverse Events, version
3.0 (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf).
The data-cutoff date was April 11, 2014, for safety and pharmacokinetics data and May 16,
2014, for efficacy data.
MOLECULAR ANALYSES
In patients with sufficient tumor tissue available, we performed additional molecular
analyses. Formalin-fixed, paraffin-embedded tumors were screened for ALK rearrangement
with the use of a break-apart FISH assay.20 Tumors were also screened for amplification of

Shaw et al. Page 4
MET, a gene encoding another proto-oncogene receptor tyrosine kinase, with the use of a
dual-color FISH probe (Repeat-Free Poseidon C-MET [7q31] probe, Kreatech) and a copy-
number control probe (centromere chromosome 7 gene [CEP7], Abbott-Vysis). In 30
patients, tumor tissue or nucleic acid was available for molecular characterization of the
ROS1 rearrangement. For 27 of these patients, we performed targeted next-generation
sequencing with the use of anchored multiplex PCR, as described previously.23 This assay
detects fusion transcripts involving ALK (exons 19 through 22), ROS1 (exons 31 through
37), and rearranged during transfection proto-oncogene (RET) (exons 8 through 13). For the
remaining 3 patients, we used RT-PCR to detect specific ROS1 fusion transcripts.17
STUDY OVERSIGHT
The study was designed jointly by the investigators and representatives of the sponsor,
Pfizer. The sponsor collected and analyzed the data. The first author wrote the first draft of
the manuscript. All the authors were involved in the data analysis and manuscript
preparation and vouch for the completeness and accuracy of the data and analyses and for
the adherence of the study to the protocol. No one who is not listed as an author contributed
to the writing of the manuscript.
STATISTICAL ANALYSIS
We initially determined that we would need to enroll 30 patients in order to achieve a power
of at least 85% to test the null hypothesis that the rate of response to crizotinib would be
10% or less, versus the alternative hypothesis that the response rate would be more than
10%, at a one-sided alpha level of 0.05 and with the use of a single-stage design. For the
alternative hypothesis, the response rate was assumed to be 30%. As of April 2012, there
were eight responses (among 14 patients who could be evaluated), which exceeded the six
responses required to reject the null hypothesis.14 To permit a more accurate assessment of
the efficacy and safety of crizotinib in this population, we expanded the sample size to a
maximum of 50 patients. The overall response rate was similar for the first 30 patients who
were enrolled (67%) and the additional 20 patients who were enrolled (80%).
We used a Kaplan–Meier analysis of time-to-event data to estimate median event times and
the Brookmeyer–Crowley method to calculate two-sided 95% confidence intervals. All
analyses were performed with the use of SAS statistical software, version 9.2 (SAS
Institute).
RESULTS
PATIENTS
From October 2010 through August 2013, we enrolled 50 patients with advanced NSCLC in
the ROS1 expansion cohort of the phase 1 study of crizotinib. Table 1 summarizes the
clinicopathological characteristics of all 50 patients. The majority of patients had never
smoked and had histologic features of adenocarcinoma. Most patients (86%) had received at
least one previous line of standard therapy for advanced NSCLC (Table S1 in the
Supplementary Appendix).

