Expression of estrogen receptor beta predicts a clinical response and longer progression-free survival after treatment with EGFR-TKI for adenocarcinoma of the lung

Purpose

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (EGFR-TKI) demonstrates a dramatic clinical response for the lung adenocarcinoma patients harboring a somatic mutation of EGFR. Such EGFR mutations are frequently found in adenocarcinoma with a strong expression of estrogen receptor (ER) beta, which has been shown to correlate with a favorable prognosis for the patients with EGFR mutations. The aim of this study is to elucidate the correlation between expression of ER beta and the therapeutic effect of EGFR-TKI in adenocarcinoma of the lung.

Patients and methods

Forty-three patients who were treated with EGFR-TKI for adenocarcinoma of the lung were evaluated. The expression of ER beta and the EGFR mutation were evaluated by immunohistochemistry and the polymerase chain reaction, respectively. Patients divided into two groups by the nuclear expression of ER beta. The clinical response and survival data were compared between the two groups.

Result

Strong (S) and weak (W) expression of ER beta was observed in 21 and 22 patients, respectively. EGFR mutations were detected in 30 (69.8%) cases. The S group had more frequent EGFR mutations than the W group (85.7%, 54.5%, p = 0.045). The S group had better response rate (p = 0.006) and longer progression-free survival (PFS; p = 0.001) than the W group. Even in a limited analysis in the patients with EGFR mutations, the S group had tended to have a better response rate (77.8%, 41.7%, p = 0.063), and significant longer PFS (p = 0.012) than the W group.

Conclusion

A strong expression of ER beta predicts a good clinical outcome for patients with adenocarcinoma of the lung after treatment with EGFR-TKI. This suggests that the expression status of ER beta can be a candidate surrogate marker for EGFR-TKI treatment of patients with adenocarcinoma of the lung. Further investigation will be necessary to identify biomarkers using a larger cohort of patients in a prospective study.     

Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs

Background

Epithelial-to-mesenchymal transition (EMT), which was related with an acquired resistance to gefitinib, was found in the A549 lung cancer cell line. However, the clinical feasibility of this finding is still questionable. Here, we investigated whether EMT could be detected in a more clinically suitable situation using patient's tumor and cells with deletion mutation on exon 19 of EGFR gene.

Methods

HCC827 cell line was used to establish the subline resistant to EGFR-TKIs. The induction of EMT was analyzed by immunostainings and Western blots in resistant cells and biopsied tissue from a patient with acquired resistance to erlotinib. Migration and invasion assay was performed to characterize the resistant cells. EMT-related genes expression was evaluated by cDNA microarray. Phospho-receptor tyrosine kinase array analysis was carried out to find bypass activating signals such as MET.

Results

We found that EMT developed in a lung cancer patient who had an acquired resistance to erlotinib while there were no known resistant mechanisms such as T790M and MET amplification. CL-387,785-resistant cells (HCC827/CLR) were obtained by long-term exposure to increasing concentrations of CL-387,785 (an irreversible EGFR-TKI). The morphological and molecular maker changes compatible with EMT were also found in HCC827/CLR cells. However, there were also no secondary T790M mutation and MET amplification. Furthermore, the activity of most of tested RTKs including receptor HER family was decreased suggesting that there was no bypass activating signal leading to resistance. These cells showed an enhanced capability for migration (∼1.6-fold) and invasion (∼2.8-fold).

Conclusion

EMT should be considered as one of possible mechanisms for the acquired resistance to EGFR-TKIs in lung cancer cells.     

Association between plasma hepatocyte growth factor and gefitinib resistance in patients with advanced non-small cell lung cancer

Purpose

It has been suggested that hepatocyte growth factor (HGF) and insulin-like growth factor binding protein (IGFBP)-3 are associated with gefitinib resistance in non-small cell lung cancer (NSCLC). We investigated the predictive and prognostic roles of these proteins in NSCLC patients treated with gefitinib.

Patients and methods

Of 106 patients enrolled in a randomized phase II study of gefitinib, 97 had plasma samples available for ELISA testing. Of these samples, seven and eight, respectively, had HGF and IGFBP-3 values that could not be measured. Therefore, the correlations between clinical outcomes and plasma levels of HGF and IGFBP-3 were evaluated in 90 and 89 patients, respectively.

