- Email: [email protected]
Large Cell Neuroendocrine Carcinoma Transformation and EGFR-T790M Mutation as Coexisting Mechanisms of Acquired Resistance to EGFR-TKIs in Lung Cancer
CASE REPORT
Large Cell Neuroendocrine Carcinoma Transformation and EGFR-T790M Mutation as Coexisting Mechanisms of Acquired Resistance to EGFR-TKIs in Lung Cancer Sara Baglivo, MSc; Vienna Ludovini, PhD; Angelo Sidoni, MD; Giulio Metro, MD; Biagio Ricciuti, MD; Annamaria Siggillino, MSc; Alberto Rebonato, MD, PhD; Salvatore Messina, MD; Lucio Crinò, MD; and Rita Chiari, MD, PhD Abstract Acquired resistance to tyrosine kinase inhibitors (TKIs) represents the Achilles’ heel of targeted treatment in lung cancer. Epidermal growth factor receptor (EGFR)-TKIs are considered the standard first-line treatment for patients with EGFR mutant nonesmall cell lung cancer; however, after a median of 9 to 12 months, virtually all patients develop acquired resistance, which is mediated by the development of an EGFR-T790M secondary mutation in approximately 60% of cases. Different mechanisms of acquired resistance have also been described with lower incidence, including mutations in other driver oncogenes or phenotypic transformation. Herein, we report the first case of a patient with EGFR-mutant lung adenocarcinoma with a long-lasting response to first-line erlotinib treatment who acquired resistance to treatment because of acquisition of both EGFR-T790M mutation and “high-grade” large cell neuroendocrine transformation. This case also shows how resistance to third-generation EGFR-TKI osimertinib can be mediated by the development of phenotypic neuroendocrine transformation, which in the present case occurred during first-line treatment with erlotinib. In addition, our report highlights the pivotal role of rebiopsy and of molecular profiling at the time of progression to guide clinicians to choose the right therapy for the right patient. ª 2017 Mayo Foundation for Medical Education and Research
From the Medical Oncology Division (S.B., V.L., G.M., B.R., A.S., R.C.) and Nuclear Medicine Division (S.M.), Azienda Ospedaliera di Perugia, Perugia, Italy; Department of Experimental Medicine, Section of Pathological Anatomy and Histology, Medical School , University of Perugia, Perugia, Italy (A.S.); Radiology Unit, Department of Surgical and Biomedical Science, Santa Maria della Misericordia University Hospital, Perugia University, Perugia, Italy (A.R.); and Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy (L.C.).
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ung cancer is the leading cause of cancer-related deaths worldwide in both men and women, representing 27% of all cancer deaths.1 Recent advances in the understanding of molecular mechanisms underlying the development of none small cell lung cancer (NSCLC) have led to a classification based on the biological features of each tumor, such that NSCLC becomes a group of different diseases, each with its own specific clinical and biological characteristics. The discovery of sensitizing epidermal growth factor receptor (EGFR) mutations as a predictive marker of sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs) has revolutionized the therapeutic approach to this subgroup of patients with advanced NSCLC who represent 10% to 15% of white patients and 40% to 60% of Asian patients.2 First- and second-
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generation EGFR-TKIs have become the standard first-line treatment for patients with advanced EGFR-mutant NSCLC, having definitely shown in phase III trials a higher efficacy compared with platinum-based chemotherapy.3 Unfortunately, acquired resistance to treatment invariably occurs within 9 to 12 months and is primarily mediated by the EGFRT790M secondary mutation that accounts for approximately 60% of cases. Other acquired resistance mechanisms have also been described, including mutations in other driver oncogenes or phenotypic transformation.4-7 Herein, we report the first case of a patient with EGFR-mutant lung adenocarcinoma with a long-lasting response to first-line erlotinib treatment who acquired resistance to EGFR-TKIs because of the development of
Mayo Clin Proc. n August 2017;92(8):1304-1311 n http://dx.doi.org/10.1016/j.mayocp.2017.03.022 www.mayoclinicproceedings.org n ª 2017 Mayo Foundation for Medical Education and Research
TWO MECHANISMS OF ACQUIRED RESISTANCE TO ERLOTINIB IN LUNG CANCER
both EGFR-T790M mutation and “high-grade” large cell neuroendocrine carcinoma (LCNEC) transformation coexisting in the same adrenal metastatic lesion. RESULTS We present the case of a 57-year-old male patient who never smoked and was diagnosed in November 2011 with stage IV (cT1b N2M1b [bone and brain] tumor, node, metastasis staging version 7.0), grade 3, thyroid transcription factor 1 (TTF1) positive lung adenocarcinoma (Figure 1A(a) and B(a)). Sequence analysis on the transbronchial lung agobiopsy sample obtained at diagnosis scored positive for EGFR exon 19 deletion (2236_ 2252del18/L747_S752del) (Table), and the patient received erlotinib 150 mg once daily as first-line treatment. One month later the patient achieved a partial response that turned into a radiologically complete response after 4 months (Figure 1B). The patient had been treated with erlotinib until January 2014 when he had a relapse in the posterior basal segment of the right lower lobe, which was present as a single lesion. The patient was considered eligible for radical surgery and underwent atypical lung resection (Figure 1A(d)). The microscopical examination confirmed the same histology of the primary tumor (adenocarcinoma Periodic AcideSchiff diastase stain positive (PAS-Dþ), TTF1þ, Cytokeratin 7 positive (CK 7þ), Cytokeratin 20 negative (CK 20), napsinþ, and Ki-67 60%) (Figure 2, A and B1), whereas EGFR genotyping (exon 18, 19, 20, and 21) revealed the presence of exon 19 deletion together with the presence of T790M resistance (2369C>T) mutation. After lung atypical resection, treatment with erlotinib was pursued for 24 months until January 2016 when the patient had disease progression at both cranial (multiple lesions of 3-4 mm in diameter) and extracranial (mediastinal node and left adrenal gland) sites (Figure 1A(f)). Subsequently, the patient underwent whole-brain radiotherapy (total dose, 30 Gy/10 f), which obtained complete response in the brain that was still ongoing until the last available brain magnetic resonance imaging scan (>9 months) (Figure 1C). At the same time, the patient underwent ultrasound-guided biopsy of the mediastinal lymph node that confirmed adenocarcinoma histology along with the Mayo Clin Proc. n August 2017;92(8):1304-1311 www.mayoclinicproceedings.org