Shaw et al. Page 5
In 49 of the 50 patients (98%), we used a break-apart FISH assay to identify the presence of
a ROS1 rearrangement. In 1 of these 49 patients, an atypical ROS1 FISH pattern was noted
(isolated 5′ green signal), and next-generation sequencing subsequently revealed normal,
nonrearranged ROS1. In another patient, the tumor was positive for both ROS1 and ALK
rearrangement on the basis of FISH, but next-generation sequencing revealed only an
EML4-ALK fusion and no ROS1 rearrangement. Among an additional 32 ROS1-positive
tumors tested for ALK rearrangement, none were positive. Among 15 ROS1 FISH-positive
tumors tested for MET amplification with the use of FISH, 1 was positive, with a MET-to-
CEP7 ratio of 5.34, whereas the remaining 14 were negative. (A ratio of MET to the control
of more than 2.0 is an indicator of copy-number gain.)
EFFICACY
Among the 50 study patients, 3 patients (6%) had a complete response, 33 patients (66%)
had a partial response, and 9 patients (18%) had stable disease as their best response (Fig.
1A, and Table S2 in the Supplementary Appendix). The overall response rate was 72%
(95% confidence interval [CI], 58 to 84). The median time to the first response was 7.9
weeks (range, 4.3 to 32.0) (Fig. 1B, and Table S2 in the Supplementary Appendix). At the
time of data cutoff, 23 of the 36 responses (64%) were ongoing (Fig. 1C). The estimated
median duration of response was 17.6 months (95% CI, 14.5 to not reached [NR]) (Table S2
in the Supplementary Appendix).
Three of the 50 patients (6%) had evidence of progressive disease on the first restaging
scans. For 1 of the 3 patients, results on FISH were atypical, and next-generation sequencing
was negative for ROS1 rearrangement, as noted above. A second patient, whose tumor was
positive for ROS1 rearrangement, had discontinued crizotinib for 6 weeks before the first
restaging scans because of bowel perforation that was thought to be related to glucocorticoid
use and preexisting diverticular disease. This patient was later able to resume treatment with
crizotinib and subsequently had a 62% reduction in the tumor burden. For the third patient,
FISH results were positive for ROS1 rearrangement, but the first restaging scans showed an
increase in the tumor burden of 26%.
Among the 50 patients, the median duration of treatment was 64.5 weeks (range, 2.3 to
182.0), and 30 patients (60%) continued to receive crizotinib after the data cutoff date.
Median progression-free survival was 19.2 months (95% CI, 14.4 to NR) (Fig. 2). Data for
27 patients (54%) were censored, including data for 25 patients (50%) undergoing follow-up
for progression. Median follow-up for overall survival was 16.4 months (95% CI, 13.8 to
19.8). Nine of the 50 patients (18%) had died by the time of data cutoff. The overall survival
rate at 12 months was 85% (95% CI, 72 to 93); the median had not been reached.
ADVERSE EVENTS
The safety profile of crizotinib in this study was similar to that reported previously.22,24
Treatment-related adverse events (as determined by the investigators) that were seen in at
least 10% of the patients are listed in Table 2; the most common events were visual
impairment (82%), diarrhea (44%), nausea (40%), peripheral edema (40%), constipation
(34%), vomiting (34%), an elevated aspartate aminotransferase level (22%), fatigue (20%),

Shaw et al. Page 6
dysgeusia (18%), and dizziness (16%). Of the 388 treatment-related adverse events that
were reported, 365 (94%) were grade 1 or 2. Of the 42 visual-impairment events that were
reported, all were grade 1; they were often described as brief image persistence triggered by
dark-to-light adaptation, as reported previously.22 One patient (2%) discontinued crizotinib

because of treatment-related nausea.
The most common treatment-related grade 3 adverse events, reported in at least 4% of the
patients, were hypophosphatemia (10%), neutropenia (10%), and an elevated alanine
aminotransferase level (4%). There were no treatment-related grade 4 or five adverse events.
Grade 4 adverse events that were not deemed to have been related to treatment were
reported in 4 patients: pulmonary embolism, hypoxemia, hypotension, and pericardial
effusion. There were five deaths, all of which were due to disease progression and were
considered to be unrelated to treatment. There were no serious adverse events or deaths in
the 5-week period between the cutoff date for safety data and the cutoff date for efficacy
data.
MOLECULAR CHARACTERIZATION OF ROS1 REARRANGEMENTS

