Subset Of NSCLC Patients Research Articles

Abstract 657: Impact of therapy induced APOBEC3A mutagenesis on tumor evolution in non small cell lung cancer

Abstract Acquired drug resistance to even the most effective anti cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. We have seen that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug tolerant cancer cells persisting during therapy. Preventing therapy induced A3A mutagenesis by gene deletion delayed the emergence of drug resistance. Here, we show that therapy induced A3A mutagenesis contributes to tumor evolution in NSCLC. Whole genome sequencing revealed that resistant clones that evolved from persistent drug tolerant cells (late clones) harbored more A3A mutations compared to the parental cell population than pre existing resistant clones (early clones). In a subset of NSCLC patients who received targeted therapies, we observed A3A mutations accompanied clonal evolution during treatment. Comparison of APOBEC mutation fractions in short vs long term responders suggests that short responders with acquired resistance mechanisms that evolved from pre existing resistant clones have less accumulation of APOBEC mutations. Collectively, these findings insist that an increase in mutagenic processes drives tumor evolution during targeted therapy treatment and leads to acquired resistance. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to halt the evolution of resistant clones and prevent acquired resistance to lung cancer targeted therapy. Citation Format: Hideko Isozaki, Ammal Abbasi, Naveed Nikpour, Marcello Stanzione, Ramin Sakhtemani, Susanna L. Monroe, Alice T. Shaw, Jessica J. Lin, Lecia V. Sequist, Zofia Piotrowska, Rémi Buisson, Michael S. Lawrence, Aaron N. Hata. Impact of therapy induced APOBEC3A mutagenesis on tumor evolution in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 657.

PD-1/PD-L1 inhibitor monotherapy or in combination with chemotherapy as upfront treatment for advanced NSCLC with PD-L1 expression ≥ 50%: Selecting the best strategy.

Treatment strategies for advanced NSCLC patients without driver molecular alterations are influenced by PD-L1 expression. If PD-L1 is <50 %, the preferred upfront treatment is the association of chemotherapy and immunotherapy, while immunotherapy alone is an additional option if PD-L1 ≥ 50 %. Both treatments demonstrated their superiority over chemotherapy in this subset of NSCLC patients, with comparable efficacy outcomes but less safety concerns for immunotherapy alone. Nevertheless, a significant difference in terms of early progression-free survival rate emerges by analyzing and comparing the survival curves of the two strategies, reflecting a non-negligible loss of patients due to early disease progression at 3 and 6 months from treatment initiation with immunotherapy alone as compared to its association with chemotherapy. We deeply analyzed efficacy similarities and differences of the two approaches in advanced NSCLC with PD-L1 expression ≥50 %, trying to suggest clinical and biologic aspects to be considered when facing the treatment choice.

BRIGHTSTAR: A pilot trial of local consolidative therapy (LCT) with brigatinib in tyrosine kinase inhibitor (TKI)-naïve ALK-rearranged advanced NSCLC.

9624 Background: Approximately, 95% of patients who have an initial response to ALK-TKIs exhibit an incomplete response resulting in residual disease that enables the emergence of acquired resistance. Eliminating residual disease using LCT may delay resistance emergence and improve clinical outcomes. Methods: This is a single center investigator-initiated trial that assesses the safety, feasibility and efficacy of brigatinib with LCT. Eligible patients have TKI-naïve ALK rearranged advanced NSCLC with any number of metastases. Patients treated with brigatinib for an induction period of 8 weeks followed by LCT with radiation and/or surgery. Results: Between 12/2018 and 01/2020, 17 out of 24 planned patients were enrolled. Median age 55 (range 33-73). At study entry, 15 patients had polymetastatic disease ( &gt; 3 sites) while 2 had oligometastatic disease. As of February 1, 2020, 16 patients were evaluated for response and completed LCT while 1 patient remained on induction brigatinib. The disease control rate was 100% with an objective response rate of 94% (n = 15). Median follow up was 8 months (range 3-13) with no patients with disease progression to date. LCT used was radiation (n = 11), surgery (n = 3), surgery and radiation (n = 2). Among 5 patients who had surgery, 4 had lobectomy and mediastinal lymph node dissection (MLND), 1 had wedge resection with MLND, and 1 had adrenalectomy. Of these, 2 had complete pathological response and 1 had complete pathological response at the primary tumor. There were no grade ≥2 adverse events (AEs) related to LCT, including in 7 patients treated with concurrent brigatinib and radiation, and 6 patients treated with radiation while brigatinib was held. All patients continued brigatinib after LCT. Brigatinib-related severe AEs included grade 3: increased blood levels of creatine kinase, lipase, alanine aminotransferase, amylase (n = 1 each) and nausea (n = 1). One patient had grade 2 pneumonitis after 2 weeks of starting brigatinib, this resolved with steroids and brigatinib was resumed at a lower dose. Conclusions: Brigatinib with LCT is safe and feasible in patients with ALK-rearranged advanced NSCLC irrespective of number of metastatic sites. Brigatinib and LCT may be an effective therapeutic strategy in this subset of NSCLC patients. Clinical trial information: NCT03707938 .

