V O L U M E 2 8 䡠 N U M B E R 5 䡠 F E B R U A R Y 1 0 2 0 1 0
`
`JOURNAL OF CLINICAL ONCOLOGY
`
`O R I G I N A L R E P O R T
`
`From the Center for Sarcoma and Bone
`Oncology and Ludwig Center for
`Cancer Research; and Department of
`Radiology, Dana-Farber Cancer Insti-
`tute; Harvard Medical School; Division
`of Translational Medicine, Departments
`of Medicine and of Pathology, Brigham
`and Women’s Hospital; and Center for
`Molecular Oncologic Pathology, Dana-
`Farber Cancer Institute/Brigham and
`Women’s Cancer Center, Boston, MA;
`and Department of Pathology and
`Melanoma and Sarcoma Service,
`Department of Medicine, Memorial
`Sloan-Kettering Cancer Center, New
`York, NY.
`
`Submitted July 24, 2009; accepted
`September 25, 2009; published online
`ahead of print at www.jco.org on
`January 4, 2010.
`
`Supported in part by the sPECial Fund
`for PEComa Research and by the
`Virginia and D.K. Ludwig Fund for
`Cancer Research (to the Dana-Farber/
`Harvard Ludwig Center).
`
`Presented in part at the 14th Annual
`Meeting of the Connective Tissue
`Oncologic Society,November 13-15,
`2008, London, United Kingdom.
`
`Authors’ disclosures of potential con-
`flicts of interest and author contribu-
`tions are found at the end of this
`article.
`
`Corresponding author: Andrew J.
`Wagner, MD, PhD, Center for Sarcoma
`and Bone Oncology, Dana-Farber
`Cancer Institute, 44 Binney St, Boston,
`MA 02115; e-mail: Andrew_Wagner@
`dfci.harvard.edu.
`
`© 2010 by American Society of Clinical
`Oncology
`
`0732-183X/10/2805-835/$20.00
`
`DOI: 10.1200/JCO.2009.25.2981
`
`Clinical Activity of mTOR Inhibition With Sirolimus in
`Malignant Perivascular Epithelioid Cell Tumors: Targeting
`the Pathogenic Activation of mTORC1 in Tumors
`Andrew J. Wagner, Izabela Malinowska-Kolodziej, Jeffrey A. Morgan, Wei Qin, Christopher D.M. Fletcher,
`Natalie Vena, Azra H. Ligon, Cristina R. Antonescu, Nikhil H. Ramaiya, George D. Demetri,
`David J. Kwiatkowski, and Robert G. Maki
`
`A
`
`B
`
`S
`
`T
`
`R
`
`A
`
`C
`
`T
`
`Purpose
`Perivascular epithelioid cell tumors (PEComas) represent a family of mesenchymal neoplasms,
`mechanistically linked through activation of the mTOR signaling pathway. There is no known
`effective therapy for PEComa, and the molecular pathophysiology of aberrant mTOR signaling
`provided us with a scientific rationale to target this pathway therapeutically. On this mechanistic
`basis, we treated three consecutive patients with metastatic PEComa with an oral mTOR
`inhibitor, sirolimus.
`
`Patients and Methods
`Patients with advanced PEComa were treated with sirolimus and consented to retrospective
`collection of data from their medical records and analysis of archival tumor specimens. Tumor
`response was determined by computed tomography scans obtained at the clinical discretion of the
`treating physicians. Tumors were assessed for immunohistochemical evidence of mTORC1
`activation and genetic evidence of alterations in TSC1 and TSC2.
`
`Results
`Radiographic responses to sirolimus were observed in all patients. PEComas demonstrated loss
`of TSC2 protein expression and evidence of baseline mTORC1 activation. Homozygous loss of
`TSC1 was identified in one PEComa.
`
`Conclusion
`Inhibition of mTORC1, pathologically activated by loss of the TSC1/TSC2 tumor suppressor
`complex, is a rational mechanistic target for therapy in PEComas. The clinical activity of sirolimus
`in PEComa additionally strengthens the pathobiologic similarities linking PEComas to other
`neoplasms related to the tuberous sclerosis complex.
