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Durable Response to Afatinib in Lung Adenocarcinoma Harboring NRG1 Gene Fusions
Accepted Manuscript Durable Response to Afatinib in Lung Adenocarcinoma Harboring NRG1 Gene Fusions Nathan D. Gay, M.D., Ying Wang, M.D., Carol Beadling, Ph.D., Andrea Warrick., Tanaya Neff., Christopher L. Corless, M.D., Ph.D., Khaled Tolba, M.D. PII:
S1556-0864(17)30359-3
DOI:
10.1016/j.jtho.2017.04.025
Reference:
JTHO 581
To appear in:
Journal of Thoracic Oncology
Received Date: 25 February 2017 Revised Date:
18 April 2017
Accepted Date: 27 April 2017
Please cite this article as: Gay ND, Wang Y, Beadling C, Warrick. A, Neff. T, Corless CL, Tolba K, Durable Response to Afatinib in Lung Adenocarcinoma Harboring NRG1 Gene Fusions, Journal of Thoracic Oncology (2017), doi: 10.1016/j.jtho.2017.04.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Durable Response to Afatinib in
Nathan D. Gay, M.D.1
SC
Ying Wang, M.D.2
RI PT
Lung Adenocarcinoma Harboring NRG1 Gene Fusions
Carol Beadling, Ph.D. 2
M AN U
Andrea Warrick. 2 Tanaya Neff. 2
Christopher L. Corless, M.D., Ph.D. 2
TE D
Khaled Tolba, M.D. 1, 3
EP
Disclosures: Dr. Tolba has received honoraria as an advisory board member for Genentech Roche and Boehringer Ingelheim.
Knight Cancer Institute, Division of Hematology-Oncology.
2
Department of Pathology
AC C
1
3181 SW Sam Jackson Park Road Oregon Health & Science University Portland, Oregon
3
97239
Corresponding Author. 1
ACCEPTED MANUSCRIPT
Introduction
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Patients with invasive mucinous adenocarcinoma harbor gene fusions involving neuregulin-1 (NRG1), resulting in activation of ErB3/ErB4 (members of the ERBB family of receptor tyrosine kinases)[1]. In this report, we describe two patients with stage IV lung adenocarcinoma harboring two different NRG1 gene fusions that had a durable clinical response to the
SC
irreversible pan-ErbB inhibitor afatinib. To our knowledge these are the first examples of
M AN U
response to targeted treatment in NRG1 fusion-positive NSCLC.
Methods
404 NSCLC cases were screened for principal driver mutations with a custom next-generation sequencing (NGS) panel covering 124 cancer genes using the AmpliSeq library technology
TE D
(ThermoFisher). Sequencing was performed on an Illumina NextSeq500 and variant calling was done with pipeline based on GATK (licensed from Broad Institute, Massachusetts Institute of Technology). Sixty cases that were negative for such mutations were secondarily screened for
EP
gene fusions using a custom RNA sequencing protocol. Among the 60 cases, 2 were found to
AC C
harbor NRG1 fusions, as confirmed by bidirectional Sanger sequencing. The overall frequency of NRG1 fusions was 0.5% (2/404 cases). Case 1
A 42-year-old lifelong non-smoking Caucasian male presented with metastatic adenocarcinoma, positive for CK7 and TTF1 with involvement of the liver and left temporal bone (Figure 1A). Initial mutational analysis was negative for alterations in EGFR, ALK, ROS-1 and KRAS. He was treated with 4 cycles of cisplatin/pemetrexed followed by maintenance pemetrexed until the
2
ACCEPTED MANUSCRIPT
time of progression. NGS revealed a TP53 mutation and decrease in TP53 copy number. RNA from the same biopsy was screened for gene fusions and revealed an SLC3A2-NRG1 fusion[2]. Patient started on afatinib 40 mg once daily. Within 2-3 days of start of treatment, cough and
RI PT
shortness of breath resolved and follow-up chest CT scan documented objective response (Figure 2). An MRI of the brain showed decrease in the frontotemporal osseous metastasis (Figure 3). Twelve months after initiation of afatinib, he progressed and biopsy was positive for the
SC
SLC3A2-NRG1 gene fusion and reduced copy number for the tumor suppressor CDKN2A. Patient continued with afatinib alone for 6 months before enrolling on a clinical trial with
M AN U
MEK1/2 inhinitor.