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presence of EGFR-del19 and T790M mutations (Figure 2C, and Table). Erlotinib was discontinued, and second-line treatment with osimertinib (AZD9291) was initiated in January 2016. One month later, the mediastinal lymph node disappeared but disease progression occurred in the left adrenal gland (Figure 1, A(g)). The patient underwent left adrenalectomy, and histological examination revealed 2 coexisting distinct components: adenocarcinoma (TTF1þ and CK-7þ) and LCNEC (synaptophysinþ, TTF-1þ, and CK-7 “dot-like” positive) (Figure 2D). Both the components, which were separately examined for EGFR mutations, harbored an EGFR exon 19 deletion mutation with no evidence of EGFR-T790M. After an accurate revision of the sample from the atypical lung resection of the right lower lobe (January 2014), we found that phenotypic transformation into LCNEC was already present as a small focus in that surgical sample, which at the time was assumed to be a specimen of adenocarcinoma bearing exon 19 and EGFR-T790M mutation (Figure 2B2). After surgery the patient underwent radiotherapy on the site of left adrenalectomy (48.4 Gy/22 f þ SYB 55 Gy/22 f) and resumed osimertinib. Unfortunately, 2 months later (May 2016) a radiologic revaluation exhibited disease progression at multiple bone sites (Figure 1A(h)), a picture that was consistent with rapidly progressing high-grade neuroendocrine tumor. Therefore, considering the histological features and the radiological evidence of widespread disease, the patient began receiving cisplatin-etoposide combination chemotherapy that led to a partial remission after 2 cycles, which was maintained until the end of the sixth cycle. Unfortunately, after 2 months from the end of platinum-etoposide chemotherapy, the patient had disease progression at multiple bone sites. Therefore, in light of the rapid worsening of his clinical condition, we decided to go on with best supportive care and the patient died after more than 61 months from diagnosis. During the course of his disease, we had the opportunity to collect 3 tumor samples (2 surgical samples and 1 small biopsy specimen) of erlotinib-refractory metastatic lesions, all of which harbored an EGFR exon 19 deletion mutation. At microscopic examination, the 2 surgical samples included both LCNEC
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FIGURE 1. (continued).
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FIGURE 1. A, Patient’s positron emission tomographyecomputed tomography (PET-CT) during the course of the patient’s disease: type of systemic and locoregional treatments (namely, erlotinib, osimertinib, and chemotherapy with cisplatin/etoposide, surgery, and extracranial radiotherapy) that the patient received over the time are depicted as well as a trasversal cut, showing lung relapse in January 2014 when he underwent atypical resection of the right lower lobe. PET-TC (a) before, (b) after 1 month, (c) after 4 months and (d) after 25 months of erlotinib treatment showing first PD; (e) CR after resection, (f) after 50 months of erlotinib therapy, (g) after 1 month, and (h) after 4 months of osimertinib treatment; (i) after 3 months from the beginning of chemotherapy showing PR. The timeline of treatment has different scales of measurement. B, Brain contrast-enhanced T1-weighted magnetic resonance imaging before (a), at 3 months (b), and at 6 months (c) after the beginning of first-line erlotinib treatment, showing the progressive disappearance of the centimetric cortical metastasis in the parietal left lobe (white arrow). C, Brain contrast-enhanced 3-dimensional T1-weighted spoiled gradient recalled-echo magnetic resonance imaging performed 4 years after the beginning of first-line erlotinib treatment, showing brain disease progression with 1 right cerebellar (a), 1 right occipital (b), and 2 parietal (c and d) metastases (white arrows). At 3- (data not shown) and 6-month follow-up after WBRT, contrast-enhanced T1-weighted magnetic resonance imaging confirmed the disappearance of all the lesions (e, f, g, and h). BSC ¼ best supportive care; CR ¼ complete response; PD ¼ progression disease; PR ¼ partial response; SD ¼ stable disease; WBRT ¼ whole-brain radiotherapy.
and adenocarcinoma histology coexisting in the same lesion. The adenocarcinoma component of the surgical sample of 2014 scored positive for EGFR-T790M, whereas both the adenocarcinoma and LCNEC components of the surgical lesion of 2016 were EGFRT790M negative at both direct sequencing and therascreen analysis. We subjected all the available tumor samples to digital polymerase chain reaction and next-generation sequencing (NGS) for a more Mayo Clin Proc. n August 2017;92(8):1304-1311 www.mayoclinicproceedings.org
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sensitive analysis and large spectrum mutation investigation and obtained the same results as described above. Moreover, no MET amplification was found in any samples by using digital polymerase chain reaction (QuantStudio 3D Digital PCR System, Life Technologies) analysis, and NGS analysis (Ion Torrent Personal Genome Machine (PGM), AmpliSeq Colon and Lung Research Panel v2, Life Technologies) did not reveal any mutations in the hotspot positions
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ND WT (CNV) WT ND WT (CNV) WT ND WT ND WT (CNV) WT ND WT (CNV) WT ND WT (CNV) WT ND ND WT ND ND WT ND WT ND ND WT ND ND WT ND ND WT ND ND WT ND ND WT ND WT ND ND WT ND ND WT ND ND WT ND ND WT ND ND WT ND WT ND ND WT ND ND WT ND ND WT cfDNA NA Blood Second erlotinib PD January 2016
Surgical sample Metastatic lesion LCNEC Adrenal gland February 2016 D
Osimertinib PD
February 2016 D
Osimertinib PD
Adrenal gland
ADC
Biopsy Metastatic lesion Surgical sample Metastatic lesion ADC Lymph node January 2016 C
Second erlotinib PD
Surgical sample Metastatic lesion ADC Right lung First erlotinib PD January 2014 B
NA Mayo Clin Proc.