We used a targeted next-generation sequencing assay or an RT-PCR assay to identify ROS1
fusion partners in available tumor specimens. A total of 30 samples were tested, 27 with the
use of next-generation sequencing and 3 with the use of RT-PCR. Of the 27 samples tested
by means of next-generation sequencing, 22 had a specific ROS1 rearrangement. Among the
remaining 5 samples, the assay failed in 1 and was negative for ROS1 rearrangement in 4. In
1 of the 4 negative samples, a different oncogenic fusion gene, EML4-ALK, was identified,
and FISH results were positive for ALK, suggesting that results on FISH were falsely
positive for ROS1. In a second negative sample, FISH results were atypical and were
probably not indicative of a ROS1 rearrangement. In the remaining two samples, the failure
to detect ROS1 fusions may have been due to the limited quantity of tumor material.
The most common ROS1 fusion partner that we identified was the gene encoding CD74,
which was present in 11 of 25 samples (44%); other partner genes included SDC4 (in 4
tumors), EZR (in 4 tumors), SLC34A2 (in 3 tumors), and TPM3 (in 1 tumor), all of which
have previously been identified as ROS1 fusion partners. Using next-generation sequencing,
we also discovered 2 novel partners, LIMA1 (LIM domain and actin binding 1) and MSN
(moesin).23,25,26 The predicted structures of both novel ROS1 fusion proteins are shown in
Figure S1 in the Supplementary Appendix. Tumor responses were observed regardless of the
ROS1 fusion partner (Fig. S2 in the Supplementary Appendix). There was also no apparent
correlation between the specific ROS1 rearrangement and the duration of crizotinib
treatment (Fig. 3). However, given the number of different ROS1 fusions, the relationship
between ROS1 fusion and the response to crizotinib is difficult to assess on the basis of this
small study.
DISCUSSION
We found that crizotinib had potent antitumor activity in patients who had advanced NSCLC
with a ROS1 rearrangement. These results validate ROS1 as a therapeutic target in ROS1-
rearranged lung cancers.

Shaw et al. Page 7
In preclinical studies, cell lines expressing oncogenic fusions of either ALK or ROS1 were
highly sensitive to crizotinib.12,14,16 The dual inhibition of ALK and ROS1 by the same
small molecule is probably due to structural similarities between these two closely related
tyrosine kinases. The three-dimensional structures of the sites of crizotinib binding with
ALK and ROS1 are similar (Fig. S3 in the Supplementary Appendix).27,28 Most of the
amino acid differences between ALK and ROS1 are conservative or do not contact
crizotinib. Only one difference, a valine-to-leucine difference at codon 1180 of ALK and
codon 2010 of ROS1, is predicted to have an effect on binding, since the larger leucine in
ROS1 extends closer to and makes more direct contact with crizotinib. The functional
significance of this and other amino acid differences has not yet been studied.
Clinically, although ALK and ROS1 rearrangements define different subgroups of NSCLC,
there are several important similarities between the two disease subtypes. Patients with ALK-
rearranged NSCLC and those with ROS1-rearranged NSCLC have similar
clinicopathological features. In addition, the ALK-rearranged and ROS1-rearranged disease
subtypes were both highly responsive to crizotinib, with similar times to the first response
(median, 7.9 weeks for both) and similar response rates (61% and 72%, respectively).22 For
both ALK-rearranged NSCLC and ROS1-rearranged NSCLC, responses were observed
independently of the specific type of rearrangement.20
One apparent difference between ALK rearrangement and ROS1 rearrangement in patients
with NSCLC may lie in the durability of the response to crizotinib. In the ALK expansion
cohort of 143 patients, the median duration of response was 49.1 weeks, and the median
progression-free survival was 9.7 months.22 In contrast, the estimated median duration of
response in the ROS1 cohort was longer, at 17.6 months (75.9 weeks), and the median
progression-free survival was 19.2 months. This estimate is still preliminary, since half the
patients remain in follow-up for progression. The apparent difference in efficacy is not
attributable to differences in drug exposure, since the mean trough plasma levels of
crizotinib were similar in patients with ALK rearrangements and in those with ROS1
rearrangements (Fig. S4 in the Supplementary Appendix).
Several factors may account for the longer responses observed in ROS1-rearranged NSCLC.
First, crizotinib may be a more potent inhibitor of ROS1 than of ALK, leading to more
effective target inhibition and more durable responses. In support of this hypothesis, in vitro
measurements of the equilibrium dissociation constant (Kd) with the use of isothermal
titration calorimetry indicated that crizotinib binds significantly more tightly to ROS1 than
to ALK, with values of 0.4 nM and 4.4 nM, respectively.13 This finding is consistent with
cell-viability assays showing that crizotinib is approximately five times as potent against
ROS1 as against ALK in Ba/F3 cells engineered to express either CD74-ROS1 or EML4-
ALK.14 Second, ROS1 rearrangement could in theory confer a more favorable prognosis
regardless of treatment, perhaps because of the intrinsic biology of ROS1-positive NSCLC.
However, in several small series, overall survival among patients with ROS1 rearrangement
was similar to that among patients without ROS1 rearrangement.12,29
As in patients with ALK-rearranged NSCLC, resistance to crizotinib eventually develops in

patients with ROS1-rearranged NSCLC. As of the data cutoff date, disease progression or