Isolated metastasis of an EGFR-L858R-mutated NSCLC of the meninges: the potential impact of CXCL12/CXCR4 axis in EGFRmut NSCLC in diagnosis, follow-up and treatment.

Brain and leptomeningeal metastasis (LMM) of non-small cell lung cancer is still associated with poor prognosis. Moreover, the current diagnostic standard for LMM often yields false negative results and the scientific progress in this field is still unsatisfying.We present a case of a 71-year old patient with an isolated LMM. While standard diagnostics could only diagnose a cancer of unknown primary, the use of [68Ga]-Pentixafor-PET/CT (CXCR4-PET/CT, a radiotracer targeting CXCR4) and a liquid biopsy of the cerebrospinal fluid revealed the primary NSCLC. The detection of L858R-EGFR, a common driver mutation in NSCLC, enabled us to treat the patient with Afatinib and monitor treatment using [68Ga]-Pentixafor PET/CT. To estimate the impact of CXCR4 signaling and its ligands in NSCLC brain metastasis we looked at their expression and correlation with EGFR mutations in a primary and brain metastasis data set and investigated the previously described binding of extracellular ubiquitin to CXCR4.In conclusion, we describe a novel approach to improve diagnostics towards LMM and underline the impact of the CXCL12/CXCR4 axis in brain metastasis in a subset of NSCLC patients. We cannot confirm a correlation of CXCR4 expression with EGFR mutations or the binding of extracellular ubiquitin as previously reported.

Long-term clinical benefit from salvage EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer patients with EGFR wild-type tumors.

Erlotinib has been approved for the management of NSCLC patients after failure of the first or subsequent line of chemotherapy. Although the efficacy of erlotinib is clearly associated with the presence of EGFR mutations, there is a subset of patients with EGFR wild-type (EGFRwt) tumors who impressively respond. Patients with EGFRwt NSCLC who received salvage (≥2nd line) treatment with erlotinib for a prolonged period (>6months), were sought from the database of the Hellenic Oncology Research Group. We retrospectively analyzed the clinical, pathological and molecular characteristics of the patients with available tumor material. Forty-four patients that received erlotinib for >6months (median 10.1months) were enrolled in the study. The majority of them were male, never-smokers with adenocarcinoma histology and a good performance status. KRAS and PIK3CA mutations were detected in 21% (9/42 tested) and 13% (4/30 tested) of the patients, respectively. The ALK-EML4 translocation was found in 10% (2/20 tested); there was no patient with HER2 or BRAF mutated tumor. Twelve (54.5%) tumor specimens were considered positive for EGFR-overexpression. Eleven patients experienced a partial response (objective response rate 25%; 95% CI 12-38%) and the remaining 33 had stable disease. The median progression-free survival and overall survival were 10.1 (95% CI 8.6-11.6months) and 24.1 (95% CI 11.2-37months), respectively. Treatment with erlotinib significantly improves the clinical outcome in a subset of NSCLC patients with EGFRwt tumors. Further molecular analysis of such tumor specimens could provide a more comprehensive characterization of this particular group of patients. Nevertheless, the presence of other mutations should not prevent the treating physician from using erlotinib at later lines of salvage therapy for NSCLC patients.

