Small Cell Lung Cancer Transformation Research Articles

430P - Resistance mechanisms causing first-line epidermal growth factor receptor-tyrosine kinase inhibitor treatment failure

Background: Patients with epidermal growth factor receptor (EGFR) mutant advanced non-small cell lung cancer receiving first-line EGFR-tyrosine kinase inhibitor (TKI) inevitably developed disease progression after 9-13 months. Methods: Before 1st January 2017, patients were investigated for resistance mechanisms upon failure of first-line EGFR-TKI by means of tissue re-biopsy or liquid biopsy to detect secondary T790M mutation in plasma cell free tumor-DNA (cfDNA) if tissue re-biopsy could not be performed. After that, liquid biopsy followed by tumor re-biopsy if cfDNA was negative for T790M mutation or if the patients had rapidly enlarging tumors. Results: Of 45 patients who were tested, 31(68.9%) underwent tissue re-biopsy and 14 (31.1%) underwent liquid biopsy as the first investigation to determine the presence of T790M mutation. For the latter group, 4 (8.9%) subsequently also had tumor re-biopsy. T790M mutation was detected in 30 (66.7%) of the 45 patients. C-Met amplification was tested in 7 T790M mutation-negative patients for possible recruitment into a clinical trial with 4 showing c-Met amplification. Small cell lung cancer transformation and ALK rearrangement were detected in 2 (5.7%) and in 1 (2.9%) of the re-biopsy tissue specimens, respectively. The resistance mechanisms in 8 patients (17.8%) was unknown. In short, two-third (66.7%) of our patients had T790M mutation upon first-line EGFR-TKI failure; while another one-third (33.3%) failed first-line EGFR-TKI due to other resistance mechanisms. The demographic, clinical and treatment characteristics were equally distributed among these 2 groups of patients. (Table)Table: 430P Demographic, clinical and treatment characteristics of 45 patients with first-line EGFR-TKI treatment failureCharacteristicTotal number of patients (n = 45)Patients with T790M mutation (n = 30)Resistance mechanism other than T790M (n = 15)P value of univariate analysis*Gender, No. (%) Male Female18 (40.0) 27 (60.0)13 (43.3) 17 (56.7)5 (33.3) 10 (66.7)0.780Smoking history, No. (%) Never smoker Previous or current smoker39 (80.0) 9 (20.0)22 (73.3) 8 (26.7)14 (93.3) 1 (6.7)0.963EGFR mutation subtype, No. (%) Exon 19 deletion Exon 21 L858R mutation Others Unsure26 (57.8) 16 (35.6) 2 (4.4) 1 (2.2)17 (56.7) 12 (40.0) 1 (3.3) 09 (60.0) 4 (26.7) 1 (6.7) 1 (6.7)0.999Treatment received, No. (%) EGFR-TKI EGFR-TKI followed by chemotherapy34 (75.5) 11 (24.5)22 (73.3) 8 (26.7)12 (80.0) 5 (20.0)0.484Best tumour response, No. (%) Partial response Stable disease Progression of disease38 (84.4) 6 (13.3) 1 (2.2)27 (90.0) 2 (6.7) 1 (3.3)11 (73.3) 4 (26.7) -0.419Progression-free-survival on first-line EGFR-TKI, months Median13.013.011.70.538 Open table in a new tab Conclusions: T790M mutation is the commonest acquired resistance mechanism causing first-line EGFR-TKI treatment failure. There was no difference in the clinical and treatment characteristics between patients with and without acquired T790M mutation as causes of resistance to first-line EGFR-TKI treatment. Legal entity responsible for the study: Chai Chee Shee, Liam Chong Kin Funding: None Disclosure: All authors have declared no conflicts of interest.

MTE 10.01 Bridging the Gap between Genomics and Clinics

MTE 10.01 Bridging the Gap between Genomics and Clinics

Abstract 4583: Heterogeneity in immune biomarker expression: Detailed analysis of a case with SCLC transformation after EGFR-TKI treatment