ADC ¼ adenocarcinoma; cfDNA ¼ circulating free DNA; CNV ¼ copy number variation; dPCR ¼ digital polymerase chain reaction; EGFR ¼ epidermal growth factor receptor; LCNEC ¼ large cell neuroendocrine carcinoma; NA ¼ not applicable; ND ¼ not done; NGS ¼ next-generation sequencing; PD ¼ progression disease; WT ¼ wild type.
MET PTEN TP53 PIK3CA EGFR
WT ND WT ND ND WT ND WT ND ND WT ND ND WT ND ND WT Sanger sequencing dPCR NGS Therascreen dPCR NGS Therascreen NGS Therascreen dPCR NGS Therascreen dPCR NGS Therascreen dPCR NGS
Method Sample type
Transbronchial lung biopsy ADC
Histology Organ
Right lung Diagnosis
Status Time
November 2011 A
Lesion
TABLE. Summary of Primary/Metastatic Lesions and cfDNA Molecular Analysis
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L747_S752del del19 L747_S752del del19, T790M del19, T790M L747_S752del, T790M del19, T790M L747_S752del, T790M del19 del19 L747_S752del del19 del19 L747_S752del del19 del19 L747_S752del
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for 21 cancer-related genes analyzed by the specific NGS panel, including PIK3CA, PTEN, NOTCH1, TP53, and FGFR (for expansion of gene symbols, see www.genenames.org). Analysis of circulating tumor DNA at different time points from January 2016 (second progression during erlotinib treatment) and during osimertinib and chemotherapy treatment was positive for EGFR-del19, with no evidence either of EGFR-T790M or of any mutations in the 21 genes analyzed by the same NGS panel used for tumor tissue analysis (Table). DISCUSSION Several different mechanisms explaining acquired resistance to EGFR-TKIs in NSCLC have been identified, including on-target EGFR (60%) and off-target EGFR (20%) molecular alterations.4-7 The EGFR-T790M secondary mutation represents the most common mechanism of on-target EGFR acquired resistance. Among off-target EGFR molecular alterations, bypass track pathway activation, such as MET amplification (5%), and histological transformation to high-grade neuroendocrine tumors (10%) have been reported.7,8 More commonly, neuroendocrine transformation is represented by transition to small cell lung cancer (SCLC) but histological transformation into LCNEC has also been identified and described in a few reports.9-11 Herein, we report the case of a patient who developed, after a long-lasting response to erlotinib (50 months), both EGFR-T790M mutation and LCNEC transformation as mechanisms of acquired resistance to firstgeneration EGFR-TKIs. We were able to document the presence of both components (EGFR-T790M and LCNEC) after 25 months of erlotinib treatment in the same lung lesion and consequently in the same neoplastic clone that probably at that time had no metastatic potential and was eradicated by a locoregional treatment (right lower lobe atypical resection). On the contrary, after 50 months of erlotinib treatment, 3 components originating by the same neoplastic clone were present: (1) 1 clone bearing the EGFR-T790M gatekeeper mutation in the mediastinal lymph node that responded to the third-generation EGFR-TKI osimertinib and 2 other clones: (2) LCNEC and (3) T790M-negative adenocarcinoma components with high metastatic potential coexisting in
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adrenal metastasis that were both positive for EGFR-del19. Intriguingly, these last 2 components were both EGFR-T790M negative and insensitive to osimertinib. To our knowledge, 6 patients with SCLC transformation during treatment with EGFR-TKIs who had data for multiple
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metastatic lesions have been reported so far. In 3 of them, all metastatic lesions exhibited SCLC transformation without evidence of the EGFR-T790M mutation, whereas in the other 3, some lesions were T790M positive and others had SCLC phenotype, thus suggesting that SCLC transformation and EGFR-T790M
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mutation occur in a reciprocal manner in a single patient upon acquisition of resistance to EGFR-TKIs.4,12-16 Herein, we presented the first case in which EGFR-T790M mutation and neuroendocrine transformation exist together in the same lesion (lung 2014). Both these components had been documented in vivo more than 2 years before widespread disease progression, which was correlated with the aforementioned components sharing the original EGFR-activating mutation (del19) that we found in adrenal metastasis: (1) the highgrade neuroendocrine component responding to platinum-etoposide combination and (2) the adenocarcinoma EGFR-T790Menegative component rapidly progressing after platinum/etoposide debulking. In both these components, EGFR expression was silenced (immunohistochemistry: data not shown) as previously reported for EGFR-mutated lung adenocarcinoma and SCLC with primary resistance to EGFR-TKIs.16 This could explain the resistance to both erlotinib and osimertinib. Except for EGFR mutation, no other co-occurring mutations have been identified in any samples collected, neither in TP53 gene nor in other known driver oncogenes such as PIK3CA or MET. No EGFR-C797S third-generation TKI acquired resistance mutation was found, either on tissue biopsies or on liquid biopsies. This case highlights the importance of rebiopsy and of molecular analysis of sequential samples of the same patient during the course of disease to help clinicians