Shaw et al. Page 8
death had occurred in 23 of 50 patients (46%). Two distinct mechanisms of resistance to

crizotinib in ROS1-rearranged NSCLC have been described: a secondary mutation that
hinders drug binding27 and activation of epidermal growth factor receptor, which enables
cancer cells to bypass crizotinib-mediated inhibition of ROS1 signaling.30 Recently, more

potent, structurally distinct, next-generation ALK inhibitors have been shown to effectively
overcome crizotinib resistance in ALK-rearranged NSCLC.31–33 Some, but not all, of these
new ALK inhibitors also target ROS1.33,34 Whether ROS1-rearranged NSCLC will also be
susceptible to sequential therapy with increasingly potent inhibitors remains to be
determined.
In conclusion, ROS1 rearrangement defines a second molecular subgroup of NSCLC for

which crizotinib is highly active. In the majority of patients, crizotinib induced durable
clinical responses and was associated with grade 2 or lower toxic effects. These results
highlight the importance of screening for this genetic alteration in patients with advanced
NSCLC. Although FISH was used in this study, other diagnostic methods have been
proposed, and further work is required to establish the most effective screening strategy for
ROS1 rearrangement.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Supported by Pfizer, by grants from the National Cancer Institute (R21CA161590, to Dr. Iafrate; K12CA086913, to
Dr. Doebele; P50CA058187, to Drs. Doebele and Camidge; and P30CA046934, to Dr. Varella-Garcia), by a
research grant from Uniting against Lung Cancer (to Dr. Shaw), by the Swedish Research Council (postdoctoral
fellowship 350-2012-368, to Dr. Zheng), and by Be a Piece of the Solution.
We thank the participating patients and their families, as well as the research nurses, study coordinators, and
operations staff; and Roger Wild at ACUMED, Tytherington, United Kingdom, for his assistance in the preparation
of original versions of the figures.
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28. Huang Q, Johnson TW, Bailey S, et al. Design of potent and selective inhibitors to overcome
clinical anaplastic lymphoma kinase mutations resistant to crizotinib. J Med Chem. 2014;
57:1170–1187. [PubMed: 24432909]
29. 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; 37:554–562. [PubMed: 23426121]

Shaw et al. Page 10
30. Davies KD, Mahale S, Astling DP, et al. Resistance to ROS1 inhibition mediated by EGFR
pathway activation in non-small cell lung cancer. PLoS One. 2013; 8(12):e82236. [PubMed:
24349229]
31. Shaw AT, Kim DW, Mehra R, et al. Ceritinib in ALK-rearranged non–small-cell lung cancer. N
Engl J Med. 2014; 370:1189–1197. [PubMed: 24670165]
32. Gadgeel SM, Gandhi L, Riely GJ, et al. Safety and activity of alectinib against systemic disease
and brain metastases in patients with crizotinib-resistant ALK-rearranged non-small-cell lung
cancer (AF-002JG): results from the dose-finding portion of a phase 1/2 study. Lancet Oncol.
2014; 15:1119–1128. [PubMed: 25153538]
33. Gettinger, SN.; Bazhenova, L.; Salgia, R., et al. Updated efficacy and safety of the ALK inhibitor
AP26113 in patients with advanced malignancies, including ALK+ non-small cell lung cancer
(NSCLC). Presented at the Annual Meeting of the American Society of Clinical Oncology; May
30–June 3, 2014; Chicago. abstract.
34. Marsilje TH, Pei W, Chen B, et al. Synthesis, structure-activity relationships, and in vivo efficacy
of the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5-chloro-N2-(2-
isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-
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Shaw et al. Page 11
Figure 1. Tumor Responses to Crizotinib in ROS1-Rearranged Non–Small-Cell Lung Cancer