Abstract Introduction: The expression status of immune markers are of current research interest due to their potential roles as predictive biomarkers for immunotherapy in cancers. Currently there is little information on how the microenvironment of tumors and/or therapy-inducible histological transformation may affect the expression of these immune markers. Methods: A 76-year-old never-smoking female with epidermal growth factor receptor (EGFR) mutated lung adenocarcinoma (AC) acquired resistance to gefitinib. After her death, autopsy revealed small-cell lung cancer (SCLC) transformation and EGFR T790M secondary mutation as mutually exclusive resistance mechanisms. Two liver metastases (SCLC vs. AC with T790M) and two lymph node metastases (SCLC vs. AC with T790M) were analyzed to compare the expression status of immune markers by immunohistochemistry (IHC) and an immune-oncology gene expression panel (HTG EdgeSeq Immuno-Oncology Assay). Results: IHC analysis revealed that PD-L1 was expressed in 5% of tumor cells with AC histology (T790M) but not in tumor cells with SCLC transformation (Table 1). The liver metastasis with SCLC transformation showed negative stromal PD-L1 expression and scant tumor infiltrating lymphocytes, while the other lesions demonstrated stromal PD-L1 staining and infiltration of CD8-positive T cells. PD-1 was positive in lymphocytes from lymph node metastases, but not in those from liver metastases. Data generated using an IO panel indicated higher expression of galectin 9 and higher level of T cell stimulatory checkpoints in lesions with SCLC transformation. Bioinformatic analysis also demonstrated lower expression of type I interferon regulated genes in lesions with SCLC transformation. Conclusion: These data highlight the heterogeneity of expression of immune markers depending on the metastatic sites and histological transformation, and indicate that a biopsy from one lesion may not be representative of immune marker status for all lesions. Summary of immune marker IHC staining in all specimensSpecimensPD-L1 (tumor)PD-L1 (stroma)TILsCD8 (T cells)PD-1 (T cells)Liver metastasis (AC / T790M)5%5%+++negativeLiver metastasis (SCLC)negativenegativescantn.a.n.a.Lymph node metastasis (AC / T790M)5%5%++++Lymph node metastasis (SCLC)negative10%++++ Citation Format: Kenichi Suda, Isao Murakami, Hui Yu, Jihye Kim, Kim Ellison, Christopher J. Rivard, Tetsuya Mitsudomi, Fred R. Hirsch. Heterogeneity in immune biomarker expression: Detailed analysis of a case with SCLC transformation after EGFR-TKI treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4583. doi:10.1158/1538-7445.AM2017-4583

JCES01.24 Molecular Mechanism of Transformation from Adenocarcinoma to Small-Cell Lung Cancer after EGFR-TKI

In patients with advanced non–small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) activating mutations, EGFR-tyrosine kinase inhibitors (TKIs) are recommended as first-line treatment due to favorable clinical efficacy. However, acquired resistance inevitably develops after median progression-free survival (PFS) of 9-14 months. Among the mechanisms of acquired resistance, small-cell lung cancer (SCLC) transformation was reported to account for nearly 5%. However, the molecular details underlying this histological change and resistance to EGFR-TKI therapy remain unclear. 15 out of 233 (6.4%) patients were confirmed to develop SCLC transformation after failure to EGFR-TKI. We analyzed the clinical parameters of these patients by using chi-square test and Kaplan-Meier analysis. To explore gene alterations that might contribute to SCLC transformation, next generation sequencing (NGS) was performed on four pairs of matched pre- and post-transformation tumor tissue samples. We further performed NGS on 11 matched circulating tumor DNA (ctDNA) to explore the potential mechanism of resistance to EGFR-TKI. The median age of SCLC transformed patients was 53 years. 93.3% (14/15) patients harbored EGFR exon19 deletion. The median PFS and overall survival (OS) of SCLC-transformed patients treated with EGFR-TKI compared to those without transformation were 11.7 versus 11.9 months (P=0.473) and 29.4 versus 24.3 months (P=0.664), respectively. All 4 patients developed loss of heterozygosity of TP53/RB1 after transformation. Besides, increased copy number of five proto-oncogenes were identified in post-transformation tissue samples. Three patients developed EGFR T790M mutation in the post-transformation ctDNA rather than their tissue samples. SCLC transformation was commonly seen in patients harboring EGFR exon 19 deletion. The clinical outcomes of TKI and OS in SCLC transformed patients were similar to non-transformed patients. The loss of heterozygosity of TP53 and RB1along with increased copy number of proto-oncogenes may lead to the SCLC transformation. The mechanisms of acquired resistance to TKI during SCLC transformation might be the emergence of classic drug resistance mutations, which was undetectable due to the intra-tumor heterogeneity.