`
`J Clin Oncol 28:835-840. © 2010 by American Society of Clinical Oncology
`
`INTRODUCTION
`
`Sarcomas are a heterogeneous collection of tu-
`mors sharing a common mesenchymal origin. His-
`torically, treatment studies of pooled subtypes of
`sarcoma have shown overall poor responses to con-
`ventional chemotherapeutic agents.1 However, in
`recent years, identification of molecular subtypes of
`sarcoma has led to the application of effective tar-
`geted therapies, such as imatinib mesylate for the
`treatment of gastrointestinal stromal tumors that
`usually harbor activating mutations in the KIT re-
`ceptor tyrosine kinase. Identification of molecular
`alterations in other sarcoma subtypes may lead to
`more effective therapies for this otherwise difficult-
`to-treat group of diseases.
`
`tumor
`The perivascular epithelioid cell
`(PEComa) family of tumors consists of related mes-
`enchymal neoplasms that exhibit myomelanocytic
`differentiation and share a distinctive cell type,
`the perivascular epithelioid cell, or PEC.2-4 The ma-
`jor members of this family include lymphangio-
`leiomyomatosis (LAM), a disease predominantly
`presenting as numerous nodular and interstitial pul-
`monary lesions in premenopausal women; angio-
`myolipoma (AML), commonly identified as an
`asymptomatic renal lesion with evidence of vas-
`cular, muscle, and adipocytic differentiation; and
`PEComa, an epithelioid malignancy with clear-to-
`granular eosinophilic cytoplasm typically arising in
`the gastrointestinal tract, retroperitoneum, uterus,
`or somatic soft tissues, composed of nests and sheets
`
`© 2010 by American Society of Clinical Oncology
`Information downloaded from jco.ascopubs.org and provided by at Univ of Chicago Library on February 25, 2016 from
`
`205.208.61.64Copyright © 2010 American Society of Clinical Oncology. All rights reserved.
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`Wagner et al
`
`of epithelioid or occasionally spindled cells, intimately related to blood
`vessel walls.
`Tumors of the PEComa family are rare and usually occur
`sporadically. LAM and AML also are seen at high frequency in
`patients with tuberous sclerosis complex (TSC), a disorder caused
`by mutation of TSC1 or TSC2, for which the gene products nega-
`tively regulate mTORC1 through inhibition of the mTOR kinase
`activator RHEB. Recently Bissler et al5 described modest, transient
`improvement in lung function and reduction in size of AML in a
`trial of 25 patients with LAM and AML treated with sirolimus, an
`inhibitor of mTORC1. PEComas have rarely been reported in
`association with TSC.6
`Most PEComas are benign tumors and do not recur after com-
`plete surgical resection. However, a subset of PEComas exhibit malig-
`nant behavior, with either locally invasive recurrences or development
`of distant metastases, most commonly in the lung. No effective ther-
`apy for malignant PEComa has been described. Because PEComas
`share activation of the mTOR pathway with LAM and AML in many
`instances,6,7 we treated three consecutive patients at two institutions
`with sirolimus.
`
`PATIENTS AND METHODS
`
`Patient Selection, Treatment, and Clinical Assessments
`Three consecutive patients with PEComa who presented to Dana-
`Farber Cancer Institute or Memorial Sloan-Kettering Cancer Center were
`offered off-label treatment with sirolimus by their treating physicians.
`None of the patients exhibited clinical signs or a history of TSC. There was
`no significant family history of other cancers in any of the index patients.
`All patients provided informed consent for treatment with sirolimus as
`well as retrospective review of medical records and evaluation of archival
`tumor specimens according to institutional review board–approved pro-
`tocols. The dosage of sirolimus (range, 1 mg every other day to 8 mg daily)
`was determined by the treating physician and was adjusted on the basis of
`trough sirolimus levels or patient tolerance. Disease status was assessed by
`CT scans at baseline and at intervals recommended by the treating physi-
`cian. [18F]fluorodeoxyglucose–positron emission tomography scans were
`not performed as part of tumor assessment.
