- Email: [email protected]
Complete Response to Immunotherapy Plus Chemotherapy After an Unusual Clinical Response to Afatinib and Stereotactic Radiosurgery in a Patient With Metastatic EGFR-Mutant Non–Small-Cell Lung Cancer
Journal Pre-proof Case Report: Complete response to immunotherapy plus chemotherapy following an unusual clinical response to Afatinib and stereotactic radiosurgery in a metastatic EGFR-mutant NSCLC patient Gonzalo Pizarro, Mauricio P. Pinto, Matías Muñoz Medel, Miguel Cordova-Delgado, M. Loreto Bravo, Bruno Nervi, César Sánchez, Carolina Ibañez, José Peña, Benjamín Walbaum, Jorge Madrid, Juan Briones, Erica Koch, Jose Valbuena, Sergio Gonzalez, Roger Gejman, Francisco Acevedo, Sebastian Mondaca, Marcelo Garrido, Eugenio Vines, Hector Galindo PII:
S1525-7304(20)30012-7
DOI:
https://doi.org/10.1016/j.cllc.2020.01.012
Reference:
CLLC 1077
To appear in:
Clinical Lung Cancer
Received Date: 9 August 2019 Revised Date:
27 December 2019
Accepted Date: 20 January 2020
Please cite this article as: Pizarro G, Pinto MP, Medel MM, Cordova-Delgado M, Bravo ML, Nervi B, Sánchez C, Ibañez C, Peña J, Walbaum B, Madrid J, Briones J, Koch E, Valbuena J, Gonzalez S, Gejman R, Acevedo F, Mondaca S, Garrido M, Vines E, Galindo H, Case Report: Complete response to immunotherapy plus chemotherapy following an unusual clinical response to Afatinib and stereotactic radiosurgery in a metastatic EGFR-mutant NSCLC patient, Clinical Lung Cancer (2020), doi: https:// doi.org/10.1016/j.cllc.2020.01.012. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Elsevier Inc. All rights reserved.
Case Report Case Report: Complete response to immunotherapy plus chemotherapy following an unusual clinical response to Afatinib and stereotactic radiosurgery in a metastatic EGFR-mutant NSCLC patient Running title: Unusual complete response to immunotherapy
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Authors: Gonzalo Pizarro *, Mauricio P. Pinto *, Matías Muñoz Medel , Miguel Cordova-Delgado , M. 1 1 1 1 1 1 Loreto Bravo , Bruno Nervi , César Sánchez , Carolina Ibañez , José Peña , Benjamín Walbaum , 1 1 1 2 2 2 Jorge Madrid , Juan Briones , Erica Koch , Jose Valbuena , Sergio Gonzalez , Roger Gejman , 1 1 1 1 1 Francisco Acevedo , Sebastian Mondaca , Marcelo Garrido , Eugenio Vines , Hector Galindo . Affiliations: 1 Departamento de Hematología y Oncología. Facultad de Medicina. Pontificia Universidad Católica de Chile 2 Departamento de Anatomía Patológica. Facultad de Medicina. Pontificia Universidad Católica de Chile
Correspondence: Hector Galindo, MD Departamento de Hematología y Oncología Facultad de Medicina Pontificia Universidad Católica de Chile Diagonal Paraguay 362, 6 piso, Postal code: 8330077 Phone: +56 (2) 23549034 [email protected] * These authors contributed equally to this work
List of abbreviations: CPI: Checkpoint inhibitor; LC: Lung Cancer; MRI: Magnetic Resonance Imaging; NGS: Next Generation Sequencing; PCR: Polymerase Chain Reaction; PDL1: Programmed Death-Ligand 1; PET/CT: Positron Emission Tomography/Computed Tomography; TMB: Tumor Mutational Burden; TKI: Tyrosine Kinase Inhibitor; SBRT: Stereotactic Body Radiation Therapy; SCNA: Somatic Copy Number Alteration
Conflicts of interest None.
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Abstract Background: Checkpoint inhibitors (CPIs) such as Pembrolizumab have recently become the standard treatment for Non-Small Cell Lung Cancers (NSCLC) in patients without Epidermal Growth Factor (EGFR) gene alterations. Previous reports suggest no significant benefit from CPI treatments among EGFR mutant NSCLC patients and recommend the use of Tyrosine Kinase Inhibitors such as Afatinib. Case presentation: Herein, we report a rare case of a 39-year old, light-smoker woman with a metastatic EGFR mutant NSCLC (exon-19 deletion) that exhibited rapid progression and an unusual clinical response to Afatinib without changes in tumor histology. Tumor was negative for PDL1. Biopsy analyses by NGS prior and after Afatinib treatment indicated an increase in focal somatic copy number alterations (fSCNAs) but no T790M or c-MET amplification, and a low Tumor Mutational Burden (TMB). Given her rapid progression and deterioration, and despite its PDL1- status and her low TMB patient started treatment with Pembrolizumab plus chemotherapy. Surprisingly, patient displayed a complete response after just 4 cycles and is soon to complete a year in remission. Conclusions: We speculate intratumoral heterogeneity, the combination of radiotherapy plus immunotherapy or the concomitant use of chemotherapy plus immunotherapy could explain the observed response in this patient. Also, based in our data we suggest the SBRT/CPI combination could be an option for Tyrosine Kinase Inhibitor-refractory EGFR mutant NSCLC patients or as a second-line treatment. Keywords: checkpoint inhibitors, immunotherapy, lung cancer, radiation therapy, complete response, unusual clinical response, somatic copy number alterations.