P1.06-005 An International Cohort of Patients with Small Cell Lung Cancer after a Non-Small Cell Lung Carcinoma Oncogene or Non-Oncogene Addicted

Phenotypic transformation from Non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is a resistance mechanism in tyrosine kinase inhibitors (TKIs) treated EGFR mutant tumors. SCLC is also however less frequently diagnosed in patients without EGFR mutations treated with chemotherapy. These transformations are rare and little is known about the clinical and therapeutic characteristics of these patients. In this study we describe and compare the characteristics of SCLC arising from mutant or non-mutant NSCLC. We performed a multicentric retrospective collection (27 centers in France and Italy) of cases. Between 2005 and 2015, patients with stage III or IV NSCLC with a secondary transformation to SCLC with histological proof were included. Forty seven cases of SCLC transformation were collected, 34 in EGFR mutant and 13 in non. Most of the patients (n=37, 82%) were stage IV, (n=27, 57%) female and (n=26, 76%) had an exon 19 mutation. The last treatment before transformation was a TKI in 23 (68%) cases in the mutant group and in 3 (23%) patients (erlotinib) in the non-mutant. Median time to SCLC transformation was 17 [IQR, 11-29] months in the mutant group and 26 [IQR 23-36] months in the other (p=0.03). Molecular analyses were not performed in the non-mutant group, 25 (74%) had molecular analyses in the EGFR mutant. A driver mutation was identified in 22/25 (88%) patients: in most of the cases the same as the initial, 1 case of ALK fusion and 1 of PI3K mutation. Thirty patients (88%) received at least one line of treatment after transformation in the mutant group, in all cases a platinum-etoposide (P-E) chemotherapy. Median survival from initial diagnosis in the EGFR mutant group was significantly worse 28 [17-41] months vs 49 [36-118] months in the non EGFR mutant group (p=0.01). After transformation, the same tendency was observed with a median survival of 8 [3-12] months for the EGFR mutated patients vs 13 [6-15] months for the non EGFR mutated patients (p=0.06). SCLC that occurs in EGFR mutant treated by TKIs is more aggressive than classic SCLC, and differs on epidemiological characteristics. These transformed SCLC are not fully explained and we need to define the molecular characteristics of this cohort, before and after transformation and if funded the whole genome sequencing of the tumors to understand this TKIs mechanism of resistant.

SC04.02 Management of Resistance to EGFR Tyrosine Kinase Inhibitors

Discovery of activating mutations of the EGFR gene in adenocarcinoma of the lung in 2004 opened the door to a new era for personalized therapy in thoracic oncology. Lung cancers with EGFR mutation are highly sensitive to EGFR-tyrosine kinase inhibitors (TKI) such as gefitinib, erlotinib, or afatinib, resulting in significantly prolonged progression free survival compared with those treated with platinum doublet chemotherapy. However, acquired resistance inevitably develops usually after a median of 10∼12 months. The mechanisms for this resistance have been extensively studied and can be classified into 1) target gene alteration, 2) activation of bypass / accessory pathway, and 3) histologic transformation (Fig). The most common (50∼60%) mechanism for acquired resistance to the EGFR-TKI is a missense mutation at codon 790 of the EGFR gene resulting in substitution of threonine to methionine (T790M). This amino acid change reduces affinity between EGFR kinase and EGFR-TKI compared with that between EGFR-kinase and ATP, leading to reactivation of down-stream pathways. L747S, D761Y, and T854A are also known as secondary mutations that cause acquired resistance, but they are very rare. In these cases, cancer cells are still addicted to or dependent on EGFR pathway. Amplification of the MET gene which codes for a receptor of hepatocyte growth factor (HGF) was the first that was identified as a bypass track resistance mechanism against EGFR-TKI. Following this report, aberrant activation of other receptor tyrosine kinases such as HER2, HER3, AXL, IGF1R, have been reported. It is also shown that some ligands for the receptor tyrosine kinases such as HGF, FGF or IGF cause acquired resistance to EGFR-TKIs. Similarly, alteration of downstream molecule cause resistance. These molecules include BRAF, PTEN, JAK2, CRKL, DAPK, NF-kB, or PUMA. The third mechanism of acquired resistance is histologic transformation that includes small cell lung cancer transformation and epithelial-mesenchymal transition EMT). Exact mechanisms of these histologic changes are not fully understood. However, AXL, Notch-1, TGFb pathway activation as well as down regulation of MED12 ((Mediator Complex Subunit 12) have been proposed as mechanisms of EMT. Then, How are we able to cope with these resistance? For T790M gatekeeper mutations, the third generation EGFR inhibitors that selectively inhibit EGFR-T790M while sparing the wild-type EGFR are active. One of these drugs, osimertinib is already approved and gives a response rate of ∼60% and progression free survival of ∼11 months. Therefore, identification of T790M at the time of disease progression by rebiopsy is important. We have recently found that three other secondary EGFR mutations implicated in acquired resistance are also sensitive to osimertinib. Tumor resistance caused by activation of accessory pathways can be theoretically coped with by combination of the inhibitor of EGFR and involved molecules. However, because of rarity of each mechanism, there is no clear evidence whether these combination therapies will actually improve patient outcome In other cases, cytotoxic chemotherapy is still an important strategy. According to the IMPRESS study, median progression free survival for patients without T790M who received cisplatin plus pemetrexed was 5.4 months. Even with these strategies, cancer cells are smart enough to escape from the therapy using other mechanisms. Heterogeneities in terms of resistant mechanisms within a single patient become evident when specific therapeutic pressure persists. Therefore, we also need to have armamentarium that utilizes other mechanisms to cure lung cancer. Recent advances of immunotherapy targeting immune checkpoints appear attractive in this respect. These mechanism-driven therapeutic approaches will convert this fatal disease into a more chronic disorder, and eventually into a curable disease with the least patient burden. Adenocarcinoma, EGFR, Resistance, T790M