Panel A shows the best response of patients treated with crizotinib in the ROS1 expansion
cohort. The bars indicate best percent change in the target tumor burden from baseline. Two
patients died within 6 weeks after receiving the first dose of crizotinib, so the tumor
response was unknown. The asterisk indicates the tumor burden in a patient who had an
atypical result on fluorescence in situ hybridization (FISH) for ROS1 (an isolated 5′ green
signal). Since this tumor was subsequently shown to be negative for ROS1 rearrangement on
next-generation sequencing, an isolated green signal is probably not indicative of a ROS1

Shaw et al. Page 12
rearrangement. The letter A denotes a FISH-positive tumor that was negative for ROS1
rearrangement on next-generation sequencing but positive for ALK rearrangement on FISH
and next-generation sequencing. The letter M denotes a FISH-positive tumor that was also
positive for MET amplification on FISH (MET-to-CEP7 ratio, 5.34). Panel B shows
positron-emission tomographic scans obtained at baseline (left panel) and after 7 weeks of
crizotinib treatment (right panel) in a representative patient. On the basis of Response
Evaluation Criteria in Solid Tumors, this patient had a partial response (a decrease in tumor
burden of 46%), which was ongoing at the time of data cutoff. Panel C shows the duration of
response among the 36 patients with a partial or complete response. Arrows indicate patients
who had an ongoing response at the time of data cutoff. The letter A indicates that the
patient’s tumor was positive for ALK rearrangement.

Shaw et al. Page 13
Figure 2. Progression-free Survival

Shown is the Kaplan–Meier curve for estimated progression-free survival in the ROS1
cohort of patients treated with crizotinib. Progression-free survival was defined as the time
from the administration of the first dose of crizotinib to objective disease progression or
death from any cause. Data from 27 patients were censored; of these patients, 25 remained
in follow-up for progression-free survival at the time of data cutoff. The shaded area
represents the 95% Hall–Wellner confidence limits. Vertical lines on the survival curve
indicate censoring of data.

Shaw et al. Page 14
Figure 3. Duration of Treatment and ROS1 Fusion Partners

The duration of crizotinib treatment is shown for the 25 patients in whom the ROS1 fusion
partner was identified with the use of either a next-generation sequencing assay or a reverse-
transcriptase–polymerase-chain-reaction assay. Patients are grouped according to the ROS1
fusion partner, as indicated on the left. The four patients with negative results on next-
generation sequencing and the one patient in whom next-generation sequencing failed are
indicated by gray bars. One of the four patients with negative results was positive for EML4-
ALK rearrangement, as indicated. One patient had negative results on next-generation
sequencing and had an atypical FISH pattern (as indicated by an asterisk). The arrows
indicate patients who were continuing to receive crizotinib at the time of data cutoff.

Shaw et al. Page 15
Table 1
Characteristics of the Patients at Baseline.

ROS1 Cohort
Characteristic (N = 50)
Age — yr
Median 53
Range 25–77
Sex — no. (%)
Male 22 (44)
Female 28 (56)
Race — no. (%)*

White 27 (54)
Asian 21 (42)
Other 2 (4)
Smoking status — no. (%)
Never smoked 39 (78)

Former smoker 11 (22)
Histologic type — no. (%)
Adenocarcinoma 49 (98)
Squamous-cell carcinoma 1 (2)
ECOG performance status — no. (%)†
0 22 (44)
1 27 (54)
2 1 (2)
Previous regimens for advanced disease — no. (%)
0 7 (14)
1 21 (42)
>1 22 (44)
*
Race was determined by the investigators.
†
Eastern Cooperative Oncology Group (ECOG) performance status ranges from 0 to 5, with higher numbers indicating increasing impairment in
activities of daily living.