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Introduction The development of checkpoint inhibitors (CPIs) has revolutionized the field of oncology. 1 2 3 4 5 Reports in melanoma , kidney , prostate , gastric and lung cancers have demonstrated its efficacy alone or in combination with chemotherapy. Despite this, a substantial portion of patients remains 5–7 unresponsive or display modest responses . In this context, high throughput sequencing such as Next Generation Sequencing (NGS) could help to elucidate molecular profiles in order to identify potential CPI-responders. Here, we describe a rare case of a metastatic Non-Small Cell Lung Cancer (NSCLC) patient diagnosed with exon-19 EGFR deletion that displayed an “unusual clinical response” to Afatinib, a Tyrosine Kinase Inhibitor (TKI). Subsequently, patient was switched to the Pemetrexed/Carboplatin plus Pembrolizumab combination and SBRT, demonstrating a complete response after 4 cycles. As pointed, a substantial portion of patients that receive immunotherapy remain unresponsive 8 or display modest responses, especially in EGFR mutant NSCLC patients . Biopsy samples were taken prior and after TKI and were analyzed by NGS indicating the acquisition of a number of new somatic alterations. These data could help to define a predictive biomarker for immunotherapy response in EGFR mutant NSCLC. We also discuss the utility of chemo or radiotherapy prior to immunotherapy in patients that fail TKIs. Case report 9
Figure 1 shows a timeline of the case, 39-year-old, light-smoker (5 packs-a-year) woman came to the clinic due to back pain. A spine MRI showed tumor lesions at L2 and L5 and a bone fracture at L2. A CT Scan revealed a lung tumor in the upper left lobe and several adenopathies, liver and adrenal metastases. Brain MRI found a 10-mm left frontal subcortical lesion and local edema (Fig. 1). Exon-19 deletion on EGFR was confirmed by liquid biopsy. Pathology indicated a Non-Small Cell lung adenocarcinoma. Further analyses confirmed exon-19 deletion and absence of PDL1. A PET/CT Scan confirmed a 65 mm lung mass associated to satellite nodes and mediastinal, hilar and cervical adenopathies, also liver, right adrenal, frontal left brain and bone metastases. Following diagnosis, the patient started oral Afatinib (TKI) and SBRT to control a left frontal brain metastasis. Six weeks after the patient returned to the clinic referring pain in the upper left arm, edema and an increase in cervical volume. Echography showed deep venous thrombosis in the left internal jugular vein and a second scan confirmed an increase in cervical and left supraclavicular lymphadenopathies along with novel secondary lymphadenopathies. At the same time, liver 10 metastasis and the lung lesion were notoriously reduced suggesting a paradoxical response . Three weeks later the patient manifested abdominal pain, anorexia and asthenia. MRI showed progression of liver metastasis and increase in secondary lymphadenopathies. Given the rapid disease progression despite the use of TKI therapy a second cervical biopsy was taken from the left lymphadenectomy that confirmed a NSCL adenocarcinoma. A NGS study compared cervical biopsies taken at diagnosis and after progression (March/May 2018). Results are summarized in Figure 2. Both biopsies demonstrated EGFR amplification, exon-19 deletion and low TMB. However, second biopsy also indicated novel alterations, including KIT, FGFR1, PDGFRA, KDR amplification and notably no T790M (Fig. 2C). Following progression, the patient started Pembrolizumab plus chemotherapy. Also, SBRT was applied for supraclavicular metastasis (40 Gy, 5 fractions) and spinal lesion (8 Gy 1 fraction, palliative). After 4 cycles, scan showed resolution on adenopathies, and decrease in lung mass, liver and spinal lesions. Brain MRI showed no lesions or novel metastases. Liquid biopsy for EGFR mutations was negative. Patient continues Pembrolizumab plus pemetrexed and is soon to complete a year in remission.