make the best treatment choice as possible. Until recently, upon documented disease progression on first- or second-generation EGFR TKIs, chemotherapy was the mainstay of second-line treatment for patients with EGFR-mutant NSCLC. Nonetheless, recent advances in the understanding of the molecular mechanism underlying the development of resistance to EGFR-TKIs revealed that T790M secondary mutation is responsible for approximately 60% of cases of acquired resistance. To overcome T790M-mediated resistance, novel third-generation EGFR-TKIs have been developed. Among these, osimertinib showed in phase I/II trials (clinicaltrials. gov Identifier: NCT01802632 and NCT02094261) promising results in pretreated patients with EGFR-mutant NSCLC.17 More recently, in a phase III trial (clinicaltrials.gov Identifier: NCT02151981), osimertinib was definitely proved to be superior to chemotherapy in terms of objective response rate and progression-free survival as second-line therapy for patients with EGFRmutant NSCLC harboring T790M secondary mutation who had progressed on or following a first-line EGFR-TKI treatment with particular evidence in brain metastasis control, which seems to improve with osimertinib because of the passage of the drug across the bloodbrain barrier.18 Unfortunately, resistance to third-generation EGFR-TKIs occurs in virtually all patients. Although the tertiary EGFR mutation C797S has recently been reported as one of the possible mechanisms of resistance, in most cases a specific mechanism
FIGURE 2. Histopathological and immunophenotypic evolution. A, Morphological picture of the transbronchial biopsy performed in November 2011: adenocarcinoma (right) and infiltrating lung parenchyma (left) (hematoxylin-eosin, original magnification 200). B, In the atypical resection of the right inferior lobe (January 2014) at the periphery of the adenocarcinoma, a small focus of undifferentiated carcinoma (left side of the inset B1: hematoxylin-eosin, original magnification 200) was present, showing a strong positivity for synaptophysin (inset B2: peroxidase, original magnification 200). C, Cell block obtained by fine needle aspiration cytology from a mediastinal lymph node (January 2016), showing fragments of adenocarcinoma (hematoxylin-eosin, original magnification 400). D, Adrenalectomy (February 2016) revealed a synchronous metastasis of adenocarcinoma (left) and undifferentiated large cells carcinoma (right) in cortical metastasis, with extension of the latter component into the retroperitoneal adipose tissue (hematoxylin-eosin, original magnification 200). E, Immunohistochemical staining for synaptophysin in the same area revealed strong positivity in the large cell component (right inset); the adenocarcinomatous component was completely negative (left inset) (peroxidase, original magnification 200; insets: original magnification 400). LCNEC ¼ large cell neuroendocrine carcinoma.
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cannot be identified. Our case suggests that phenotypic transformation into LCNEC might represent a novel mechanism of resistance to osimertinib, however, also being maintained in the LCNEC phenotype the presence of the EGFR-del19 mutation. Notably, upon documentation of such phenotypic transformation, in the absence of any potentially targetable genetic alteration, the patient was treated with an “LCNEC-like” chemotherapy, obtaining a partial response. This highlights the fundamental role of rebiopsy at disease progression that in our case allowed a proper molecularly tailored treatment of the disease, which translated into a clinically meaningful survival benefit of more than 61 months from diagnosis. Because biopsies at disease progression may not always be feasible, circulating tumor DNA analysis might provide a fast and noninvasive diagnostic tool. However, because of technical issues and low sensitivity, plasma genotyping should be considered complementary to traditional biopsies.19 CONCLUSION To our knowledge this is the first evidence of EGFR-T790Mepositive lung adenocarcinoma and LCNEC transformation existing together in the same lesion as shown in the resected first lung cancer relapse of the disease, with the subsequent T790M-positive adenocarcinoma being the putative intermediate step that precedes the development of the mixed metastatic phenotype consisting in LCNEC and adenocarcinoma (both EGFR-del19 positive) present in the adrenal gland. We do not have any formal proof that this means that EGFRT790Mepositive, EGFR-T790Menegative, and LCNEC components are subclones deriving from the same cell, but this is far more reasonable than simple intrametastatic heterogeneity. Abbreviations and Acronyms: EGFR = epidermal growth factor receptor; LCNEC = large cell neuroendocrine carcinoma; NGS = next-generation sequencing; NSCLC = none small cell lung cancer; SCLC = small cell lung cancer; TKI = tyrosine kinase inhibitor Grant Support: The work was supported by the Italian Association for Cancer Research (AIRC; grant number 9979) and the Umbria Association Against Cancer (AUCC). Correspondence: Address to Sara Baglivo, MSc, Medical Oncology Division, Azienda Ospedaliera di Perugia, 06132 Perugia, Italy ([email protected]). Mayo Clin Proc. n August 2017;92(8):1304-1311 www.mayoclinicproceedings.org