`
`Histologic and Immunohistochemical Evaluations
`Hemotoxylin and eosin staining and immunohistochemical stains of
`sections of archival formalin-fixed paraffin-embedded tissue were per-
`formed according to standard techniques for routine clinical pathologic
`evaluation. For additional immunohistochemical analyses, slides were
`processed through a xylene and ethanol series, subjected to Dako Antigen
`Retrieval procedure (Dako, Carpinteria, CA), and stained with a monoclo-
`nal mouse antiphospho-S6 antibody (S240/244, clone DAK-S6-240; Dako,
`Carpinteria, CA) or a polyclonal rabbit anti-TSC2 antibody (gift of Vijaya
`Ramesh, Massachusetts General Hospital, Boston, MA) followed by horse-
`radish peroxidase detection.
`
`Genetic Analyses
`Genomic DNA was extracted from paraffin-embedded tumor tissue or
`peripheral-blood mononuclear cells and was assessed for copy number
`changes by mulitplex ligation-dependent probe amplification (MLPA) with
`nine exonic probes for TSC1 and TSC2, respectively, and five control probes
`from other genomic locations.8 Signals were normalized to values obtained
`with DNA extracted from control tissues. A value of 1 represented two copies
`of the assayed gene, 0.5 represented one copy, and 0 represented loss of
`both copies.
`
`Fluorescence in situ hybridization (FISH) was performed by using
`interphase nuclei isolated from archival paraffin-embedded tissue and
`fosmid DNA probes corresponding to TSC1 (G248P87270H4 and
`G248P87503E5) and TSC2 (G248P89119C12 and G248P84443G9) with
`4⬘,6-diamidino-2-phenylindole counterstaining, according to standard
`methods.9 Genomic DNA from PEComas was amplified for PIK3CA (ex-
`ons 10 and 21), AKT1 (exon 3), and KRAS (exons 2 and 3), and then was
`sequenced by standard dideoxy sequencing.
`
`A
`
`B
`
`C
`
`Fig 1. Computed tomography images of perivascular epithelioid cell tumors
`(PEComas;
`left panels) at baseline and (right panels) after treatment with
`sirolimus. All
`images are chosen to show the maximal size of the lesions
`indicated by arrows; other lesions may be out of phase in the two studies. (A)
`Retroperitoneal PEComa before and after 1 year of treatment. (B) Renal PEComa
`before and after 9 months of treatment. (C, top) Metastatic uterine PEComa in
`lung before and after 6 weeks of treatment; some lesions are improved, whereas
`others remain stable. (C, bottom, left) Before and (right) 3 weeks after treatment
`with sorafenib.
`
`836
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`JOURNAL OF CLINICAL ONCOLOGY
`© 2010 by American Society of Clinical Oncology
`Information downloaded from jco.ascopubs.org and provided by at Univ of Chicago Library on February 25, 2016 from
`
`205.208.61.64Copyright © 2010 American Society of Clinical Oncology. All rights reserved.
`
`Ex. 1100-0002
`
`

`
`Sirolimus for Malignant PEComa
`
`RESULTS
`
`Patient outcomes with systemic therapy are summarized in narrative
`format in this Results section. Appendix Table A1 indicates the out-
`comes of systemic therapy for index patients as well.
`
`Patient 1
`Patient 1 is a 65-year-old man who underwent resection of a
`20-cm retroperitoneal PEComa in 2005, complicated by preoper-
`ative tumor rupture. Multifocal retroperitoneal recurrences were
`noted on surveillance imaging, and all sites of disease were resected
`in 2007. Additional sites of disease were noted on surveillance scans
`3 months later.
`He participated in a phase I study of an oral inhibitor of MET and
`developed rapid disease progression. In 2008, he began treatment with
`sirolimus 8 mg daily and achieved a serum trough level of 36 ng/mL;
`sirolimus has been well tolerated except for mild fatigue. Significant
`reduction in size of all tumors was noted on serial CT scans (Fig 1A),
`with near complete disappearance at 1 year. Both treatment and re-
`sponse are ongoing currently at 16 months of follow-up.