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Discussion Numerous studies have demonstrated the effectiveness of TKIs against NSCLCs that display 11 activating EGFR mutations . In general, TKIs in these patients has substantial benefits, an initial 70% 11 response rate and 1-3 year overall survival (OS) . Despite this, a portion of EGFR mutant TKI-failed 10 12 NSCLC patients acquire novel alterations including the T790M or c-MET amplification . Surprisingly, this was not the case. Instead, TKI-failure correlated with emergence of several alterations. 10 Initially, this case suggested a paradoxical response , including partial response on liver/lung lesions, however clinical and radiological evidence clearly indicated rapid disease progression following Afatinib. Therefore, we opted for the term “unusual clinical response” to describe this rare 13 case. Afatinib resistance in EGFR mutant NSCLCs is rare (<10% of cases) . Herein, we speculate Afatinib triggered the expansion of subclonal mutations. Interestingly, post-Afatinib tissue biopsy confirmed persistence of the exon-19del on EGFR along with FGFR1, KDR, KIT and PDGFRA 14 amplifications which could induce TKI-resistance via activation of bypass kinases . Unfortunately, EGFR status at this timepoint was not tested. Subsequent liquid biopsy (September 2018) demonstrated absence of EGFR alterations, suggesting an effect of Afatinib even though it was discontinued. Following progression and based on the available evidence we opted for the KEYNOTE189 15 schema (Pembrolizumab plus chemotherapy) . Notably, this study excluded patients with EGFR/ALK alterations. More recently, IMPOWER150 validated the use of Atezolizumab plus chemotherapy in 16 EGFR mutant NSCLCs . Unfortunately, at the time the patient failed Afatinib the results of the IMPOWER150 were not available. A recent analysis found that NSCLC patients with uncommon EGFR mutations and without T790M had better response to immunotherapy versus common mutations such as Exon19del or 17 L858R . Within this context, it is interesting to speculate on a “signature” for immunotherapy response. Our analysis showed IKZF1 amplification that persisted after TKI-failure. A study found that IKZF1 overexpression can be utilized to sensitize immune-resistant tumors for anti-PD1 or anti-CTLA4 18 treatments , a finding that would support the observed response. Similarly, our analysis showed a CSF1R deletion. A study in pancreatic cancer demonstrated that CSF1/CSF1R blockade enhances 19 antigen presentation and T-cell activation, thereby reducing immunosuppression and supporting a role of CSF1R deletion. We also found acquisition of INHBA amplification following progression, this gene encodes for inhibin subunit beta-A; a marker of M1-like Tumor-Associated Macrophages (TAMs) polarization. Unlike M2 macrophages, these are pro-inflammatory cells that display cytotoxic 20 properties . We could speculate INHBA amplification induced M1-TAM polarization causing infiltration of immunocompetent cells, potentiating an antitumoral response. Intriguingly, Afatinib also triggered GPR124 amplification, a known Wnt signaling pathway activator. Aberrant Wnt signaling 21 plays a role in immune evasion, consequently high-Wnt tumors display lower immune infiltration . This would argue against immunotherapy response in this case. In search for mechanisms, we speculate the response could be attributed to at least four: first, tumor heterogeneity; a prospective study reports a preliminary analysis assessing the role of 22 intratumoral heterogeneity in clinical outcomes . Investigators assessed clonal and subclonal mutations and somatic copy number alterations (SCNAs). Results demonstrated extensive intratumoral heterogeneity among NSCLC patients with a median of 30% and 48% for subclonal somatic mutations and subclonal copy-number alterations, respectively. In fact, >75% of tumors 22 carried subclonal driver alterations in genes like PIK3CA, KRAS, TP53 and NOTCH . Intratumoral heterogeneity serves as a substrate for tumor evolution and the development of drug resistance. Hence, tumor cells subjected to a selective pressure (TKI) trigger the emergence of alternative pathways in a subset of cells. Supporting this notion, an in vitro study found FGFR1 pathway 23 activation in Afatinib-resistant lung cancer cell lines . Recently, a study demonstrated that strong 24 PDL1+ correlates with de novo resistance to TKI among EGFR mutant NSCLCs characterized by KRAS mutations and c-MET amplification. Remarkably, this patient was PDL1- and lacked RAS/MET
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alterations. Second, response could be explained by the immunotherapy/chemotherapy and SBRT combination. The Abscopal effect describes the spontaneous shrinkage of metastases following a localized antitumor treatment. Although this effect has been documented in several malignancies it is 25 rare in lung adenocarcinomas . A Phase I preliminary report indicated low toxicity, a rather modest PFS/OS benefit, and a 13% Objective Response Rate (ORR) in advanced solid tumor using this 26 strategy . On the other hand, previous studies have postulated a synergistic effect of immunotherapy 27 and RT. Currently, clinical trials are exploring the CPI-RT combination in NSCLCs . Regarding this case, we could speculate that RT and/or the chemotherapy released previously unexposed tumor antigens potentiating an anti-tumoral response. Third, the response could be simply due to chemotherapy. A systematic review suggests that pemetrexed-based therapies display a favorable 28 efficacy on EGFR mutant NSCLCs that have failed TKI-treatments . Fourth, studies have hypothesized on the synergistic effect of immunotherapy, radiotherapy and chemotherapy especially 29 in lung cancer , however these results should be interpreted carefully due to potential treatment derived toxicities in real-world cases. Finally, a recent case report demonstrates complete response to second-line Pembrolizumab 30 in a metastatic HER2/PDL1-negative, MMR proficient, low TMB gastric cancer patient . Authors found that focal somatic copy number alterations (fSCNA) correlated with immunotherapy response. Similarly, our data showed an increase in fSCNAs especially in Copy Number Variations. Future clinical trials should determine if fSCNA or CNV changes correlate with immunotherapy response and therefore could serve as predictive biomarkers for response. Conclusion We speculate intratumoral heterogeneity could explain the unusual TKI-response in a reduced subset of EGFR mutant NSCLCs. CPI plus SBRT or CPI plus chemotherapy (pemetrexed and carboplatin) combinations might be an option for these patients. Also, pending its assessment and validation by clinical trials, changes in fSCNAs could serve as a predictor of immunotherapy response in low-TMB, PDL1- patients.