Results

Plasma HGF levels were significantly higher in older patients, male patients, patients with squamous cell carcinoma, current smokers, and patients with epidermal growth factor receptor (EGFR) wild-type tumors. Low HGF levels were significantly associated with higher response rate, and longer progression-free survival (PFS) and overall survival (OS) irrespective of EGFR mutation status. In a multivariate analysis, the presence of EGFR mutations (P = 0.002) and low HGF levels (P = 0.031) were independently predictive of longer PFS, and an ECOG PS of 0 (P = 0.001) and low HGF levels (P = 0.002) were independently predictive of longer OS. No statistically significant differences were found for IGFBP-3.

Conclusion

High HGF levels are significantly associated with resistance to gefitinib and can be used as a predictive marker for the differential outcome of gefitinib treatment in NSCLC irrespective of EGFR mutation status.     

Abnormality of the hepatocyte growth factor/MET pathway in pulmonary adenocarcinogenesis

Background

Signaling mediated by hepatocyte growth factor (HGF)/MET promotes multiple biological activities, including cell proliferation, motility, invasion, angiogenesis, and morphogenesis. Overexpression of HGF and MET and an increase of the MET gene copy number have recently been found in various cancers that had a poor outcome. Here we investigated the copy number of the MET gene and expression of MET and HGF in small pulmonary adenocarcinomas.

Methods

Tumor tissues were obtained from 106 pulmonary small adenocarcinomas 2 cm or less in diameter. MET gene copy number, and the expression of MET and HGF, were analyzed using fluorescence in situ hybridization (FISH) and immunohistochemistry, respectively.

Results

MET FISH-positive signals were observed in 11 (10.4%) of 106 cases. One case (0.9%) showed gene amplification and 10 (9.4%) exhibited high polysomy. High immunoreactivity for MET and HGF in tumor cells was found in 30 (28.3%) and 19 cases (17.9%), respectively. HGF was also expressed in stromal cells in 32 cases (30.2%). No cases of non-invasive adenocarcinoma (adenocarcinoma in situ, localized bronchioloalveolar carcinoma) showed MET FISH-positive signals or high expression of HGF in the tumor cells. Expression of both MET and stromal HGF was stronger in invasive than in non-invasive adenocarcinoma. MET FISH-positive signals and high immunoreactivity for MET and HGF in tumor cells were associated with factors indicative of poor prognosis such as pleural invasion, vascular invasion, lymphatic permeation, lymph node metastasis, and nuclear grading. Univariate and multivariate analyses that included these factors showed that all statuses except for MET and HGF immunoreactivity were significantly associated with an increased risk of death. However, multivariate analysis revealed no independent factors related to poor prognosis.

Conclusion

Our results suggest that abnormality of the HGF/MET pathway occurs during the course of progression from non-invasive to invasive pulmonary adenocarcinoma. An increased MET gene copy number is indicative of a poor outcome in patients with small pulmonary adenocarcinomas.     

Clinical significance of tumor suppressor PTEN in colorectal carcinoma

Background

It has been demonstrated that the deletion, mutation, hypermethylation and subcellular location of the tumor suppressor phosphatase and tensin homologue (PTEN) are closely correlated with carcinogenesis, progression and prognosis of malignancy. Both mutation and the microsatellite instability of the PTEN gene influence regulation of the PI3K/Akt signaling pathway. This study investigated whether loss of nuclear PTEN is correlated with chemosensitivity, clinicopathological parameters and survival.

Methods

Intracellular levels of PTEN of multiple cell lines of colorectal carcinoma (CRC) were evaluated by Western blotting and immunocytochemistry. The chemosensitivity of cell lines with various expression levels of PTEN was evaluated using 5-flurouracil (5-FU), oxaliplatin and irinotecan (CPT), and clinical significance was evaluated by immunohistochemical analysis of 133 CRC specimens.

Results

Colon cancer cell lines HT-29, LoVo and SW480 differed in expression of PTEN, with high, moderate and low levels, respectively. HT-29 and LoVo PTEN expression was suppressed by a low concentration of 5-FU and oxaliplatin; however, SW480 was insensitive to these chemotherapeutic agents. Nuclear PTEN was overexpressed in most (>80%) normal colon mucosa samples, but the incidence significantly decreased (89.2% → 53.4%) in the CRC group. PTEN in the nucleus was negatively correlated with tumor size and vascular invasion in CRC, and CRC patients with negative PTEN expression in the nucleus exhibited poor survival.