`
`Patient 2
`Patient 2 is a 70-year-old man who presented with hematuria
`in 2001. A radical nephrectomy was performed for removal of a
`9-cm renal mass, and the pathology was initially interpreted as a
`poorly differentiated sarcomatoid variant of clear cell carcinoma.
`Five years later, a local recurrence was detected and resected. One
`year later, the patient developed flank pain and again was noted to
`have developed a locoregional recurrence. Treatment with sunitinib
`
`was initiated but was stopped with evidence of disease progression
`after 6 weeks.
`In 2008, the patient presented to one of our institutions, and the
`pathologic diagnosis was reclassified as PEComa. Sirolimus 4 mg daily
`was initiated but was complicated by diarrhea and fatigue. The dose
`was reduced to 1 mg every other day, and improvement in the adverse
`effect profile and reduction in flank pain were noted. Restaging studies
`demonstrated reduction in size of the tumor (Fig 1B), with 40%
`reduction in the longest diameter and continued disease control for 10
`months before evidence of progression despite a sirolimus level of 9.4
`ng/mL. He subsequently had an additional surgical resection and
`remains alive with disease.
`
`Patient 3
`Patient 3 was a 61-year-old woman who presented with uter-
`ine bleeding in 2007. A total abdominal hysterectomy was per-
`formed, which identified a 9-cm PEComa with malignant features
`arising from the cervix. Staging studies demonstrated numerous
`bilateral pulmonary metastases. Sirolimus 4 mg orally each day was
`initiated. Restaging studies performed after 6 weeks of treatment
`revealed interval reduction in size and central cavitation of most of
`the pulmonary nodules (Fig 1C, top). Repeat evaluation at 3
`months demonstrated significant progression of disease; a serum
`trough sirolimus level was 5 ng/mL. The dose of sirolimus was
`increased to 8 mg daily, but restaging studies 1 month later showed
`additional progression of disease, and the serum trough sirolimus
`level was 7 ng/mL. The sirolimus dose was decreased to 2 mg daily,
`and clarithromycin 500 mg daily was added to inhibit CYP3A4, the
`major enzyme responsible for metabolism of sirolimus. The serum
`
`Retroperitoneal
`
`Renal
`
`Uterine
`
`A
`
`B
`
`C
`
`Fig 2. Perivascular epithelioid cell
`tumor (PEComa) histology and immuno-
`histochemical stains. Formalin-fixed paraffin-
`embedded sections stained with (A)
`hematoxylin and eosin, (B) antiphospho-S6
`antibody, and (C) anti-TSC2 antibody.
`
`www.jco.org
`
`© 2010 by American Society of Clinical Oncology
`Information downloaded from jco.ascopubs.org and provided by at Univ of Chicago Library on February 25, 2016 from
`
`205.208.61.64Copyright © 2010 American Society of Clinical Oncology. All rights reserved.
`
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`Wagner et al
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`3’
`
`
`
`E22 C5-chr4C4-chr2C3-chr11C2-chr17C1-chr22
`
`
`
`
`Control Probes
`
`5’
`
`E1
`
`E6
`
`E12
`
`E16
`
`E23
`
`E27
`
`E34
`
`E39
`
`TSC2
`
`1.0
`
`0.5
`
`0
`
`1.0
`
`0.5
`
`0
`
`1.0
`
`0.5
`
`0
`
`1.0
`
`0.5
`
`0
`
`A
`
`Assessment Value
`Genomic Copy No.
`
`B
`
`Assessment Value
`Genomic Copy No.
`
`C
`
`Assessment Value
`Genomic Copy No.
`
`D
`
`Assessment Value
`Genomic Copy No.