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Declarations Ethics approval and consent to participate Patient was incorporated as a part of a lung cancer patient registry approved by the Ethics committee (Approval #171004005 dated November 7th, 2017). Patient also signed a consent to participate Consent for publication The patient signed a consent form for publication of data Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding Boehringer Ingelheim Chile provided financial support for open access publication costs. Acknowledgements Authors wish to thank the nurses and all other medical staff at the Cancer Center “Nuestra Señora de la Esperanza”.
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Legends to figures and tables Figure 1. Timeline of a metastatic EGFR mutant NSCLC patient’s unusual clinical response to TKI and subsequent complete response to CPI plus chemotherapy. Evolution of metastatic disease is shown by whole body PET/CT scans and brain, abdominal and spinal MRI at various time points. NGS analyses were performed at the time of diagnosis (March 2018) and after the unusual clinical response to TKI (May 2018). SBRT sessions applied and the systemic treatments performed on the patient are also indicated: Afatinib was initially applied for 2 months, followed by CPI + chemotherapy. Figure 2. Radiological, histopathological analyses and molecular changes at diagnosis versus progression. A: PET imaging show biopsy sites (arrowheads) at left cervical lymphadenopathy. Also, note the size reduction in lung tumor and hepatic metastasis by TKI-treatment. B: Histological analysis of biopsy by Hematoxylin & Eosin stains. Upper left: Low power magnification (4x) shows lymph node tissue is extensively replaced by neoplasm. Upper right: High power magnification (40x) shows large neoplastic cells, with vesicular nuclei, conspicuous nucleoli, and abundant pale eosinophilic cytoplasm. Lower left: 40x magnification of the cell block of group III cervical lymph node showing clusters of cohesive neoplastic cells with glandular differentiation. Lower right: 40x image showing necrotic tumor cells. C: Changes in clinically relevant variants by NGS. Left (at diagnosis): EGFR gene amplification and exon-19 deletion, microsatellite stability and low TMB (4 Mut/Mb). Right (at progression): In addition to EGFR alterations, MS and TMB status, acquisition of 8 additional alterations (ZNF703, MYST3, PDGFRA, KIT, KDR and FGFR1 amplifications). D: Changes in Variants of Unknown Significance (VUS) by NGS. Left (at diagnosis): Twelve VUS were found. Right (at progression): acquisition of 7 additional VUS.
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SBRT
AFATINIB CARBOPLATIN PEMETREXED PEMBROLIZUMAB
2018 JANUARY FEBRUARY MRI
PET/CT
SBRT
MARCH Brain MRI
APRIL Brain MRI
MAY Brain MRI
JUNE
SBRT
AUGUST
Abdominal MRI
SEPTEMBER Brain MRI
2019
JANUARY
APRIL
Brain MRI
Brain MRI
Liquid BIOPSY -No EGFR mutation
Liquid BIOPSY -No EGFR mutation
L2
L5 Spinal lesion
Cervical BIOPSY: -NSCL AdenoCA -TTF1 (+) -P40 (-) -GATA3 (-) -Exon19del EGFR -PDL1 (-) FoundationOne -EGFR Amp -Exon-19del EGFR Liquid BIOPSY -Exon-19del EGFR
Cervical BIOPSY FoundationOne: -EGFR Amp -Exon-19del EGFR -FGFR1 Amp -KDR Amp -KIT Amp -PDGFRA Amp -KDM6A dupl -MYST3 Amp -RUNX1 del -ZNF703 Amp
Spinal PET/CT L2
L5
Liquid BIOPSY -No EGFR mutation
JULY Liquid BIOPSY -No EGFR mutation
A
B
C
March, 2018
May, 2018 Genetic alteration type: Amplification (CNV) Non-frameshift Deletion
EGFR ZNF703 RUNX1 MYST3
Duplication
March, 2018
KDM6A PDGFRA KIT KDR FGFR1
D
March, 2018 ZNF217 TSC2
4x
40x
PALB2 NTRK1 MUTYH MRE11A MLL2 IKZF1 HNF1A
May, 2018
CSF1R AXIN1 TP53 PRKCI NPM1 INHBA GPR124 GATA3
40x
40x
CTNNB1 RICTOR
May, 2018 Genetic alteration type: Missense Amplification (CNV) Non-frameshift Deletion Rearrangement
Clinical Practice Points -Tyrosine Kinase Inhibitors (TKI) are the standard of care for Epidermal Growth Factor (EGFR)mutant Non-Small Cell Lung Cancer (NSCLC) patients. Studies suggest no significant benefit of immune Checkpoint Inhibitors (CPI) for these patients. -We report an unusual clinical response to Afatinib in an advanced EGFR-mutant NSCLC patient, including rapid disease progression. Subsequent treatment with Pembrolizumab (CPI) plus chemotherapy (Pemetrexed and Carboplatin) and concomitant Stereotactic Body Radiotherapy (SBRT) triggered a complete and durable response. -We suggest the combination of CPI plus SBRT, or CPI plus chemotherapy could be an option for TKI-refractory EGFR mutant NSCLC patients, or as a second-line treatment.