Case 2
A 62-year-old non-smoking, HIV-positive Caucasian male presented with persistent cough.
TE D
Diagnostic work-up revealed an early stage lung cancer for which he underwent right lower lobectomy with mediastinal lymph node dissection. Final pathology revealed mucinous adenocarcinoma diffusely positive for CDX2 and CK7, but negative for napsin, TTF1, and p63.
EP
A few months later, he was found to have a left adrenal metastasis. NGS from the resected lung mass showed a TP53 exon 9 splice site mutation with associated gene copy loss, as well as MYC
AC C
gene amplification but no detectable alterations within the KRAS or the ERBB family. RNA sequencing revealed a CD74-NRG1 fusion[3]. The patient was started on afatinib 40 mg once daily and achieved a durable response for 10 months, when a follow-up CT showed recurrent disease in the lung, liver and skull base. A liver biopsy confirmed metastatic pulmonary adenocarcinoma that continued to express the CD74-NRG1 fusion gene and showed the same TP53 and MYC alterations. There was no evidence for mutation or amplification of ERBB2, ERBB3 or other commonly targeted receptor tyrosine kinases. Patient continued treatment with 3
ACCEPTED MANUSCRIPT
afatinib due to lack of a suitable alternative, while the bone metastases where managed by palliative radiation and a bisphosphonate. Discussion
RI PT
The human genome encodes four neuregulin genes (NRG1-4), and through alternative splicing of their mRNA, at least 20 different neuregulin isoforms are expressed. Most of these isoforms start as transmembrane molecules whereby a biologically active EGF-like module is tethered to a
SC
trans-membrane domain[4]. The membrane-tethered EGF-like domain of NRG1 protein
mediates autocrine and juxtacrine signaling through HER2:HER3 and HER3:HER4 and forms
M AN U
the basis for therapeutic targeting of the ErbB family[5], Figure 5.
In this report, we describe two patients with NRG-1 chromosomal translocations using different fusion partners. Both patients were of Caucasian ethnicity, non-smokers who developed exuberant bone metastases within the skull. Only the second patient had detectable mucinous
TE D
adenocarcinoma.
Afatinib is an oral, irreversible ErbB-family inhibitor that selectively blocks signaling from EGFR, HER2 and HER4. Given the lack of specific inhibitors for ErbB3 in the clinic, we elected
EP
to use afatinib for its ability to irreversibly inhibit signaling from the remaining three ErbB family members that serve as dimerization partners for activated HER3. We documented durable
AC C
responses of 10 and 12 months to afatinib, comparable to first line afatinib response in EGFRmutant NSCLC. Neither of our two patients possessed an activating mutation or copy number alteration in an ErbB receptor prior to or after afatinib treatment, raising the possibility of an epigenetic event as an escape mechanism. In this context, it is conceivable that ErbB resistance to afatinib might proceed along the epigenetic MLL2/FOXO/c-Myc pathway recently reported in HER2+ breast cancer cells with acquired resistance to lapatinib[6]. In summary, our experience
4
ACCEPTED MANUSCRIPT
suggests that patients with NRG1 fusion-positive lung adenocarcinoma may benefit from
AC C
EP
TE D
M AN U
SC
RI PT
treatment with afatinib and possibly other ErbB-targeted therapies.