P3.02b-116 Molecular Mechanism of Transformation from Adenocarcinoma to Small-Cell Lung Cancer after EGFR-TKI: Topic: EGFR RES

In patients with advanced non–small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) activating mutations, EGFR-tyrosine kinase inhibitors (TKIs) are recommended as first-line treatment due to favorable clinical efficacy. However, acquired resistance inevitably develops after median progression-free survival (PFS) of 9-14 months. Among the mechanisms of acquired resistance, small-cell lung cancer (SCLC) transformation was reported to account for nearly 5%. However, the molecular details underlying this histological change and resistance to EGFR-TKI therapy remain unclear. 15 out of 233 (6.4%) patients were confirmed to develop SCLC transformation after failure to EGFR-TKI. We analyzed the clinical parameters of these patients by using chi-square test and Kaplan-Meier analysis. To explore gene alterations that might contribute to SCLC transformation, next generation sequencing (NGS) was performed on four pairs of matched pre- and post-transformation tumor tissue samples. We further performed NGS on 11 matched circulating tumor DNA (ctDNA) to explore the potential mechanism of resistance to EGFR-TKI. The median age of SCLC transformed patients was 53 years. 93.3% (14/15) patients harbored EGFR exon19 deletion. The median PFS and overall survival (OS) of SCLC-transformed patients treated with EGFR-TKI compared to those without transformation were 11.7 versus 11.9 months (P=0.473) and 29.4 versus 24.3 months (P=0.664), respectively. All 4 patients developed loss of heterozygosity of TP53/RB1 after transformation. Besides, increased copy number of five proto-oncogenes were identified in post-transformation tissue samples. Three patients developed EGFR T790M mutation in the post-transformation ctDNA rather than their tissue samples. SCLC transformation was commonly seen in patients harboring EGFR exon 19 deletion. The clinical outcomes of TKI and OS in SCLC transformed patients were similar to non-transformed patients. The loss of heterozygosity of TP53 and RB1 along with increased copy number of proto-oncogenes may lead to the SCLC transformation. The mechanisms of acquired resistance to TKI during SCLC transformation might be the emergence of classic drug resistance mutations, which was undetectable due to the intra-tumor heterogeneity.

A case treated with nivolumab after small cell lung cancer transformation of mutant EGFR non-small cell lung cancer

A case treated with nivolumab after small cell lung cancer transformation of mutant EGFR non-small cell lung cancer

Small cell lung cancer transformation and the T790M mutation: A case report of two acquired mechanisms of TKI resistance detected in a tumor rebiopsy and plasma sample of EGFR-mutant lung adenocarcinoma.

The present study describes the case of a 45-year-old man diagnosed with metastatic lung adenocarcinoma, which harbored a deletion within exon 19 of the epidermal growth factor receptor (EGFR) gene. The patient was subsequently treated with gefitinib (250 mg/day orally from May 2013 to March 2014), but developed acquired resistance to the drug following 11 months of treatment. Tumor burden molecular analysis was performed on a tumor rebiopsy and plasma sample, and histological analysis was also performed on the tumor rebiopsy. A small cell transformation retaining the original EGFR mutation was detected in the tumor rebiopsy, while the T790M mutation together with the activating ex19del mutation were identified only in the plasma sample. The patient was treated with cytotoxic chemotherapy (off-label schedule with epirubicin 80 mg/mq and paclitaxel 160 mg/mq every 21 days for 6 cycles) and radiation (50.4 Gy administered in 28 fractions of 1.8 Gy once daily for 5.5 weeks) specific for small cell lung cancer, and may also have benefitted from treatment with a third generation T790M-specific EGFR-TKI. To better describe the mechanisms of resistance to TKI inhibitors and to optimize therapeutic regimens, the simultaneous analysis of tumor biopsies and circulating tumor DNA should be considered.