S1525-7304(20)30012-7
DOI:
https://doi.org/10.1016/j.cllc.2020.01.012
Reference:
CLLC 1077
To appear in:
Clinical Lung Cancer
Received Date: 9 August 2019 Revised Date:
27 December 2019
Accepted Date: 20 January 2020
Please cite this article as: Pizarro G, Pinto MP, Medel MM, Cordova-Delgado M, Bravo ML, Nervi B, Sánchez C, Ibañez C, Peña J, Walbaum B, Madrid J, Briones J, Koch E, Valbuena J, Gonzalez S, Gejman R, Acevedo F, Mondaca S, Garrido M, Vines E, Galindo H, Case Report: Complete response to immunotherapy plus chemotherapy following an unusual clinical response to Afatinib and stereotactic radiosurgery in a metastatic EGFR-mutant NSCLC patient, Clinical Lung Cancer (2020), doi: https:// doi.org/10.1016/j.cllc.2020.01.012. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Elsevier Inc. All rights reserved.
Case Report Case Report: Complete response to immunotherapy plus chemotherapy following an unusual clinical response to Afatinib and stereotactic radiosurgery in a metastatic EGFR-mutant NSCLC patient Running title: Unusual complete response to immunotherapy
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Authors: Gonzalo Pizarro *, Mauricio P. Pinto *, Matías Muñoz Medel , Miguel Cordova-Delgado , M. 1 1 1 1 1 1 Loreto Bravo , Bruno Nervi , César Sánchez , Carolina Ibañez , José Peña , Benjamín Walbaum , 1 1 1 2 2 2 Jorge Madrid , Juan Briones , Erica Koch , Jose Valbuena , Sergio Gonzalez , Roger Gejman , 1 1 1 1 1 Francisco Acevedo , Sebastian Mondaca , Marcelo Garrido , Eugenio Vines , Hector Galindo . Affiliations: 1 Departamento de Hematología y Oncología. Facultad de Medicina. Pontificia Universidad Católica de Chile 2 Departamento de Anatomía Patológica. Facultad de Medicina. Pontificia Universidad Católica de Chile
Correspondence: Hector Galindo, MD Departamento de Hematología y Oncología Facultad de Medicina Pontificia Universidad Católica de Chile Diagonal Paraguay 362, 6 piso, Postal code: 8330077 Phone: +56 (2) 23549034 [email protected] * These authors contributed equally to this work
List of abbreviations: CPI: Checkpoint inhibitor; LC: Lung Cancer; MRI: Magnetic Resonance Imaging; NGS: Next Generation Sequencing; PCR: Polymerase Chain Reaction; PDL1: Programmed Death-Ligand 1; PET/CT: Positron Emission Tomography/Computed Tomography; TMB: Tumor Mutational Burden; TKI: Tyrosine Kinase Inhibitor; SBRT: Stereotactic Body Radiation Therapy; SCNA: Somatic Copy Number Alteration
Conflicts of interest None.
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Abstract Background: Checkpoint inhibitors (CPIs) such as Pembrolizumab have recently become the standard treatment for Non-Small Cell Lung Cancers (NSCLC) in patients without Epidermal Growth Factor (EGFR) gene alterations. Previous reports suggest no significant benefit from CPI treatments among EGFR mutant NSCLC patients and recommend the use of Tyrosine Kinase Inhibitors such as Afatinib. Case presentation: Herein, we report a rare case of a 39-year old, light-smoker woman with a metastatic EGFR mutant NSCLC (exon-19 deletion) that exhibited rapid progression and an unusual clinical response to Afatinib without changes in tumor histology. Tumor was negative for PDL1. Biopsy analyses by NGS prior and after Afatinib treatment indicated an increase in focal somatic copy number alterations (fSCNAs) but no T790M or c-MET amplification, and a low Tumor Mutational Burden (TMB). Given her rapid progression and deterioration, and despite its PDL1- status and her low TMB patient started treatment with Pembrolizumab plus chemotherapy. Surprisingly, patient displayed a complete response after just 4 cycles and is soon to complete a year in remission. Conclusions: We speculate intratumoral heterogeneity, the combination of radiotherapy plus immunotherapy or the concomitant use of chemotherapy plus immunotherapy could explain the observed response in this patient. Also, based in our data we suggest the SBRT/CPI combination could be an option for Tyrosine Kinase Inhibitor-refractory EGFR mutant NSCLC patients or as a second-line treatment. Keywords: checkpoint inhibitors, immunotherapy, lung cancer, radiation therapy, complete response, unusual clinical response, somatic copy number alterations.