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REFERENCES 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30. 2. Pirker R, Filipits M. Personalized treatment of advanced nonsmall-cell lung cancer in routine clinical practice. Cancer Metastasis Rev. 2016;35(1):141-150. 3. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatinpaclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009; 361(10):947-957. 4. Sequist LV, Waltman BA, Dias-Santagata D, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 2011;3(75):75ra26. 5. Oser MG, Niederst MJ, Sequist LV, Engelman JA. Transformation from non-small-cell lung cancer to small-cell lung cancer: molecular drivers and cells of origin. Lancet Oncol. 2015;16(4): e165-e172. 6. Oxnard GR, Arcila ME, Sima CS, et al. Acquired resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant lung cancer: distinct natural history of patients with tumors harboring the T790M mutation. Clin Cancer Res. 2011;17(6):1616-1622. 7. Camidge DR, Pao W, Sequist LV. Acquired resistance to TKIs in solid tumours: learning from lung cancer. Nat Rev Clin Oncol. 2014;11(8):473-481. 8. Yu HA, Arcila ME, Rekhtman N, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013;19(8):2240-2247. 9. Popat S, Wotherspoon A, Nutting CM, Gonzalez D, Nicholson AG, O’Brien M. Transformation to “high grade” neuroendocrine carcinoma as an acquired drug resistance mechanism in EGFR-mutant lung adenocarcinoma. Lung Cancer. 2013;80(1):1-4. 10. Lim JU, Woo IS, Jung YH, et al. Transformation into large-cell neuroendocrine carcinoma associated with acquired resistance to erlotinib in nonsmall cell lung cancer. Korean J Intern Med. 2014;29(6):830-833. 11. Kogo M, Shimizu R, Uehara K, et al. Transformation to large cell neuroendocrine carcinoma as acquired resistance mechanism of EGFR tyrosine kinase inhibitor. Lung Cancer. 2015;90(2):364-368. 12. Zakowski MF, Ladanyi M, Kris MG; Memorial Sloan-Kettering Cancer Center Lung Cancer OncoGenome Group. EGFR mutations in small-cell lung cancers in patients who have never smoked. N Engl J Med. 2006;355(2):213-215. 13. Morinaga R, Okamoto I, Furuta K, et al. Sequential occurrence of non-small cell and small cell lung cancer with the same EGFR mutation. Lung Cancer. 2007;58(3):411-413. 14. Fallet V, Ruppert AM, Poulot V, et al. Secondary resistance to erlotinib: acquired T790M mutation and small-cell lung cancer transformation in the same patient. J Thorac Oncol. 2012;7(6): 1061-1063. 15. Suda K, Murukami I, Sakai K, et al. Small cell lung cancer transformation and T790M mutation: complimentary roles in acquired resistance to kinase inhibitors in lung cancer. Sci Rep. 2015;5:14447. 16. Niederst MJ, Sequist LV, Poirier JT, et al. RB loss in resistant EGFR mutant lung adenocarcinomas that transform to smallcell lung cancer. Nat Commun. 2015;6:6377. 17. Yang J, Ramalingam SS, Jänne PA, Cantarini M, Mitsudomi T. LBA2_PR: osimertinib (AZD9291) in pre-treated pts with T790M-positive advanced NSCLC: updated Phase 1 (P1) and pooled Phase 2 (P2) results. J Thorac Oncol. 2016;11(4 suppl): S152-S153. 18. Mok TS, Wu YL, Ahn MJ, et al; AURA3 Investigators. Osimertinib or platinum-pemetrexed in EGFR T790M-positive lung cancer. N Engl J Med. 2017;376(7):629-640. 19. Oxnard GR, Thress KS, Alden RS, et al. Association between plasma genotyping and outcomes of treatment with osimertinib (AZD9291) in advanced non-small-cell lung cancer. J Clin Oncol. 2016;34(28):3375-3382.
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Large Cell Neuroendocrine Carcinoma Transformation and EGFR-T790M Mutation as Coexisting Mechanisms of Acquired Resistance to EGFR-TKIs in Lung Cancer Sara Baglivo, MSc; Vienna Ludovini, PhD; Angelo Sidoni, MD; Giulio Metro, MD; Biagio Ricciuti, MD; Annamaria Siggillino, MSc; Alberto Rebonato, MD, PhD; Salvatore Messina, MD; Lucio Crinò, MD; and Rita Chiari, MD, PhD Abstract Acquired resistance to tyrosine kinase inhibitors (TKIs) represents the Achilles’ heel of targeted treatment in lung cancer. Epidermal growth factor receptor (EGFR)-TKIs are considered the standard first-line treatment for patients with EGFR mutant nonesmall cell lung cancer; however, after a median of 9 to 12 months, virtually all patients develop acquired resistance, which is mediated by the development of an EGFR-T790M secondary mutation in approximately 60% of cases. Different mechanisms of acquired resistance have also been described with lower incidence, including mutations in other driver oncogenes or phenotypic transformation. Herein, we report the first case of a patient with EGFR-mutant lung adenocarcinoma with a long-lasting response to first-line erlotinib treatment who acquired resistance to treatment because of acquisition of both EGFR-T790M mutation and “high-grade” large cell neuroendocrine transformation. This case also shows how resistance to third-generation EGFR-TKI osimertinib can be mediated by the development of phenotypic neuroendocrine transformation, which in the present case occurred during first-line treatment with erlotinib. In addition, our report highlights the pivotal role of rebiopsy and of molecular profiling at the time of progression to guide clinicians to choose the right therapy for the right patient. ª 2017 Mayo Foundation for Medical Education and Research
From the Medical Oncology Division (S.B., V.L., G.M., B.R., A.S., R.C.) and Nuclear Medicine Division (S.M.), Azienda Ospedaliera di Perugia, Perugia, Italy; Department of Experimental Medicine, Section of Pathological Anatomy and Histology, Medical School , University of Perugia, Perugia, Italy (A.S.); Radiology Unit, Department of Surgical and Biomedical Science, Santa Maria della Misericordia University Hospital, Perugia University, Perugia, Italy (A.R.); and Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy (L.C.).