Bronchoscopic Re-biopsy for Mutational Analysis of Non-small Cell Lung Cancer.

Currently, several acquired resistance mechanisms and rare driver oncogenes are identified in non-small cell lung cancer (NSCLC) relapses. Re-biopsy increases valuable information to guide treatment strategies, but the utility and feasibility of bronchoscopic re-biopsy has not been investigated. We studied 70 patients who underwent bronchoscopic for re-biopsy of NSCLC that was resistant to at least one regimen of chemotherapy or molecular-targeted therapy between January 2013 and December 2014. We assessed clinical data, technical success rate, and mutational analysis. Procedures performed were transbronchial biopsy (n=52) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) (n=18). Overall detection rate of re-biopsy for malignant cells was 87% (83% for TBB and 100% for EBUS-TBNA). Mutational analysis was possible in almost all technically successful cases; likewise, acquired-resistant mutations (55% of EGFR mutants) and small cell lung cancer transformation were identified from the bronchoscopy specimens. Other driver mutations were seen in four cases, including ALK fusion gene (n=2) and ROS1 fusion gene (n=2). There were no associated severe complications. This study shows that bronchoscopic re-biopsy for NSCLC is feasible and provides adequate samples that enable identification of resistance mutations and rare driver oncogenes.

The mechanism of acquired resistance to irreversible EGFR tyrosine kinase inhibitor-afatinib in lung adenocarcinoma patients.

IntroductionEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are associated with favorable response in EGFR mutant lung cancer. Acquired resistance to reversible EGFR TKIs remains a significant barrier, and acquired EGFR T790M-mutation is the major mechanism. Second-generation irreversible EGFR TKI, afatinib, had also been approved for treating EGFR mutant lung cancer patients, but the mechanism of acquired resistance to afatinib has not been well studied.ResultsForty-two patients had tissue specimens taken after acquiring resistance to afatinib. The sensitizing EGFR mutation were all consistent between pre- and post-afatinib tissues. Twenty patients (47.6%) had acquired T790M mutation. T790M rate was not different between first-generation EGFR TKI-naïve patients (50%) and first-generation EGFR TKI-treated patients (46.4%) (p = 0.827). No clinical characteristics or EGFR mutation types were associated with the development of acquired T790M. No other second-site EGFR mutations were detected. There were no small cell or squamous cell lung cancer transformation. Other genetic mutations were not identified in PIK3CA, BRAF, HER2, KRAS, NRAS, MEK1, AKT2, LKB1 and JAK2.MethodsAfatinib-prescription record of our department of pharmacy from January 2007 and December 2014 was retrieved. We investigated patients with tissue specimens available after acquiring resistance to afatinib. Enrolled patients should have partial response or durable stable disease of treatment response to afatinib. Various mechanisms of acquired resistance to first-generation EGFR TKIs were evaluated. Histology and cytology were reviewed. EGFR, PIK3CA, BRAF, HER2, KRAS, NRAS, MEK1, AKT2, LKB1 and JAK2 genetic alterations were evaluated by sequencing. Statistical analysis was performed using Chi-square test and Kaplan-Meier method.ConclusionsT790M was detected in half of the lung adenocarcinoma after acquiring resistance to afatinib. T790M is still the major acquired resistance mechanism. First-generation EGFR TKI exposure did not influence the prevalence of T790M in lung cancer acquired resistance to afatinib.

Small cell lung cancer transformation and T790M mutation: complimentary roles in acquired resistance to kinase inhibitors in lung cancer

Lung cancers often harbour a mutation in the epidermal growth factor receptor (EGFR) gene. Because proliferation and survival of lung cancers with EGFR mutation solely depend on aberrant signalling from the mutated EGFR, these tumours often show dramatic responses to EGFR tyrosine kinase inhibitors (TKIs). However, acquiring resistance to these drugs is almost inevitable, thus a better understanding of the underlying resistance mechanisms is critical. Small cell lung cancer (SCLC) transformation is a relatively rare acquired resistance mechanism that has lately attracted considerable attention. In the present study, through an in-depth analysis of multiple EGFR-TKI refractory lesions obtained from an autopsy case, we observed a complementary relationship between SCLC transformation and EGFR T790M secondary mutation (resistance mutation). We also identified analogies and differences in genetic aberration between a TKI-refractory lesion with SCLC transformation and one with EGFR T790M mutation. In particular, target sequencing revealed a TP53 P151S mutation in all pre- and post-treatment lesions. PTEN M264I mutation was identified only in a TKI-refractory lesion with SCLC transformation, while PIK3CA and RB1 mutations were identified only in pre-treatment primary tumour samples. These results provide the groundwork for understanding acquired resistance to EGFR-TKIs via SCLC transformation.