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Introduction The development of checkpoint inhibitors (CPIs) has revolutionized the field of oncology. 1 2 3 4 5 Reports in melanoma , kidney , prostate , gastric and lung cancers have demonstrated its efficacy alone or in combination with chemotherapy. Despite this, a substantial portion of patients remains 5–7 unresponsive or display modest responses . In this context, high throughput sequencing such as Next Generation Sequencing (NGS) could help to elucidate molecular profiles in order to identify potential CPI-responders. Here, we describe a rare case of a metastatic Non-Small Cell Lung Cancer (NSCLC) patient diagnosed with exon-19 EGFR deletion that displayed an “unusual clinical response” to Afatinib, a Tyrosine Kinase Inhibitor (TKI). Subsequently, patient was switched to the Pemetrexed/Carboplatin plus Pembrolizumab combination and SBRT, demonstrating a complete response after 4 cycles. As pointed, a substantial portion of patients that receive immunotherapy remain unresponsive 8 or display modest responses, especially in EGFR mutant NSCLC patients . Biopsy samples were taken prior and after TKI and were analyzed by NGS indicating the acquisition of a number of new somatic alterations. These data could help to define a predictive biomarker for immunotherapy response in EGFR mutant NSCLC. We also discuss the utility of chemo or radiotherapy prior to immunotherapy in patients that fail TKIs. Case report 9
Figure 1 shows a timeline of the case, 39-year-old, light-smoker (5 packs-a-year) woman came to the clinic due to back pain. A spine MRI showed tumor lesions at L2 and L5 and a bone fracture at L2. A CT Scan revealed a lung tumor in the upper left lobe and several adenopathies, liver and adrenal metastases. Brain MRI found a 10-mm left frontal subcortical lesion and local edema (Fig. 1). Exon-19 deletion on EGFR was confirmed by liquid biopsy. Pathology indicated a Non-Small Cell lung adenocarcinoma. Further analyses confirmed exon-19 deletion and absence of PDL1. A PET/CT Scan confirmed a 65 mm lung mass associated to satellite nodes and mediastinal, hilar and cervical adenopathies, also liver, right adrenal, frontal left brain and bone metastases. Following diagnosis, the patient started oral Afatinib (TKI) and SBRT to control a left frontal brain metastasis. Six weeks after the patient returned to the clinic referring pain in the upper left arm, edema and an increase in cervical volume. Echography showed deep venous thrombosis in the left internal jugular vein and a second scan confirmed an increase in cervical and left supraclavicular lymphadenopathies along with novel secondary lymphadenopathies. At the same time, liver 10 metastasis and the lung lesion were notoriously reduced suggesting a paradoxical response . Three weeks later the patient manifested abdominal pain, anorexia and asthenia. MRI showed progression of liver metastasis and increase in secondary lymphadenopathies. Given the rapid disease progression despite the use of TKI therapy a second cervical biopsy was taken from the left lymphadenectomy that confirmed a NSCL adenocarcinoma. A NGS study compared cervical biopsies taken at diagnosis and after progression (March/May 2018). Results are summarized in Figure 2. Both biopsies demonstrated EGFR amplification, exon-19 deletion and low TMB. However, second biopsy also indicated novel alterations, including KIT, FGFR1, PDGFRA, KDR amplification and notably no T790M (Fig. 2C). Following progression, the patient started Pembrolizumab plus chemotherapy. Also, SBRT was applied for supraclavicular metastasis (40 Gy, 5 fractions) and spinal lesion (8 Gy 1 fraction, palliative). After 4 cycles, scan showed resolution on adenopathies, and decrease in lung mass, liver and spinal lesions. Brain MRI showed no lesions or novel metastases. Liquid biopsy for EGFR mutations was negative. Patient continues Pembrolizumab plus pemetrexed and is soon to complete a year in remission.