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ung cancer is the leading cause of cancer-related deaths worldwide in both men and women, representing 27% of all cancer deaths.1 Recent advances in the understanding of molecular mechanisms underlying the development of none small cell lung cancer (NSCLC) have led to a classification based on the biological features of each tumor, such that NSCLC becomes a group of different diseases, each with its own specific clinical and biological characteristics. The discovery of sensitizing epidermal growth factor receptor (EGFR) mutations as a predictive marker of sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs) has revolutionized the therapeutic approach to this subgroup of patients with advanced NSCLC who represent 10% to 15% of white patients and 40% to 60% of Asian patients.2 First- and second-
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generation EGFR-TKIs have become the standard first-line treatment for patients with advanced EGFR-mutant NSCLC, having definitely shown in phase III trials a higher efficacy compared with platinum-based chemotherapy.3 Unfortunately, acquired resistance to treatment invariably occurs within 9 to 12 months and is primarily mediated by the EGFRT790M secondary mutation that accounts for approximately 60% of cases. Other acquired resistance mechanisms have also been described, including mutations in other driver oncogenes or phenotypic transformation.4-7 Herein, we report the first case of a patient with EGFR-mutant lung adenocarcinoma with a long-lasting response to first-line erlotinib treatment who acquired resistance to EGFR-TKIs because of the development of
Mayo Clin Proc. n August 2017;92(8):1304-1311 n http://dx.doi.org/10.1016/j.mayocp.2017.03.022 www.mayoclinicproceedings.org n ª 2017 Mayo Foundation for Medical Education and Research
TWO MECHANISMS OF ACQUIRED RESISTANCE TO ERLOTINIB IN LUNG CANCER
both EGFR-T790M mutation and “high-grade” large cell neuroendocrine carcinoma (LCNEC) transformation coexisting in the same adrenal metastatic lesion. RESULTS We present the case of a 57-year-old male patient who never smoked and was diagnosed in November 2011 with stage IV (cT1b N2M1b [bone and brain] tumor, node, metastasis staging version 7.0), grade 3, thyroid transcription factor 1 (TTF1) positive lung adenocarcinoma (Figure 1A(a) and B(a)). Sequence analysis on the transbronchial lung agobiopsy sample obtained at diagnosis scored positive for EGFR exon 19 deletion (2236_ 2252del18/L747_S752del) (Table), and the patient received erlotinib 150 mg once daily as first-line treatment. One month later the patient achieved a partial response that turned into a radiologically complete response after 4 months (Figure 1B). The patient had been treated with erlotinib until January 2014 when he had a relapse in the posterior basal segment of the right lower lobe, which was present as a single lesion. The patient was considered eligible for radical surgery and underwent atypical lung resection (Figure 1A(d)). The microscopical examination confirmed the same histology of the primary tumor (adenocarcinoma Periodic AcideSchiff diastase stain positive (PAS-Dþ), TTF1þ, Cytokeratin 7 positive (CK 7þ), Cytokeratin 20 negative (CK 20), napsinþ, and Ki-67 60%) (Figure 2, A and B1), whereas EGFR genotyping (exon 18, 19, 20, and 21) revealed the presence of exon 19 deletion together with the presence of T790M resistance (2369C>T) mutation. After lung atypical resection, treatment with erlotinib was pursued for 24 months until January 2016 when the patient had disease progression at both cranial (multiple lesions of 3-4 mm in diameter) and extracranial (mediastinal node and left adrenal gland) sites (Figure 1A(f)). Subsequently, the patient underwent whole-brain radiotherapy (total dose, 30 Gy/10 f), which obtained complete response in the brain that was still ongoing until the last available brain magnetic resonance imaging scan (>9 months) (Figure 1C). At the same time, the patient underwent ultrasound-guided biopsy of the mediastinal lymph node that confirmed adenocarcinoma histology along with the Mayo Clin Proc. n August 2017;92(8):1304-1311 www.mayoclinicproceedings.org
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presence of EGFR-del19 and T790M mutations (Figure 2C, and Table). Erlotinib was discontinued, and second-line treatment with osimertinib (AZD9291) was initiated in January 2016. One month later, the mediastinal lymph node disappeared but disease progression occurred in the left adrenal gland (Figure 1, A(g)). The patient underwent left adrenalectomy, and histological examination revealed 2 coexisting distinct components: adenocarcinoma (TTF1þ and CK-7þ) and LCNEC (synaptophysinþ, TTF-1þ, and CK-7 “dot-like” positive) (Figure 2D). Both the components, which were separately examined for EGFR mutations, harbored an EGFR exon 19 deletion mutation with no evidence of EGFR-T790M. After an accurate revision of the sample from the atypical lung resection of the right lower lobe (January 2014), we found that phenotypic transformation into LCNEC was already present as a small focus in that surgical sample, which at the time was assumed to be a specimen of adenocarcinoma bearing exon 19 and EGFR-T790M mutation (Figure 2B2). After surgery the patient underwent radiotherapy on the site of left adrenalectomy (48.4 Gy/22 f þ SYB 55 Gy/22 f) and resumed osimertinib. Unfortunately, 2 months later (May 2016) a radiologic revaluation exhibited disease progression at multiple bone sites (Figure 1A(h)), a picture that was consistent with rapidly progressing high-grade neuroendocrine tumor. Therefore, considering the histological features and the radiological evidence of widespread disease, the patient began receiving cisplatin-etoposide combination chemotherapy that led to a partial remission after 2 cycles, which was maintained until the end of the sixth cycle. Unfortunately, after 2 months from the end of platinum-etoposide chemotherapy, the patient had disease progression at multiple bone sites. Therefore, in light of the rapid worsening of his clinical condition, we decided to go on with best supportive care and the patient died after more than 61 months from diagnosis. During the course of his disease, we had the opportunity to collect 3 tumor samples (2 surgical samples and 1 small biopsy specimen) of erlotinib-refractory metastatic lesions, all of which harbored an EGFR exon 19 deletion mutation. At microscopic examination, the 2 surgical samples included both LCNEC
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FIGURE 1. (continued).
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FIGURE 1. A, Patient’s positron emission tomographyecomputed tomography (PET-CT) during the course of the patient’s disease: type of systemic and locoregional treatments (namely, erlotinib, osimertinib, and chemotherapy with cisplatin/etoposide, surgery, and extracranial radiotherapy) that the patient received over the time are depicted as well as a trasversal cut, showing lung relapse in January 2014 when he underwent atypical resection of the right lower lobe. PET-TC (a) before, (b) after 1 month, (c) after 4 months and (d) after 25 months of erlotinib treatment showing first PD; (e) CR after resection, (f) after 50 months of erlotinib therapy, (g) after 1 month, and (h) after 4 months of osimertinib treatment; (i) after 3 months from the beginning of chemotherapy showing PR. The timeline of treatment has different scales of measurement. B, Brain contrast-enhanced T1-weighted magnetic resonance imaging before (a), at 3 months (b), and at 6 months (c) after the beginning of first-line erlotinib treatment, showing the progressive disappearance of the centimetric cortical metastasis in the parietal left lobe (white arrow). C, Brain contrast-enhanced 3-dimensional T1-weighted spoiled gradient recalled-echo magnetic resonance imaging performed 4 years after the beginning of first-line erlotinib treatment, showing brain disease progression with 1 right cerebellar (a), 1 right occipital (b), and 2 parietal (c and d) metastases (white arrows). At 3- (data not shown) and 6-month follow-up after WBRT, contrast-enhanced T1-weighted magnetic resonance imaging confirmed the disappearance of all the lesions (e, f, g, and h). BSC ¼ best supportive care; CR ¼ complete response; PD ¼ progression disease; PR ¼ partial response; SD ¼ stable disease; WBRT ¼ whole-brain radiotherapy.