5
ACCEPTED MANUSCRIPT
References
AC C
EP
TE D
M AN U
SC
RI PT
1. Riely GJ, Yu HA. EGFR: The Paradigm of an Oncogene-Driven Lung Cancer. Clin Cancer Res 2015; 21: 2221-2226. 2. Shin DH, Lee D, Hong DW et al. Oncogenic function and clinical implications of SLC3A2NRG1 fusion in invasive mucinous adenocarcinoma of the lung. Oncotarget 2016. 3. Fernandez-Cuesta L, Plenker D, Osada H et al. CD74-NRG1 fusions in lung adenocarcinoma. Cancer Discov 2014; 4: 415-422. 4. Montero JC, Rodriguez-Barrueco R, Ocana A et al. Neuregulins and cancer. Clin Cancer Res 2008; 14: 3237-3241. 5. Wilson TR, Lee DY, Berry L et al. Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers. Cancer Cell 2011; 20: 158172. 6. Matkar S, Sharma P, Gao S et al. An Epigenetic Pathway Regulates Sensitivity of Breast Cancer Cells to HER2 Inhibition via FOXO/c-Myc Axis. Cancer Cell 2015; 28: 472-485.
6
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Figure Legends:
Figure 1 A & B: Hematoxylin & eosin stained sections (one from each case) showing invasive
RI PT
adenocarcinoma. Focal mucin production is evident in B (lower right).
Figure 2: Contrast enhanced CT scans of chest in Case #1 showing significant decrease in large
SC
speculated right lower lobe mass and persistent response at 8 months after initiation of afatinib.
Figure 3: Contrast enhanced T1 weighted MRI of brain in Case #1 showing decrease in
M AN U
frontotemporal osseous metastasis.
Figure 4: CT scan of the left adrenal metastases before (A) and after (B) afatinib treatment showing stable disease, in contrast to new liver metastases appearing at the time of disease progression, before (C) and after (D).
TE D
Figure 5: Graphical depiction of DNA splice site and protein fusion products SLC3A2-NRG1 (A) and CD74-NRG1 (B) along with depiction of how they drive ErbB mediated cellular
AC C
EP
proliferation through overactive autocrine and paracrine signaling through EGF like domain.
7
RI PT
ACCEPTED MANUSCRIPT
Figure 1. A and B:
EP AC C
B
TE D
M AN U
SC
A
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ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
Figure 3
Pre-Afatinib
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ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
Figure 4
Prior to Afatinib
Eight Months on
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
B
C
AC C
EP
TE D
A
D
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S1556-0864(17)30359-3
DOI:
10.1016/j.jtho.2017.04.025
Reference:
JTHO 581
To appear in:
Journal of Thoracic Oncology
Received Date: 25 February 2017 Revised Date:
18 April 2017
Accepted Date: 27 April 2017
Please cite this article as: Gay ND, Wang Y, Beadling C, Warrick. A, Neff. T, Corless CL, Tolba K, Durable Response to Afatinib in Lung Adenocarcinoma Harboring NRG1 Gene Fusions, Journal of Thoracic Oncology (2017), doi: 10.1016/j.jtho.2017.04.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Durable Response to Afatinib in
Nathan D. Gay, M.D.1
SC
Ying Wang, M.D.2
RI PT
Lung Adenocarcinoma Harboring NRG1 Gene Fusions
Carol Beadling, Ph.D. 2
M AN U
Andrea Warrick. 2 Tanaya Neff. 2
Christopher L. Corless, M.D., Ph.D. 2
TE D
Khaled Tolba, M.D. 1, 3
EP
Disclosures: Dr. Tolba has received honoraria as an advisory board member for Genentech Roche and Boehringer Ingelheim.
Knight Cancer Institute, Division of Hematology-Oncology.
2
Department of Pathology
AC C
1
3181 SW Sam Jackson Park Road Oregon Health & Science University Portland, Oregon
3
97239
Corresponding Author. 1
ACCEPTED MANUSCRIPT
Introduction
RI PT
Patients with invasive mucinous adenocarcinoma harbor gene fusions involving neuregulin-1 (NRG1), resulting in activation of ErB3/ErB4 (members of the ERBB family of receptor tyrosine kinases)[1]. In this report, we describe two patients with stage IV lung adenocarcinoma harboring two different NRG1 gene fusions that had a durable clinical response to the
SC
irreversible pan-ErbB inhibitor afatinib. To our knowledge these are the first examples of
M AN U
response to targeted treatment in NRG1 fusion-positive NSCLC.