P1.02-048 MET Exon 14 Skipping Mutations and Gene Amplifications Are Not Simultaneous Events in NSCLC

Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, have been described with responsiveness to crizotinib. Until now, patient selection has been made in view of MET amplification/high polysomy and protein overexpression, with discrepancies in positivity criteria. We investigated the prevalence of abnormal MET mutational status and the subsequent gene copy number alterations (CNAs) in NSCLC. We routinely tested 190 lung adenocarcinomas and adenosquamous carcinomas for MET exon 14 and flanking introns mutations by PCR-direct sequencing, and for MET gene CNAs by FISH (Abbott Molecular). Amplifications were defined as mean gene by mean centromere ratio ≥2.2, and high gains as mean gene ≥5.0. RT-PCR was performed to validate mutations leading to exon 14 skipping. We collected clinical-pathological data together with EGFR and KRAS mutational status and ALK, ROS1 and RET rearrangements. MET alterations were found in 34 patients (17.9%): 11 mutated cases (5.8%), eight gene amplifications (4.2%), and 15 high gains (9.1%). Six out of 11 mutations were confirmed to lead to exon 14 skipping (3.2%). Remarkably, none of these exon 14 skipped cases had concurrent MET amplification nor high gains. Although, KRAS p.G12C and EGFR 19 exon mutations were found concomitant with MET mutation in two of these cases. Among the 14 confirmed MET altered cases (6 exon 14 skipped and 8 amplified): 13 patients were male (93%), with a median age of 65.7 years (range: 40-91), nine current smokers (64%) (40 pack-years, range: 20-60), and eight diagnosed in an advanced stage disease (III and IV) (57%). Correlation with c-MET protein expression by IHC is ongoing, and data will be presented at the meeting. As no concurrent MET mutated and amplified cases were found, our data support prospective identification of both, MET exon 14 skipping mutations and gene amplifications. These mutations define a new subset of NSCLC patients that should be analyzed independently of the status of MET gene copy number.

12P Volatolomic signatures of anaplastic lymphoma kinase gene rearrangement in adenocarcinoma

Background: Mutations in the exon 14 of the MET gene affecting RNA splice acceptor and donor sites, which lead to exon skipping, have been recently associated to responsiveness to crizotinib. Up to date, patient selection for Met inhibitors has been made according to MET amplification/high polysomy and/or protein overexpression. We have investigated the prevalence of MET exon 14 mutations and their correlation with gene copy number alterations (CNAs) in NSCLC patients. Methods: We tested 132 lung adenocarcinomas and adenosquamous carcinoma samples for mutations in MET exon 14 by Sanger sequencing and for MET gene CNAs by FISH (Abbott Molecular). We collected clinical data together with EGFR and KRASmutational status and ALK, ROS1 and RET rearrangements. Results: MET alterations were found in 23 patients (17.5%): 3 exon 14 skipping mutations (2.3%), 8 gene amplifications (6.1%), and 12 high polysomy cases (9.1%). Among the 11 MET altered cases (3 mutated and 8 amplified): 8 patients were male, with a median age of 64.5 years (range: 46–91), current or former smokers (50 pack/years, range: 30–80), and all were diagnosed in an advanced stage disease (III and IV). None of the 3 mutated cases had concurrent MET CNAs. One MET mutated case presented also a KRAS p.G12C substitution while the other two were wild type for EGFR (exons 18 to 21) and KRAS (exon 2). Interestingly, patients with MET alterations have a shorter overall survival (p < 0.001). Conclusions: We have identified alterations of the MET gene (mutations in exon 14 and CNAs) in 8.4% of NSCLC patients. Our data support the usefulness of prospective screening of MET exon 14 mutations and gene amplifications due to their prognostic and predictive role. These mutations define a new subset of NSCLC patients that should be analyzed independently of the MET gene copy number status. Legal entity responsible for the study: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Funding: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Disclosure: All authors have declared no conflicts of interest.

11P Prognostic impact of MET mutations in exon 14 and copy number alterations in a series of NSCLC patients