Abstract 1451: Modeling patient-derived lung cancer in mice: Preclinical tool for drug development

Abstract Background: Genotype directed treatment is the standard of care for patients with advanced non-small cell lung cancer (NSCLC). However, acquired drug resistance invariably develops. There is a need to develop clinically relevant animal models from patients who have developed acquired resistance in order to study mechanisms of resistance and validate new therapeutic strategies. We have generated a portfolio of clinically relevant, early passage, patient-derived xenograft (PDX) models of lung cancer. Methods: Tumor biopsies (core biopsies (n = 8), pleural effusions (n = 28) and surgical samples (n = 11)) were implanted subcutaneously (n = 39) or into the sub-renal capsule (n = 8) of NSG mice under an IRB approved protocol. After implanting 35 specimens, mostly from pleural effusions, several mice exhibited weight and fur loss. Histopathology revealed infiltration of the liver, skin and lungs by human derived T-cells consistent with graft-versus-host disease (GVHD). Subsequent pleural effusions were then subjected to CD45 depletion using a human CD45 microbead separation kit which eliminated the incidence of GVHD. Tumors from all three sources were allowed to grow and subsequently passaged only as subcutaneous implants in NSG mice. Molecular characterization of PDX models was performed using targeted next generation sequencing and tumor histology confirmed by H&E staining. Some established tumor models have also been propagated as cell lines in vitro. Results: 47 specimens were implanted with an overall take rate of ∼30%. The initial take rate was 22% but increased to 66% following CD45 depletion. We have established 11 NSCLC adenocarcinoma and 3 small-cell lung cancer (SCLC) PDX models from patient samples. The models established thus far range from erlotinib resistant EGFR mutant models with T790M mutation (n = 3), MET amplification (n = 1), SCLC transformation (n = 2) or an unknown mechanism (n = 1); to models including SCLC with an NRAS Q61K mutation (n = 1); EML4-ALK resistant to crizotinib and ceritinib (n = 1); KRAS G12C mutation (n = 1); NUT-mid line carcinoma with BRD4 rearrangement (n = 1); pan wild-type (n = 2) and a PIK3CA mutation (n = 1). We have used these models to study therapeutic interventions. Treatment of the NRAS Q61K mutant SCLC with trametinib resulted in complete tumor regression. Pharmacodynamic analyses revealed inhibition of pERK 1/2 and induction of apoptosis. Data will be presented with further characterization and platform development of the lung PDX models. Notably, >50% of the models do show pulmonary metastases from the subcutaneous implant site. Conclusions: PDXs can be established from lung cancer patients undergoing routine clinical biopsies. Establishment of models from pleural effusions is also feasible following CD45 depletion to prevent GVHD. These models will provide unique information on mechanisms of drug resistance and can be used to evaluate pre-clinical therapeutic strategies. Citation Format: Prafulla C. Gokhale, Sangeetha Palakurthi, Man Xu, Antonio Calles, Shruti D. Shah, Allison O'Connell, Melissa Messineo, Yanan Kuang, Masahiko Yanagita, Mosab Ali, Cam Anh Tran, Jihyun Choi, Atsuko Ogino, Mohit Butaney, Amanda J. Redig, Cloud Paweletz, Paul T. Kirschmeier, Pasi A. Jänne. Modeling patient-derived lung cancer in mice: Preclinical tool for drug development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1451. doi:10.1158/1538-7445.AM2015-1451

Heterogeneity Underlies the Emergence of EGFRT790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor.