Methods
404 NSCLC cases were screened for principal driver mutations with a custom next-generation sequencing (NGS) panel covering 124 cancer genes using the AmpliSeq library technology
TE D
(ThermoFisher). Sequencing was performed on an Illumina NextSeq500 and variant calling was done with pipeline based on GATK (licensed from Broad Institute, Massachusetts Institute of Technology). Sixty cases that were negative for such mutations were secondarily screened for
EP
gene fusions using a custom RNA sequencing protocol. Among the 60 cases, 2 were found to
AC C
harbor NRG1 fusions, as confirmed by bidirectional Sanger sequencing. The overall frequency of NRG1 fusions was 0.5% (2/404 cases). Case 1
A 42-year-old lifelong non-smoking Caucasian male presented with metastatic adenocarcinoma, positive for CK7 and TTF1 with involvement of the liver and left temporal bone (Figure 1A). Initial mutational analysis was negative for alterations in EGFR, ALK, ROS-1 and KRAS. He was treated with 4 cycles of cisplatin/pemetrexed followed by maintenance pemetrexed until the
2
ACCEPTED MANUSCRIPT
time of progression. NGS revealed a TP53 mutation and decrease in TP53 copy number. RNA from the same biopsy was screened for gene fusions and revealed an SLC3A2-NRG1 fusion[2]. Patient started on afatinib 40 mg once daily. Within 2-3 days of start of treatment, cough and
RI PT
shortness of breath resolved and follow-up chest CT scan documented objective response (Figure 2). An MRI of the brain showed decrease in the frontotemporal osseous metastasis (Figure 3). Twelve months after initiation of afatinib, he progressed and biopsy was positive for the
SC
SLC3A2-NRG1 gene fusion and reduced copy number for the tumor suppressor CDKN2A. Patient continued with afatinib alone for 6 months before enrolling on a clinical trial with
M AN U
MEK1/2 inhinitor.
Case 2
A 62-year-old non-smoking, HIV-positive Caucasian male presented with persistent cough.
TE D
Diagnostic work-up revealed an early stage lung cancer for which he underwent right lower lobectomy with mediastinal lymph node dissection. Final pathology revealed mucinous adenocarcinoma diffusely positive for CDX2 and CK7, but negative for napsin, TTF1, and p63.
EP
A few months later, he was found to have a left adrenal metastasis. NGS from the resected lung mass showed a TP53 exon 9 splice site mutation with associated gene copy loss, as well as MYC
AC C
gene amplification but no detectable alterations within the KRAS or the ERBB family. RNA sequencing revealed a CD74-NRG1 fusion[3]. The patient was started on afatinib 40 mg once daily and achieved a durable response for 10 months, when a follow-up CT showed recurrent disease in the lung, liver and skull base. A liver biopsy confirmed metastatic pulmonary adenocarcinoma that continued to express the CD74-NRG1 fusion gene and showed the same TP53 and MYC alterations. There was no evidence for mutation or amplification of ERBB2, ERBB3 or other commonly targeted receptor tyrosine kinases. Patient continued treatment with 3
ACCEPTED MANUSCRIPT
afatinib due to lack of a suitable alternative, while the bone metastases where managed by palliative radiation and a bisphosphonate. Discussion
RI PT
The human genome encodes four neuregulin genes (NRG1-4), and through alternative splicing of their mRNA, at least 20 different neuregulin isoforms are expressed. Most of these isoforms start as transmembrane molecules whereby a biologically active EGF-like module is tethered to a
SC
trans-membrane domain[4]. The membrane-tethered EGF-like domain of NRG1 protein
mediates autocrine and juxtacrine signaling through HER2:HER3 and HER3:HER4 and forms
M AN U
the basis for therapeutic targeting of the ErbB family[5], Figure 5.