`
`E
`
`5’
`
`E1
`
`E3
`
`E8
`
`E9
`
`E14
`
`E19
`
`TSC1
`
`Fig 3. Mulitplex ligation-dependent probe amplification (MLPA) and fluorescent
`in situ hybridization (FISH) assays of TSC1 and TSC2 in perivascular epithelioid
`cell tumor (PEComa) samples. Genomic DNA from (A-C) tumor or (D) peripheral
`blood assessed for copy number by mulitplex ligation-dependent probe amplifi-
`cation (MLPA). Signals were normalized to values from control tissues. (E) FISH
`using (green) TSC1-specific DNA probes, (red) chromosome 16 centromeric
`probes, and nuclei isolated from uterine PEComa tissue.
`
`trough sirolimus level increased to 20 ng/mL, and CT scans 1
`month later demonstrated stabilization of the majority of lesions
`and interval reduction in the size of some nodules. Additional
`progression of disease was noted 1 month later, and treatment was
`empirically changed to sorafenib 200 mg twice daily and sirolimus
`4 mg daily. Significant cavitation of lung masses developed within
`2 weeks (Fig 1C, bottom) with improvement in symptoms and
`obviation of the need for supplemental oxygen, although bilateral
`pneumothoraces also developed. After 2 additional months of
`disease control, symptomatic worsening of pulmonary parenchy-
`mal disease ensued, and the patient died.
`
`Correlative Studies
`Hematoxylin and eosin–stained tumor sections showed sheets
`of epithelioid cells with granular eosinophilic cytoplasm, inti-
`mately associated with blood vessels, and with frequent mitoses,
`pleomorphism, and necrosis suggestive of malignant behavior (Fig
`2A). Immunohistochemical stains were positive for melanocytic
`markers HMB45 and Melan-A, variably positive for smooth mus-
`cle actin and desmin, and negative for cytokeratins and S100 (not
`shown). Specimens from all three patients demonstrated strong,
`diffuse, cytoplasmic staining for phosphorylated S6 protein (Fig
`2B) consistent with activation of mTORC1. Lesional cells showed
`loss of expression of tuberin, the gene product of TSC2, whereas
`expression generally was maintained in normal vessel wall vascular
`smooth muscle cells (Fig 2C). The specificity of the anti-TSC2
`antibody was confirmed in TSC2-null and -expressing cell lines
`(Appendix Fig A1).
`The integrity of the TSC1 and TSC2 genomic loci was examined
`by MLPA8 (Fig 3). Genomic DNA was extracted from paraffin-
`embedded tumor tissue (Figs 3A, 3B, and 3C) or peripheral blood (Fig
`3D) and was assessed for copy number changes by MLPA by using
`nine exonic probes for TSC1 and TSC2, respectively, and five control
`probes from other genomic locations.
`This technique allowed genomic copy number assessment; a
`normalized value of 1 indicated two intact alleles, and values of 0.5 and
`0 indicated a single allele and complete genomic loss, respectively.
`Marked reduction in signal from the TSC1 gene was observed in the
`uterine tumor specimen from patient 3 (Figs 3C to D), consistent with
`somatic biallelic deletion of TSC1 (Fig 3A). MLPA demonstrated no
`alterations in tumor samples from the retroperitoneal PEComa (pa-
`tient 1; Fig 3A) or the renal PEComa (patient 2; Fig 3B), which suggests
`that mechanisms other than gross alteration of the TSC1 or TSC2
`genomic loci were important in loss of TSC2 expression in those
`two tumors.
`FISH, performed by using interphase nuclei isolated from
`archival paraffin-embedded tissue and probes corresponding to
`TSC1 and TSC2, confirmed homozygous loss of TSC1 (Fig 3E, green
`signal) in greater than 60% of nuclei from the uterine PEComa sam-
`ple, whereas the centromeric region of chromosome 9 (Fig 3E, red
`signal) was maintained.