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Discussion Numerous studies have demonstrated the effectiveness of TKIs against NSCLCs that display 11 activating EGFR mutations . In general, TKIs in these patients has substantial benefits, an initial 70% 11 response rate and 1-3 year overall survival (OS) . Despite this, a portion of EGFR mutant TKI-failed 10 12 NSCLC patients acquire novel alterations including the T790M or c-MET amplification . Surprisingly, this was not the case. Instead, TKI-failure correlated with emergence of several alterations. 10 Initially, this case suggested a paradoxical response , including partial response on liver/lung lesions, however clinical and radiological evidence clearly indicated rapid disease progression following Afatinib. Therefore, we opted for the term “unusual clinical response” to describe this rare 13 case. Afatinib resistance in EGFR mutant NSCLCs is rare (<10% of cases) . Herein, we speculate Afatinib triggered the expansion of subclonal mutations. Interestingly, post-Afatinib tissue biopsy confirmed persistence of the exon-19del on EGFR along with FGFR1, KDR, KIT and PDGFRA 14 amplifications which could induce TKI-resistance via activation of bypass kinases . Unfortunately, EGFR status at this timepoint was not tested. Subsequent liquid biopsy (September 2018) demonstrated absence of EGFR alterations, suggesting an effect of Afatinib even though it was discontinued. Following progression and based on the available evidence we opted for the KEYNOTE189 15 schema (Pembrolizumab plus chemotherapy) . Notably, this study excluded patients with EGFR/ALK alterations. More recently, IMPOWER150 validated the use of Atezolizumab plus chemotherapy in 16 EGFR mutant NSCLCs . Unfortunately, at the time the patient failed Afatinib the results of the IMPOWER150 were not available. A recent analysis found that NSCLC patients with uncommon EGFR mutations and without T790M had better response to immunotherapy versus common mutations such as Exon19del or 17 L858R . Within this context, it is interesting to speculate on a “signature” for immunotherapy response. Our analysis showed IKZF1 amplification that persisted after TKI-failure. A study found that IKZF1 overexpression can be utilized to sensitize immune-resistant tumors for anti-PD1 or anti-CTLA4 18 treatments , a finding that would support the observed response. Similarly, our analysis showed a CSF1R deletion. A study in pancreatic cancer demonstrated that CSF1/CSF1R blockade enhances 19 antigen presentation and T-cell activation, thereby reducing immunosuppression and supporting a role of CSF1R deletion. We also found acquisition of INHBA amplification following progression, this gene encodes for inhibin subunit beta-A; a marker of M1-like Tumor-Associated Macrophages (TAMs) polarization. Unlike M2 macrophages, these are pro-inflammatory cells that display cytotoxic 20 properties . We could speculate INHBA amplification induced M1-TAM polarization causing infiltration of immunocompetent cells, potentiating an antitumoral response. Intriguingly, Afatinib also triggered GPR124 amplification, a known Wnt signaling pathway activator. Aberrant Wnt signaling 21 plays a role in immune evasion, consequently high-Wnt tumors display lower immune infiltration . This would argue against immunotherapy response in this case. In search for mechanisms, we speculate the response could be attributed to at least four: first, tumor heterogeneity; a prospective study reports a preliminary analysis assessing the role of 22 intratumoral heterogeneity in clinical outcomes . Investigators assessed clonal and subclonal mutations and somatic copy number alterations (SCNAs). Results demonstrated extensive intratumoral heterogeneity among NSCLC patients with a median of 30% and 48% for subclonal somatic mutations and subclonal copy-number alterations, respectively. In fact, >75% of tumors 22 carried subclonal driver alterations in genes like PIK3CA, KRAS, TP53 and NOTCH . Intratumoral heterogeneity serves as a substrate for tumor evolution and the development of drug resistance. Hence, tumor cells subjected to a selective pressure (TKI) trigger the emergence of alternative pathways in a subset of cells. Supporting this notion, an in vitro study found FGFR1 pathway 23 activation in Afatinib-resistant lung cancer cell lines . Recently, a study demonstrated that strong 24 PDL1+ correlates with de novo resistance to TKI among EGFR mutant NSCLCs characterized by KRAS mutations and c-MET amplification. Remarkably, this patient was PDL1- and lacked RAS/MET
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alterations. Second, response could be explained by the immunotherapy/chemotherapy and SBRT combination. The Abscopal effect describes the spontaneous shrinkage of metastases following a localized antitumor treatment. Although this effect has been documented in several malignancies it is 25 rare in lung adenocarcinomas . A Phase I preliminary report indicated low toxicity, a rather modest PFS/OS benefit, and a 13% Objective Response Rate (ORR) in advanced solid tumor using this 26 strategy . On the other hand, previous studies have postulated a synergistic effect of immunotherapy 27 and RT. Currently, clinical trials are exploring the CPI-RT combination in NSCLCs . Regarding this case, we could speculate that RT and/or the chemotherapy released previously unexposed tumor antigens potentiating an anti-tumoral response. Third, the response could be simply due to chemotherapy. A systematic review suggests that pemetrexed-based therapies display a favorable 28 efficacy on EGFR mutant NSCLCs that have failed TKI-treatments . Fourth, studies have hypothesized on the synergistic effect of immunotherapy, radiotherapy and chemotherapy especially 29 in lung cancer , however these results should be interpreted carefully due to potential treatment derived toxicities in real-world cases. Finally, a recent case report demonstrates complete response to second-line Pembrolizumab 30 in a metastatic HER2/PDL1-negative, MMR proficient, low TMB gastric cancer patient . Authors found that focal somatic copy number alterations (fSCNA) correlated with immunotherapy response. Similarly, our data showed an increase in fSCNAs especially in Copy Number Variations. Future clinical trials should determine if fSCNA or CNV changes correlate with immunotherapy response and therefore could serve as predictive biomarkers for response. Conclusion We speculate intratumoral heterogeneity could explain the unusual TKI-response in a reduced subset of EGFR mutant NSCLCs. CPI plus SBRT or CPI plus chemotherapy (pemetrexed and carboplatin) combinations might be an option for these patients. Also, pending its assessment and validation by clinical trials, changes in fSCNAs could serve as a predictor of immunotherapy response in low-TMB, PDL1- patients.