Background: Mutations in the exon 14 of the MET gene affecting RNA splice acceptor and donor sites, which lead to exon skipping, have been recently associated to responsiveness to crizotinib. Up to date, patient selection for Met inhibitors has been made according to MET amplification/high polysomy and/or protein overexpression. We have investigated the prevalence of MET exon 14 mutations and their correlation with gene copy number alterations (CNAs) in NSCLC patients. Methods: We tested 132 lung adenocarcinomas and adenosquamous carcinoma samples for mutations in MET exon 14 by Sanger sequencing and for MET gene CNAs by FISH (Abbott Molecular). We collected clinical data together with EGFR and KRASmutational status and ALK, ROS1 and RET rearrangements. Results: MET alterations were found in 23 patients (17.5%): 3 exon 14 skipping mutations (2.3%), 8 gene amplifications (6.1%), and 12 high polysomy cases (9.1%). Among the 11 MET altered cases (3 mutated and 8 amplified): 8 patients were male, with a median age of 64.5 years (range: 46–91), current or former smokers (50 pack/years, range: 30–80), and all were diagnosed in an advanced stage disease (III and IV). None of the 3 mutated cases had concurrent MET CNAs. One MET mutated case presented also a KRAS p.G12C substitution while the other two were wild type for EGFR (exons 18 to 21) and KRAS (exon 2). Interestingly, patients with MET alterations have a shorter overall survival (p < 0.001). Conclusions: We have identified alterations of the MET gene (mutations in exon 14 and CNAs) in 8.4% of NSCLC patients. Our data support the usefulness of prospective screening of MET exon 14 mutations and gene amplifications due to their prognostic and predictive role. These mutations define a new subset of NSCLC patients that should be analyzed independently of the MET gene copy number status. Legal entity responsible for the study: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Funding: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Disclosure: All authors have declared no conflicts of interest.

The RET fusion gene and its correlation with demographic and clinicopathological features of non-small cell lung cancer: a meta-analysis

Purpose. The RET fusion gene is a novel oncogene observed in a subset of NSCLC in recent years. Nevertheless, the results of epidemiological studies concerning the gene remain unclear. Thus, a meta-analysis was conducted to evaluate the correlation of RET fusion gene with demographic and clinicopathological features of NSCLC. Methods. PubMed, Embase, and Web of Science databases were searched to identify eligible studies. The association of RET fusion gene occurrence with gender, age, smoking status, histology type and tumor stage were analyzed in meta-analysis. Subgroup analysis according to patients' location (Asian and non-Asian) was also conducted. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated to assess the correlation. Results. Nine studies with a total of 6,899 NSCLC patients met the inclusion criteria. A total of 84 patients with RET fusion gene were detected. The RET fusion gene was identified at significantly higher frequencies in female (OR = 0.55, 95%CI = 0.35–0.85) than male patients and in young (<60 ) patients (OR = 0.43, 95%CI = 0.19–0.99) than old patients (≤60 ), particularly in patients from Asian. A significant higher frequency was also identified in non-smokers (OR = 0.28, 95% CI = 0.16–0.49), and in patients with lung adenocarcinomas (OR = 3.59, 95%CI = 1.50–8.56). Additionally, no association between RET fusion gene and the TNM stage of tumor was observed. Conclusion. RET fusion gene occurred predominantly in Asian females with younger age, in non-smokers, and in lung adenocarcinomas patients. This subset of NSCLC patients might be good candidates for personalized diagnostic and therapeutic approaches.

Targeting the anti-tumor function of B cells in non-small cell lung cancer patient tumors (TUM9P.1008)

Abstract Understanding the function of tumor infiltrating lymphocytes (TILs) in NSCLC patient tumors will contribute to the development of rationally designed treatments and an increase in the current 18% survival rate. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer. We used un-manipulated, primary human B cells from fresh tumor and tumor adjacent lung tissue to determine the phenotype and function of TIL-Bs in NSCLC patients. The total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. In analyzing the markers of activation and exhaustion, we observed a spectrum of activation of TIL-Bs. Further, we observed that TIL-Bs present autologous tumor antigens to CD4 TILs in a subset of NSCLC patients. These data suggest that some patients have functional TIL-Bs and some patients have nonfunctional TIL-Bs; TIL-Bs that could not present tumor-specific antigens had higher expression of B cell exhaustion markers. Lastly, we are combining blockade of the immune inhibitory PD-1:PD-L1 pathway in our antigen presentation assays because PD-1 expression is increased on CD4 TILs in our NSCLC patient tumors. Results from this study will advance the understanding of the anti-tumor function of TIL-Bs in solid tumors and will develop a model of B cell exhaustion in human cancer. Ultimately, we will predict which TIL-B functions to target in future NSCLC immunotherapies.

Abstract 337: A NSCLC PDX with EML4-ALK fusion mutation responds to crizotinib.