`Karyotype analysis of the retroperitoneal PEComa (patient 2),
`previously performed for clinical purposes, showed two copies of
`chromosome 16 as well as an add([16p]; not shown). The add(16p13)
`involved chromosome breakage within band p13, loss of material
`distal to the break, and addition of unidentified chromosomal mate-
`rial in its place. Because TSC2 maps to 16p13, interphase FISH was
`
`838
`
`JOURNAL OF CLINICAL ONCOLOGY
`© 2010 by American Society of Clinical Oncology
`Information downloaded from jco.ascopubs.org and provided by at Univ of Chicago Library on February 25, 2016 from
`
`205.208.61.64Copyright © 2010 American Society of Clinical Oncology. All rights reserved.
`
`Ex. 1100-0004
`
`

`
`Sirolimus for Malignant PEComa
`
`performed to determine the TSC2 copy number. Three copies of TSC2
`were detected (not shown), which suggests that the breakpoint of the
`add(16) maps distal to the TSC2 probe. The possibility of small dele-
`tions of or point mutations in TSC2, which are undetectable by FISH,
`cannot be excluded. No other evidence of deletion or rearrangement
`of either TSC1 or TSC2 was observed.
`PEComa DNA samples were sequenced to search for known
`activating mutations in the PIK3CA and AKT1 genes as well as for the
`common mutations that occur in KRAS. No mutations were detected
`in any of the three PEComas in any of these three genes.
`
`DISCUSSION
`
`Metastatic PEComa is a rare form of sarcoma for which no effective
`therapy has been described previously and which has a uniformly fatal
`outcome. We have observed significant clinical responses in three
`patients treated with sirolimus, an inhibitor of mTORC1, including
`one patient with an ongoing near complete response of greater than 14
`months duration. Tumors from all three patients showed evidence of
`mTORC1 activation, and somatic deletion of TSC1 was identified as
`the likely mechanism in patient 3. TSC1 acts to stabilize TSC2, so its
`genomic loss will lead to lack of TSC2 expression.10 The mechanisms
`of mTOR activation in patients 1 and 2 are unclear, but they poten-
`tially include small deletions or inactivating or missense mutations in
`TSC1 or TSC2 that would similarly account for loss of TSC2 expres-
`sion in those tumors.
`Other evidence to support activation of the mTOR pathway in
`PEComas also has recently been described. Kenerson et al7 reported
`immunohistochemical evidence of mTORC1 activity in 15 PEComas
`and absence of AKT phosphorylation in 14 tumors, which suggests the
`loss of TSC1 or TSC2 as potential mechanisms. Similarly, Pan et al6
`described elevated phospho-p70S6K and reduced phospho-AKT in 11
`of 12 PEComas.6 Seven of these tumors had loss of heterozygosity of
`the TSC2 region, and one additionally showed loss of heterozygosity
`of TSC1.
`Our data are consistent with findings to date of the activity of
`mTOR inhibitors in tumors known to be biologically related to
`PEComas, specifically AML and LAM. After case reports of pa-
`tients with renal AML and LAM responding to sirolimus were
`published,11-13 Bissler et al5 reported on treatment of 25 patients with
`AML or LAM with sirolimus for 12 months followed by 12 months of
`observation.5 After 12 months of therapy, AML volume decreased
`53% but returned to 86% of baseline after the year of observation,
`which indicated the need for continued inhibition to maintain
`tumor shrinkage. Less-impressive improvements in respiratory
`function were observed in patients with LAM, which also reversed
`on observation alone. Interestingly, facial angiofibromas associ-
`ated with tuberous sclerosis also have significantly improved on
`sirolimus in a case report.14 There are presently no reports of
`mTOR inhibitors as treatment of clear cell “sugar” tumors of the
`pancreas or lung, another neoplasm considered to be a PEComa,
`but it is expected that such tumors would respond to mTOR-
`directed therapy as well.
`The efficacy of mTOR inhibitors has been explored in patients
`with a heterogenous mix of other metastatic sarcomas, in each case
`with only a modest response rate.15,16 However, the status of
`mTOR activation of these sarcomas is unknown, although in one
`
`study the presence of S6 phosphorylation correlated with a higher
`likelihood of disease control with an mTOR inhibitor.17 Taken to-
`gether, these observations suggest that activation of mTOR through
`loss of the TSC1/TSC2 repressor complex, or potentially by other
`means, is likely a common and critically pathogenic event in PEComas.