and adenocarcinoma histology coexisting in the same lesion. The adenocarcinoma component of the surgical sample of 2014 scored positive for EGFR-T790M, whereas both the adenocarcinoma and LCNEC components of the surgical lesion of 2016 were EGFRT790M negative at both direct sequencing and therascreen analysis. We subjected all the available tumor samples to digital polymerase chain reaction and next-generation sequencing (NGS) for a more Mayo Clin Proc. n August 2017;92(8):1304-1311 www.mayoclinicproceedings.org
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sensitive analysis and large spectrum mutation investigation and obtained the same results as described above. Moreover, no MET amplification was found in any samples by using digital polymerase chain reaction (QuantStudio 3D Digital PCR System, Life Technologies) analysis, and NGS analysis (Ion Torrent Personal Genome Machine (PGM), AmpliSeq Colon and Lung Research Panel v2, Life Technologies) did not reveal any mutations in the hotspot positions
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ND WT (CNV) WT ND WT (CNV) WT ND WT ND WT (CNV) WT ND WT (CNV) WT ND WT (CNV) WT ND ND WT ND ND WT ND WT ND ND WT ND ND WT ND ND WT ND ND WT ND ND WT ND WT ND ND WT ND ND WT ND ND WT ND ND WT ND ND WT ND WT ND ND WT ND ND WT ND ND WT cfDNA NA Blood Second erlotinib PD January 2016
Surgical sample Metastatic lesion LCNEC Adrenal gland February 2016 D
Osimertinib PD
February 2016 D
Osimertinib PD
Adrenal gland
ADC
Biopsy Metastatic lesion Surgical sample Metastatic lesion ADC Lymph node January 2016 C
Second erlotinib PD
Surgical sample Metastatic lesion ADC Right lung First erlotinib PD January 2014 B
NA Mayo Clin Proc.
ADC ¼ adenocarcinoma; cfDNA ¼ circulating free DNA; CNV ¼ copy number variation; dPCR ¼ digital polymerase chain reaction; EGFR ¼ epidermal growth factor receptor; LCNEC ¼ large cell neuroendocrine carcinoma; NA ¼ not applicable; ND ¼ not done; NGS ¼ next-generation sequencing; PD ¼ progression disease; WT ¼ wild type.
MET PTEN TP53 PIK3CA EGFR
WT ND WT ND ND WT ND WT ND ND WT ND ND WT ND ND WT Sanger sequencing dPCR NGS Therascreen dPCR NGS Therascreen NGS Therascreen dPCR NGS Therascreen dPCR NGS Therascreen dPCR NGS
Method Sample type
Transbronchial lung biopsy ADC
Histology Organ
Right lung Diagnosis
Status Time
November 2011 A
Lesion
TABLE. Summary of Primary/Metastatic Lesions and cfDNA Molecular Analysis
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L747_S752del del19 L747_S752del del19, T790M del19, T790M L747_S752del, T790M del19, T790M L747_S752del, T790M del19 del19 L747_S752del del19 del19 L747_S752del del19 del19 L747_S752del
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for 21 cancer-related genes analyzed by the specific NGS panel, including PIK3CA, PTEN, NOTCH1, TP53, and FGFR (for expansion of gene symbols, see www.genenames.org). Analysis of circulating tumor DNA at different time points from January 2016 (second progression during erlotinib treatment) and during osimertinib and chemotherapy treatment was positive for EGFR-del19, with no evidence either of EGFR-T790M or of any mutations in the 21 genes analyzed by the same NGS panel used for tumor tissue analysis (Table). DISCUSSION Several different mechanisms explaining acquired resistance to EGFR-TKIs in NSCLC have been identified, including on-target EGFR (60%) and off-target EGFR (20%) molecular alterations.4-7 The EGFR-T790M secondary mutation represents the most common mechanism of on-target EGFR acquired resistance. Among off-target EGFR molecular alterations, bypass track pathway activation, such as MET amplification (5%), and histological transformation to high-grade neuroendocrine tumors (10%) have been reported.7,8 More commonly, neuroendocrine transformation is represented by transition to small cell lung cancer (SCLC) but histological transformation into LCNEC has also been identified and described in a few reports.9-11 Herein, we report the case of a patient who developed, after a long-lasting response to erlotinib (50 months), both EGFR-T790M mutation and LCNEC transformation as mechanisms of acquired resistance to firstgeneration EGFR-TKIs. We were able to document the presence of both components (EGFR-T790M and LCNEC) after 25 months of erlotinib treatment in the same lung lesion and consequently in the same neoplastic clone that probably at that time had no metastatic potential and was eradicated by a locoregional treatment (right lower lobe atypical resection). On the contrary, after 50 months of erlotinib treatment, 3 components originating by the same neoplastic clone were present: (1) 1 clone bearing the EGFR-T790M gatekeeper mutation in the mediastinal lymph node that responded to the third-generation EGFR-TKI osimertinib and 2 other clones: (2) LCNEC and (3) T790M-negative adenocarcinoma components with high metastatic potential coexisting in
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adrenal metastasis that were both positive for EGFR-del19. Intriguingly, these last 2 components were both EGFR-T790M negative and insensitive to osimertinib. To our knowledge, 6 patients with SCLC transformation during treatment with EGFR-TKIs who had data for multiple
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metastatic lesions have been reported so far. In 3 of them, all metastatic lesions exhibited SCLC transformation without evidence of the EGFR-T790M mutation, whereas in the other 3, some lesions were T790M positive and others had SCLC phenotype, thus suggesting that SCLC transformation and EGFR-T790M