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Declarations Ethics approval and consent to participate Patient was incorporated as a part of a lung cancer patient registry approved by the Ethics committee (Approval #171004005 dated November 7th, 2017). Patient also signed a consent to participate Consent for publication The patient signed a consent form for publication of data Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding Boehringer Ingelheim Chile provided financial support for open access publication costs. Acknowledgements Authors wish to thank the nurses and all other medical staff at the Cancer Center “Nuestra Señora de la Esperanza”.
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Legends to figures and tables Figure 1. Timeline of a metastatic EGFR mutant NSCLC patient’s unusual clinical response to TKI and subsequent complete response to CPI plus chemotherapy. Evolution of metastatic disease is shown by whole body PET/CT scans and brain, abdominal and spinal MRI at various time points. NGS analyses were performed at the time of diagnosis (March 2018) and after the unusual clinical response to TKI (May 2018). SBRT sessions applied and the systemic treatments performed on the patient are also indicated: Afatinib was initially applied for 2 months, followed by CPI + chemotherapy. Figure 2. Radiological, histopathological analyses and molecular changes at diagnosis versus progression. A: PET imaging show biopsy sites (arrowheads) at left cervical lymphadenopathy. Also, note the size reduction in lung tumor and hepatic metastasis by TKI-treatment. B: Histological analysis of biopsy by Hematoxylin & Eosin stains. Upper left: Low power magnification (4x) shows lymph node tissue is extensively replaced by neoplasm. Upper right: High power magnification (40x) shows large neoplastic cells, with vesicular nuclei, conspicuous nucleoli, and abundant pale eosinophilic cytoplasm. Lower left: 40x magnification of the cell block of group III cervical lymph node showing clusters of cohesive neoplastic cells with glandular differentiation. Lower right: 40x image showing necrotic tumor cells. C: Changes in clinically relevant variants by NGS. Left (at diagnosis): EGFR gene amplification and exon-19 deletion, microsatellite stability and low TMB (4 Mut/Mb). Right (at progression): In addition to EGFR alterations, MS and TMB status, acquisition of 8 additional alterations (ZNF703, MYST3, PDGFRA, KIT, KDR and FGFR1 amplifications). D: Changes in Variants of Unknown Significance (VUS) by NGS. Left (at diagnosis): Twelve VUS were found. Right (at progression): acquisition of 7 additional VUS.
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SBRT
AFATINIB CARBOPLATIN PEMETREXED PEMBROLIZUMAB
2018 JANUARY FEBRUARY MRI
PET/CT
SBRT
MARCH Brain MRI
APRIL Brain MRI
MAY Brain MRI
JUNE
SBRT
AUGUST
Abdominal MRI
SEPTEMBER Brain MRI
2019
JANUARY
APRIL
Brain MRI
Brain MRI
Liquid BIOPSY -No EGFR mutation
Liquid BIOPSY -No EGFR mutation
L2
L5 Spinal lesion
Cervical BIOPSY: -NSCL AdenoCA -TTF1 (+) -P40 (-) -GATA3 (-) -Exon19del EGFR -PDL1 (-) FoundationOne -EGFR Amp -Exon-19del EGFR Liquid BIOPSY -Exon-19del EGFR
Cervical BIOPSY FoundationOne: -EGFR Amp -Exon-19del EGFR -FGFR1 Amp -KDR Amp -KIT Amp -PDGFRA Amp -KDM6A dupl -MYST3 Amp -RUNX1 del -ZNF703 Amp
Spinal PET/CT L2
L5
Liquid BIOPSY -No EGFR mutation
JULY Liquid BIOPSY -No EGFR mutation
A
B
C
March, 2018
May, 2018 Genetic alteration type: Amplification (CNV) Non-frameshift Deletion
EGFR ZNF703 RUNX1 MYST3
Duplication
March, 2018
KDM6A PDGFRA KIT KDR FGFR1
D
March, 2018 ZNF217 TSC2
4x
40x
PALB2 NTRK1 MUTYH MRE11A MLL2 IKZF1 HNF1A
May, 2018
CSF1R AXIN1 TP53 PRKCI NPM1 INHBA GPR124 GATA3
40x
40x
CTNNB1 RICTOR
May, 2018 Genetic alteration type: Missense Amplification (CNV) Non-frameshift Deletion Rearrangement
Clinical Practice Points -Tyrosine Kinase Inhibitors (TKI) are the standard of care for Epidermal Growth Factor (EGFR)mutant Non-Small Cell Lung Cancer (NSCLC) patients. Studies suggest no significant benefit of immune Checkpoint Inhibitors (CPI) for these patients. -We report an unusual clinical response to Afatinib in an advanced EGFR-mutant NSCLC patient, including rapid disease progression. Subsequent treatment with Pembrolizumab (CPI) plus chemotherapy (Pemetrexed and Carboplatin) and concomitant Stereotactic Body Radiotherapy (SBRT) triggered a complete and durable response. -We suggest the combination of CPI plus SBRT, or CPI plus chemotherapy could be an option for TKI-refractory EGFR mutant NSCLC patients, or as a second-line treatment.