Abstract Non-small cell lung cancer (NSCLC) has the highest mortality among all malignances while with limited treatment options. Among the most significant treatment advancement in recent years are the introduction of target therapies of EGFR-TKIs (Erlotinib and Gefitinib) for adenocarcinoma with EGFR activating mutations, and Crizotinib for NSCLC with ALK activation (EML4-ALK fusion)1. ALK activation are associated with a subset of NSCLC patients (3∼5%).Crizotinib is considered not a particularly potent ALK inhibitor, with IC50 at range of tens of nanomoles. It may not be an ideal agent for treating NSCLC with activating ALK. Therefore, a new ALK inhibitor with better potency and specificity could be of particularly medical importance. Currently, the industry is actively searching for such new agents. A relevant experimental model could also greatly facilitate evaluating and validating these new drug candidates. Patient derived xenografts (PDX) (including NSCLC PDX) mirror patients’ histopathology and genetic profiles2, thus known as “patient avatar”. It has improved predictive power as preclinical cancer models, and enables discovery of predictive biomarkers for targeted therapeutics. We have established a large collection of NSCLC PDX models (NSCLC-HuPrime®) by engrafting naïve patient tumor tissues into immune-compromised mice2. Among these models, we have identified one model, LU1656, of NSCL carcinosarcoma histology and with EML4-ALK fusion mutation. This mutation apparently causes over-expression of ALK as measured at mRNA level. This model has wild-type KRAS, c-MET and EGFR genes. Treatment of LU1656 with Crizotinib caused significant but not complete tumor response, suggesting ALK activation is one of the oncogenic drivers of this model. In addition, LU1656 also responded to Cetuximab, but respond poorly to Erlotinib. It would be interesting to examine the combination of these treatments. This model is, to our knowledge, the first reported PDX model with EML4-ALK fusion mutation. We believe that LU1656 would be a useful model to evaluate investigational ALK inhibitors, and new combination treatment regimens of Crizotinib and standard of care chemotherapies. Citation Format: Jie Cai, Mengmeng Yang, Jianyun Deng, Xiaoming Song, Xuesong Huang, Taiping Chen, Jean Pierre Wery, Yiyou Chen, Henry QX Li. A NSCLC PDX with EML4-ALK fusion mutation responds to crizotinib. [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 337. doi:10.1158/1538-7445.AM2013-337

Cooperative antitumor activity between the retinoid X receptor (RXR)-selective agonist bexarotene and EGFR-tyrosine kinase inhibitors in preclinical models of NSCLC

17073 Background: Gefitinib and erlotinib, small molecule inhibitors of the EGFR-tyrosine kinase (EGFR-TKI), can have antitumor activity in patients with advanced NSCLC. However, analysis of clinical samples and in vitro characterization of NSCLC cell lines has revealed EGFR mutations that correlate with the clinical responses. While EGFR-mutant cell lines are hypersensitive to EGFR-TKI-mediated growth inhibition and have a greater tendency to undergo apoptosis in response to EGFR inhibition, wild-type EGFR cell lines undergo growth inhibition only at high concentrations, and are resistant to apoptosis induction. Bexarotene (Targretin) is a RXR modulator with clinical activity enhancing survival in a subset of NSCLC patients. We have shown that bexarotene can suppress the growth of epithelial tumors by modulating the expression and function of the EGFR pathway. This study examined the effect of bexarotene/EGFR-TKI combinations in preclinical models of NSCLC, and evaluated the possible molecular mechanisms for cooperative antitumor activity between these agents. Results: Bexarotene enhanced the activity of gefitinib in most of the cell lines tested, and did so regardless of their EGFR mutation status or intrinsic sensitivity to gefitinib alone. Resistant lines were sensitized by the combination, which decreased gefitinib IC50s to clinically achievable concentrations. Bexarotene increased the pro-apoptotic activity of both gefitinib and erlotinib, an effect which was paralleled by enhanced repression of p-Akt and p-Her2 levels by the combinations. In ex vivo cultures of human NSCLC explant tissue, the addition of bexarotene enhanced the growth inhibitory activity of both gefitinib and erlotinib. Finally, combinations of bexarotene and EGFR-TKI produced growth inhibition of TKI-resistant A427 NSCLC xenograft tumors that was superior to either agent alone. Conclusions: These results indicate that bexarotene can sensitize NSCLC cells which are unresponsive to the effects of EGFR-TKIs, resulting in cooperative growth inhibition and apoptosis induction. Combination therapy using bexarotene in conjunction with EGFR-TKIs may provide a powerful new therapeutic strategy. [Table: see text]