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mutation occur in a reciprocal manner in a single patient upon acquisition of resistance to EGFR-TKIs.4,12-16 Herein, we presented the first case in which EGFR-T790M mutation and neuroendocrine transformation exist together in the same lesion (lung 2014). Both these components had been documented in vivo more than 2 years before widespread disease progression, which was correlated with the aforementioned components sharing the original EGFR-activating mutation (del19) that we found in adrenal metastasis: (1) the highgrade neuroendocrine component responding to platinum-etoposide combination and (2) the adenocarcinoma EGFR-T790Menegative component rapidly progressing after platinum/etoposide debulking. In both these components, EGFR expression was silenced (immunohistochemistry: data not shown) as previously reported for EGFR-mutated lung adenocarcinoma and SCLC with primary resistance to EGFR-TKIs.16 This could explain the resistance to both erlotinib and osimertinib. Except for EGFR mutation, no other co-occurring mutations have been identified in any samples collected, neither in TP53 gene nor in other known driver oncogenes such as PIK3CA or MET. No EGFR-C797S third-generation TKI acquired resistance mutation was found, either on tissue biopsies or on liquid biopsies. This case highlights the importance of rebiopsy and of molecular analysis of sequential samples of the same patient during the course of disease to help clinicians
make the best treatment choice as possible. Until recently, upon documented disease progression on first- or second-generation EGFR TKIs, chemotherapy was the mainstay of second-line treatment for patients with EGFR-mutant NSCLC. Nonetheless, recent advances in the understanding of the molecular mechanism underlying the development of resistance to EGFR-TKIs revealed that T790M secondary mutation is responsible for approximately 60% of cases of acquired resistance. To overcome T790M-mediated resistance, novel third-generation EGFR-TKIs have been developed. Among these, osimertinib showed in phase I/II trials (clinicaltrials. gov Identifier: NCT01802632 and NCT02094261) promising results in pretreated patients with EGFR-mutant NSCLC.17 More recently, in a phase III trial (clinicaltrials.gov Identifier: NCT02151981), osimertinib was definitely proved to be superior to chemotherapy in terms of objective response rate and progression-free survival as second-line therapy for patients with EGFRmutant NSCLC harboring T790M secondary mutation who had progressed on or following a first-line EGFR-TKI treatment with particular evidence in brain metastasis control, which seems to improve with osimertinib because of the passage of the drug across the bloodbrain barrier.18 Unfortunately, resistance to third-generation EGFR-TKIs occurs in virtually all patients. Although the tertiary EGFR mutation C797S has recently been reported as one of the possible mechanisms of resistance, in most cases a specific mechanism
FIGURE 2. Histopathological and immunophenotypic evolution. A, Morphological picture of the transbronchial biopsy performed in November 2011: adenocarcinoma (right) and infiltrating lung parenchyma (left) (hematoxylin-eosin, original magnification 200). B, In the atypical resection of the right inferior lobe (January 2014) at the periphery of the adenocarcinoma, a small focus of undifferentiated carcinoma (left side of the inset B1: hematoxylin-eosin, original magnification 200) was present, showing a strong positivity for synaptophysin (inset B2: peroxidase, original magnification 200). C, Cell block obtained by fine needle aspiration cytology from a mediastinal lymph node (January 2016), showing fragments of adenocarcinoma (hematoxylin-eosin, original magnification 400). D, Adrenalectomy (February 2016) revealed a synchronous metastasis of adenocarcinoma (left) and undifferentiated large cells carcinoma (right) in cortical metastasis, with extension of the latter component into the retroperitoneal adipose tissue (hematoxylin-eosin, original magnification 200). E, Immunohistochemical staining for synaptophysin in the same area revealed strong positivity in the large cell component (right inset); the adenocarcinomatous component was completely negative (left inset) (peroxidase, original magnification 200; insets: original magnification 400). LCNEC ¼ large cell neuroendocrine carcinoma.
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cannot be identified. Our case suggests that phenotypic transformation into LCNEC might represent a novel mechanism of resistance to osimertinib, however, also being maintained in the LCNEC phenotype the presence of the EGFR-del19 mutation. Notably, upon documentation of such phenotypic transformation, in the absence of any potentially targetable genetic alteration, the patient was treated with an “LCNEC-like” chemotherapy, obtaining a partial response. This highlights the fundamental role of rebiopsy at disease progression that in our case allowed a proper molecularly tailored treatment of the disease, which translated into a clinically meaningful survival benefit of more than 61 months from diagnosis. Because biopsies at disease progression may not always be feasible, circulating tumor DNA analysis might provide a fast and noninvasive diagnostic tool. However, because of technical issues and low sensitivity, plasma genotyping should be considered complementary to traditional biopsies.19 CONCLUSION To our knowledge this is the first evidence of EGFR-T790Mepositive lung adenocarcinoma and LCNEC transformation existing together in the same lesion as shown in the resected first lung cancer relapse of the disease, with the subsequent T790M-positive adenocarcinoma being the putative intermediate step that precedes the development of the mixed metastatic phenotype consisting in LCNEC and adenocarcinoma (both EGFR-del19 positive) present in the adrenal gland. We do not have any formal proof that this means that EGFRT790Mepositive, EGFR-T790Menegative, and LCNEC components are subclones deriving from the same cell, but this is far more reasonable than simple intrametastatic heterogeneity. Abbreviations and Acronyms: EGFR = epidermal growth factor receptor; LCNEC = large cell neuroendocrine carcinoma; NGS = next-generation sequencing; NSCLC = none small cell lung cancer; SCLC = small cell lung cancer; TKI = tyrosine kinase inhibitor Grant Support: The work was supported by the Italian Association for Cancer Research (AIRC; grant number 9979) and the Umbria Association Against Cancer (AUCC). Correspondence: Address to Sara Baglivo, MSc, Medical Oncology Division, Azienda Ospedaliera di Perugia, 06132 Perugia, Italy ([email protected]). Mayo Clin Proc. n August 2017;92(8):1304-1311 www.mayoclinicproceedings.org
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