Rociletinib is a third-generation EGFR inhibitor active in lung cancers with T790M, the gatekeeper mutation underlying most first-generation EGFR drug resistance. We biopsied patients at rociletinib progression to explore resistance mechanisms. Among 12 patients with T790M-positive cancers at rociletinib initiation, six had T790-wild-type rociletinib-resistant biopsies. Two T790-wild-type cancers underwent small cell lung cancer transformation; three T790M-positive cancers acquired EGFR amplification. We documented T790-wild-type and T790M-positive clones coexisting within a single pre-rociletinib biopsy. The pretreatment fraction of T790M-positive cells affected response to rociletinib. Longitudinal circulating tumor DNA (ctDNA) analysis revealed an increase in plasma EGFR-activating mutation, and T790M heralded rociletinib resistance in some patients, whereas in others the activating mutation increased but T790M remained suppressed. Together, these findings demonstrate the role of tumor heterogeneity when therapies targeting a singular resistance mechanism are used. To further improve outcomes, combination regimens that also target T790-wild-type clones are required. This report documents that half of T790M-positive EGFR-mutant lung cancers treated with rociletinib are T790-wild-type upon progression, suggesting that T790-wild-type clones can emerge as the dominant source of resistance. We show that tumor heterogeneity has important clinical implications and that plasma ctDNA analyses can sometimes predict emerging resistance mechanisms.

Transformation to small-cell lung cancer following treatment with EGFR tyrosine kinase inhibitors in a patient with lung adenocarcinoma

We report the case of a 52-year-old woman with lung adenocarcinoma treated with EGFR tyrosine kinase inhibitor (TKI) therapy. After disease progression, histological examination of a secondary biopsy specimen revealed small-cell lung cancer (SCLC) that was sensitive to standard SCLC treatment. Tumor markers, including ProGRP and NSE, were elevated. Transformation to SCLC is a mechanism for acquired resistance to EGFR-TKI therapy. Secondary biopsy is important for evaluation of genetic and histological changes and selection of appropriate treatment. Furthermore, ProGRP and NSE may be useful for early detection of SCLC transformation in cases resistant to EGFR-TKI therapy.

Abstract 4468: Cripto-1 elicits innate resistance to EGFR inhibitors in non-small cell lung cancer harboring EGFR-sensitizing mutations.

Abstract Non-small cell lung cancer (NSCLC) carrying sensitizing epidermal growth factor receptor (EGFR) mutations can initially benefit from erlotinib (EGFR inhibitor) treatment. However, patients invariably develop resistance through secondary EGFR mutations, c-MET amplification, epithelial-mesenchymal transition (EMT), small cell lung cancer transformation, PIK3CA mutation and other unknown mechanisms. Here we examined whether Cripto1, which has been shown to induce EMT, could cause erlotinib resistance. We show that innate erlotinib-resistant lung adenocarcinomas carrying sensitizing EGFR mutations exhibit higher level of Cripto1 expression than their erlotinib sensitive counterparts. Ectopic expression of Cripto1 rendered erlotinib-sensitive lung adenocarcinoma cells resistant to erlotinib both in vitro and in a xenograft model. Moreover, Cripto1 activated Zeb1/EMT signaling pathway in EGFR-TKI resistant cells. High Cripto1 expression correlated with poor prognosis of NSCLC patients. Our study provides a novel Cripto1-dependent mechanism of EGFR-TKI resistance that may pave the way for the development of therapies to overcome EGFR-TKI resistance in lung cancer patients. Citation Format: Kang-Seo Park, Giaccone Giuseppe, Yisong Wang, Trung Pham, Yongwha Moon, Ji Luo, Liam Changwoo Lee, Tetsuya Mitsudomi, Isamu Okamoto, Yasushi Yatabe, Tony Mok, Mark Raffeld, Liqiang Xi, David S. Salomon, Caterina Bianco. Cripto-1 elicits innate resistance to EGFR inhibitors in non-small cell lung cancer harboring EGFR-sensitizing mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2013-4468

Neuroendocrine Differentiation in Acquired Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

BackgroundSmall cell lung cancer (SCLC) transformation during epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment in lung cancer has been suggested as one of possible resistance mechanisms.MethodsWe evaluated whether SCLC transformation or neuroendocrine (NE) differentiation can be found in the cell line model. In addition, we also investigated its effect on responses to conventional chemotherapeutic drugs of the SCLC treatment.ResultsResistant cell lines to various kinds of EGFR-TKIs such as gefitinib, erlotinib, CL-387,785 and ZD6474 with A549, PC-9 and HCC827 lung adenocarcinoma cell lines were established. Among them, two resistant cell lines, A549/GR (resistant to gefitinib) and PC-9/ZDR (resistant to ZD6474) showed increased expressions of CD56 while increased synaptophysin, Rb, p16 and poly(ADP-ribose) polymerase were found only in A549/GR in western blotting, suggesting that NE differentiation occurred in A549/GR. A549/GR cells were more sensitive to etoposide and cisplatin, chemotherapeutic drugs for SCLC, compared to parental cells. Treatment with cAMP and IBMX induced synaptophysin and chromogranin A expression in A549 cells, which also made them more sensitive to etoposide and cisplatin than parental cells. Furthermore, we found a tissue sample from a patient which showed increased expressions of CD56 and synaptophysin after development of resistance to erlotinib.ConclusionNE differentiation can occur during acquisition of resistance to EGFR-TKI, leading to increased chemosensitivity.