`Inhibition of mTOR has resulted in significant clinical activity
`in patients with PEComa and merits additional investigation in a
`prospective study. Absence of immunohistochemical evidence of
`TSC2 expression or the less-specific presence of S6 phosphoryla-
`tion may be predictive markers for responsiveness to inhibitors
`of mTORC1. These findings additionally unify the concept of
`PEComa, AML, and LAM as closely related pathologic entities,
`from histology to genetic changes to evidence of therapeutic ben-
`efit from mTOR blockade.
`
`AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS
`OF INTEREST
`
`Although all authors completed the disclosure declaration, the following
`author(s) indicated a financial or other interest that is relevant to the subject
`matter under consideration in this article. Certain relationships marked
`with a “U” are those for which no compensation was received; those
`relationships marked with a “C” were compensated. For a detailed
`description of the disclosure categories, or for more information about
`ASCO’s conflict of interest policy, please refer to the Author Disclosure
`Declaration and the Disclosures of Potential Conflicts of Interest section in
`Information for Contributors.
`Employment or Leadership Position: None Consultant or Advisory
`Role: Andrew J. Wagner, Genentech (C); George D. Demetri, Ariad (C),
`Novartis (C), Pfizer (C), Genentech (C); Robert G. Maki, Novartis (C)
`Stock Ownership: None Honoraria: Robert G. Maki, Novartis Research
`Funding: Andrew J. Wagner, Genentech; Robert G. Maki, Novartis,
`Pfizer Expert Testimony: George D. Demetri, Ariad (U) Other
`Remuneration: None
`
`AUTHOR CONTRIBUTIONS
`
`Conception and design: Andrew J. Wagner, Jeffrey A. Morgan,
`Christopher D.M. Fletcher, Azra H. Ligon, George D. Demetri, David J.
`Kwiatkowski, Robert G. Maki
`Financial support: Andrew J. Wagner, George D. Demetri
`Administrative support: Andrew J. Wagner, George D. Demetri
`Provision of study materials or patients: Andrew J. Wagner, Jeffrey A.
`Morgan, Christopher D.M. Fletcher, Cristina R. Antonescu,
`Robert G. Maki
`Collection and assembly of data: Andrew J. Wagner, Izabela
`Malinowska-Kolodziej, Jeffrey A. Morgan, Wei Qin, Christopher D.M.
`Fletcher, Natalie Vena, Azra H. Ligon, Cristina R. Antonescu, Nikhil H.
`Ramaiya, David J. Kwiatkowski, Robert G. Maki
`Data analysis and interpretation: Andrew J. Wagner, Izabela
`Malinowska-Kolodziej, Jeffrey A. Morgan, Christopher D.M. Fletcher,
`Natalie Vena, Azra H. Ligon, Nikhil H. Ramaiya, David J. Kwiatkowski,
`Robert G. Maki
`Manuscript writing: Andrew J. Wagner, Izabela Malinowska-Kolodziej,
`Christopher D.M. Fletcher, Azra H. Ligon, George D. Demetri, David J.
`Kwiatkowski, Robert G. Maki
`Final approval of manuscript: Andrew J. Wagner, Izabela
`Malinowska-Kolodziej, Jeffrey A. Morgan, Wei Qin, Christopher D.M.
`Fletcher, Natalie Vena, Azra H. Ligon, Cristina R. Antonescu, Nikhil H.
`Ramaiya, George D. Demetri, David J. Kwiatkowski, Robert G. Maki
`
`www.jco.org
`
`© 2010 by American Society of Clinical Oncology
`Information downloaded from jco.ascopubs.org and provided by at Univ of Chicago Library on February 25, 2016 from
`
`205.208.61.64Copyright © 2010 American Society of Clinical Oncology. All rights reserved.
`
`839
`
`Ex. 1100-0005
`
`

`
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