In this report, we describe two patients with NRG-1 chromosomal translocations using different fusion partners. Both patients were of Caucasian ethnicity, non-smokers who developed exuberant bone metastases within the skull. Only the second patient had detectable mucinous
TE D
adenocarcinoma.
Afatinib is an oral, irreversible ErbB-family inhibitor that selectively blocks signaling from EGFR, HER2 and HER4. Given the lack of specific inhibitors for ErbB3 in the clinic, we elected
EP
to use afatinib for its ability to irreversibly inhibit signaling from the remaining three ErbB family members that serve as dimerization partners for activated HER3. We documented durable
AC C
responses of 10 and 12 months to afatinib, comparable to first line afatinib response in EGFRmutant NSCLC. Neither of our two patients possessed an activating mutation or copy number alteration in an ErbB receptor prior to or after afatinib treatment, raising the possibility of an epigenetic event as an escape mechanism. In this context, it is conceivable that ErbB resistance to afatinib might proceed along the epigenetic MLL2/FOXO/c-Myc pathway recently reported in HER2+ breast cancer cells with acquired resistance to lapatinib[6]. In summary, our experience
4
ACCEPTED MANUSCRIPT
suggests that patients with NRG1 fusion-positive lung adenocarcinoma may benefit from
AC C
EP
TE D
M AN U
SC
RI PT
treatment with afatinib and possibly other ErbB-targeted therapies.
5
ACCEPTED MANUSCRIPT
References
AC C
EP
TE D
M AN U
SC
RI PT
1. Riely GJ, Yu HA. EGFR: The Paradigm of an Oncogene-Driven Lung Cancer. Clin Cancer Res 2015; 21: 2221-2226. 2. Shin DH, Lee D, Hong DW et al. Oncogenic function and clinical implications of SLC3A2NRG1 fusion in invasive mucinous adenocarcinoma of the lung. Oncotarget 2016. 3. Fernandez-Cuesta L, Plenker D, Osada H et al. CD74-NRG1 fusions in lung adenocarcinoma. Cancer Discov 2014; 4: 415-422. 4. Montero JC, Rodriguez-Barrueco R, Ocana A et al. Neuregulins and cancer. Clin Cancer Res 2008; 14: 3237-3241. 5. Wilson TR, Lee DY, Berry L et al. Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers. Cancer Cell 2011; 20: 158172. 6. Matkar S, Sharma P, Gao S et al. An Epigenetic Pathway Regulates Sensitivity of Breast Cancer Cells to HER2 Inhibition via FOXO/c-Myc Axis. Cancer Cell 2015; 28: 472-485.
6
ACCEPTED MANUSCRIPT
Figure Legends:
Figure 1 A & B: Hematoxylin & eosin stained sections (one from each case) showing invasive
RI PT
adenocarcinoma. Focal mucin production is evident in B (lower right).
Figure 2: Contrast enhanced CT scans of chest in Case #1 showing significant decrease in large
SC
speculated right lower lobe mass and persistent response at 8 months after initiation of afatinib.
Figure 3: Contrast enhanced T1 weighted MRI of brain in Case #1 showing decrease in
M AN U
frontotemporal osseous metastasis.
Figure 4: CT scan of the left adrenal metastases before (A) and after (B) afatinib treatment showing stable disease, in contrast to new liver metastases appearing at the time of disease progression, before (C) and after (D).
TE D
Figure 5: Graphical depiction of DNA splice site and protein fusion products SLC3A2-NRG1 (A) and CD74-NRG1 (B) along with depiction of how they drive ErbB mediated cellular
AC C
EP
proliferation through overactive autocrine and paracrine signaling through EGF like domain.
7
RI PT
ACCEPTED MANUSCRIPT
Figure 1. A and B:
EP AC C
B
TE D
M AN U
SC
A
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
Figure 3
Pre-Afatinib
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
Figure 4
Prior to Afatinib
Eight Months on
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
B
C
AC C
EP
TE D
A
D
AC C
EP
TE D
M AN U
SC
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ACCEPTED MANUSCRIPT