Monitoring of plasma pro-GRP level during EGFR-TKI treatment.

10604 Background: Lung cancers harboring mutations in the epidermal growth factor receptor gene (EGFR) respond to EGFR tyrosine kinase inhibitors (EGFR-TKI), but drug resistance invariably emerges. The major acquired mechanisms of resistance are the EGFR T790M mutation or MET gene amplification. Transformation from NSCLC into small-cell lung cancer (SCLC) has been recently identified in acquired resistance to EGFR-TKI. However, it is difficult to predict the transformation during EGFR-TKI treatment because obtaining serial and sufficient specimens for biopsy is difficult. Pro-gastrin-releasing peptide (Pro-GRP) is a specific and sensitive tumor marker for SCLC. We evaluated the plasma Pro-GRP levels in EGFR-mutant NSCLCs and determined whether plasma Pro-GRP levels could predict SCLC transformation in resistance to EGFR-TKI. Methods: From July 2008 to December 2011, 49 patients with EGFR-mutant NSCLC who received EGFR-TKI treatment were enrolled. Plasma was obtained from these patients before EGFR-TKI treatment and when EGFR-TKI treatment failed. Pro-GRP and CEA levels were measured and compared before and after treatment. Results: Patient characteristics for 49 patients (15 men, 34 women) were as follows: median age, 62 years (41–81 years); histology, 46 adenocarcinomas (AD) and 3 non-AD tumors; and EGFR mutation type, 25 exon 19 deletions and 24 exon 21 L858R. All 49 patients had received EGFR-TKI treatment (45 with gefitinib and 4 with erlotinib); the response to EGFR-TKI treatment was PR in 39 patients, SD in 7, PD in 2, and NE in 1. Positive rate of ProGRP and CEA at pre-EGFR-TKI treatment was 2.0% and 57.2% and that at post-EGFR-TKI treatment was 6.1% and 69.4%, respectively. In 3 of 49 patients, the Pro-GRP levels had increased after treatment, but the CEA level did not increase. Objective responses to cytotoxic chemotherapy were noted in all 3 patients after EGFR-TKI treatment. Conclusions: Monitoring of plasma Pro-GRP during EGFR-TKI treatment may be useful for early detection of SCLC transformation in resistance to EGFR-TKI.

Survival pattern and cause of death in patients with multiple sclerosis: results from an epidemiological survey in north east Scotland.

The mean survival period in a series of 216 multiple sclerosis deaths, which formed part of a large prevalence sample observed in the Grampian region of Scotland, was 24.5 years, with an insignificant difference between females (25.7 years) and males (23.5 years). A third of the patients survived for over 30 years after onset. The age at death ranged between 25-80 years, with majority of the deaths occurring in the seventh decade (37%). On comparing life expectancy with the Scottish general population using life tables, only a slight reduction in the short-term (less than 10 years from onset) survival was noted in all age groups, with the exception of those with onset over the age of 50 years. The long-term life expectancy was however markedly reduced in all age groups compared with the controls. The survival period could be accurately predicted from the degree of disability at a point in time, and could be correlated with a number of clinical features, the most important of which was the age at onset. Eighty five per cent of those with onset of multiple sclerosis over the age of 50 years died within 20 years. Patients with a cerebellar disturbance at onset survived the shortest, and those with a brainstem lesion or retrobulbar neuritis the longest; those with a pyramidal dysfunction had an intermediate prognosis. Other parameters which could be correlated with the survival were: the timing and frequency of occurrence of psychiatric and urinary symptoms, interval between onset and first relapse and the course of the disease. As expected, most patients (89%) were significantly disabled (unable to walk) prior to death, only a minority, however, had become so within 10 years of the onset (10%). Sixty two per cent of the patients died of complications of multiple sclerosis. No unusual excess of any disease was noted amongst other causes. As expected, the majority of patients (55%) had bronchopneumonia as the terminal event, 11% had septicaemia, 15% had myocardial infarction and 4% had documented pulmonary embolism. This is the largest series of its kind where prognosis, judged by survival period, has been assessed amongst all multiple sclerosis patients derived from a prevalence sample and observed till death.