Shaw et al. Page 16
Table 2
Adverse Events.*
Grade Grade Grade All

Adverse Event 1 2 3 Grades
number of patients (percent)
Visual impairment 41 (82) 0 0 41 (82)
Diarrhea 21 (42) 1 (2) 0 22 (44)
Nausea 18 (36) 2 (4) 0 20 (40)
Peripheral edema 15 (30) 5 (10) 0 20 (40)
Constipation 16 (32) 1 (2) 0 17 (34)
Vomiting 15 (30) 1 (2) 1 (2) 17 (34)
Elevated aspartate aminotransferase 9 (18) 1 (2) 1 (2) 11 (22)
Fatigue 9 (18) 1 (2) 0 10 (20)

Dysgeusia 9 (18) 0 0 9 (18)
Dizziness 8 (16) 0 0 8 (16)
Elevated alanine aminotransferase 3 (6) 2 (4) 2 (4) 7 (14)
Hypophosphatemia 0 2 (4) 5 (10) 7 (14)
Decreased testosterone† 2 (9) 1 (5) 0 3 (14)
Neutropenia 1 (2) 0 5 (10) 6 (12)
Dyspepsia 5 (10) 0 0 5 (10)
Sinus bradycardia 5 (10) 0 0 5 (10)
*
Listed are adverse events that were reported in at least 10% of the 50 study patients and that were deemed by the investigators to be related to
treatment. No grade 4 or grade 5 treatment-related adverse events were reported.
†
The frequency of a decreased testosterone level was calculated in 22 men only. The protocol did not require the testing of testosterone, so not all
men were evaluated.

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N Engl J Med. 2014 November 20; 371(21): 1963–1971. doi:10.1056/NEJMoa1406766.

Crizotinib in ROS1-Rearranged Non–Small-Cell Lung Cancer

Copyright © 2014 Massachusetts Medical Society.

ROS1 rearrangements occur in approximately 1% of patients with NSCLC.11 Of the

safety of crizotinib in patients with advanced, ROS1-rearranged NSCLC.

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

STUDY DESIGN AND TREATMENT

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

dark-to-light adaptation, as reported previously.22 One patient (2%) discontinued crizotinib

MOLECULAR CHARACTERIZATION OF ROS1 REARRANGEMENTS

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

independently of the specific type of rearrangement.20

As in patients with ALK-rearranged NSCLC, resistance to crizotinib eventually develops in

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

death had occurred in 23 of 50 patients (46%). Two distinct mechanisms of resistance to

cancer cells to bypass crizotinib-mediated inhibition of ROS1 signaling.30 Recently, more

In conclusion, ROS1 rearrangement defines a second molecular subgroup of NSCLC for

2. Birchmeier C, Birnbaum D, Waitches G, Fasano O, Wigler M. Characterization of an activated

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

cancer. J Thorac Oncol. 2012; 7:1086–1090. [PubMed: 22617245]

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

Figure 1. Tumor Responses to Crizotinib in ROS1-Rearranged Non–Small-Cell Lung Cancer

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

Figure 2. Progression-free Survival

indicate censoring of data.

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

Figure 3. Duration of Treatment and ROS1 Fusion Partners

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

Characteristics of the Patients at Baseline.

Sex — no. (%)

Race — no. (%)*

Smoking status — no. (%)

Never smoked 39 (78)

Former smoker 11 (22)

Histologic type — no. (%)

Squamous-cell carcinoma 1 (2)

ECOG performance status — no. (%)†

Previous regimens for advanced disease — no. (%)

N Engl J Med. Author manuscript; available in PMC 2015 May 20.

Grade Grade Grade All

Diarrhea 21 (42) 1 (2) 0 22 (44)

Nausea 18 (36) 2 (4) 0 20 (40)

Peripheral edema 15 (30) 5 (10) 0 20 (40)

Constipation 16 (32) 1 (2) 0 17 (34)

Vomiting 15 (30) 1 (2) 1 (2) 17 (34)

Elevated aspartate aminotransferase 9 (18) 1 (2) 1 (2) 11 (22)

Fatigue 9 (18) 1 (2) 0 10 (20)

Dysgeusia 9 (18) 0 0 9 (18)

Dizziness 8 (16) 0 0 8 (16)

Elevated alanine aminotransferase 3 (6) 2 (4) 2 (4) 7 (14)

Hypophosphatemia 0 2 (4) 5 (10) 7 (14)

Decreased testosterone† 2 (9) 1 (5) 0 3 (14)

Neutropenia 1 (2) 0 5 (10) 6 (12)

Dyspepsia 5 (10) 0 0 5 (10)