Conclusion

Cell lines with a high expression of PTEN are sensitive to chemotherapy with 5-FU and oxaliplatin. Nuclear PTEN expression gradually decreases after malignant transformation, and loss of PTEN expression in the nucleus is associated with tumor progression and poor clinical outcome in CRC.     

Association Between EGFR T790M Status and Progression Patterns During Initial EGFR-TKI Treatment in Patients Harboring EGFR Mutation

Background

Emergence of the T790M point mutation in exon 20 of the epidermal growth factor receptor (EGFR) is the most common mechanism of resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The aim of this study was to investigate the association between T790M mutation status and the progression patterns during EGFR-TKI treatment.

Overcoming erlotinib resistance with tailored treatment regimen in patient‐derived xenografts from naïve Asian NSCLC patients

Overall benefits of EGFR‐TKIs are limited because these treatments are largely only for adenocarcinoma (ADC) with EGFR activating mutation. The treatments also usually lead to development of resistances. We have established a panel of patient‐derived xenografts (PDXs) from treatment naïve Asian NSCLC patients, including those containing “classic” EGFR activating mutations. Some of these EGFR‐mutated PDXs do not respond to erlotinib: LU1868 containing L858R/T790M mutations, and LU0858 having L858R mutation as well as c‐MET gene amplification, both squamous cell carcinoma (SCC). Treatment of LU0858 with crizotinib, a small molecule inhibitor for ALK and c‐MET, inhibited tumor growth and c‐MET activity. Combination of erlotinib and crizotinib caused complete response, indicating the activation of both EGFR and c‐MET promote its growth/survival. LU2503 and LU1901, both with wild‐type EGFR and c‐MET gene amplification, showed complete response to crizotinib alone, suggesting that c‐MET gene amplification, not EGFR signaling, is the main oncogenic driver. Interestingly, LU1868 with the EGFR L858R/T790M, but without c‐met amplification, had a complete response to cetuximab. Our data offer novel practical approaches to overcome the two most common resistances to EGFR‐TKIs seen in the clinic using marketed target therapies.     

A novel method for detection of mutation in epidermal growth factor receptor

Background

For the rapid and sensitive screening of epidermal growth factor receptor (EGFR) hot-spot mutations, we developed a novel method combining mutant-enriched PCR with amplification refractory mutation system (ARMS) TaqMan real-time PCR in a one-step reaction tube.

Methods and results

We designed two pairs of primers to enrich and genotyping each mutation (E746_A750del and L858R): nest primers and ARMS primers. Before the PCR assays were carried out, the restriction enzymes were used to cut wild alleles. The results showed that this method could detect mutant alleles mixed samples containing 0.1% with a cutoff ΔCt value of 12. We used this method in a survey of 73 non-small cell lung cancer (NSCLC) samples, detecting 14 mutant samples of E746_A750del and 12 mutant samples of L858R. The results well agreed with the results of DxS. All unmatched samples were identified by sequencing and the results showed that our method has high specificty.

Conclusion

The mutant-enriched ARMS TaqMan PCR could be useful in the detection of mutation in clinical samples containing only a small number of mutant alleles.     

Comparison of gefitinib and erlotinib in advanced NSCLC and the effect of EGFR mutations

Introduction

Erlotinib and gefitinib are tyrosine kinase (TK) inhibitors of epidermal growth factor receptor (EGFR) that are effective in treating non-small cell lung cancer (NSCLC). This study aimed to compare their clinical uses and the influence of EGFR mutation.

Methods

The usages of erlotinib and gefitinib in advanced NSCLC were analyzed. Clinical data and EGFR mutational status of tumors were collected.

Results

Seven hundred and sixteen (716) patients received gefitinib (n = 440) or erlotinib (n = 276) for stage IIIb or IV NSCLC. Erlotinib was prescribed more frequently than gefitinib in males (58.2% vs. 41.8%, p < 0.001), smokers (60.5% vs. 39.5%, p < 0.001), and non-adenocarcinoma (70.6% vs. 29.4%, p < 0.001). Of the 716 study patients, 327 underwent testing for EGFR mutations (170 with mutant EGFR and 157 with wild-type EGFR). Adenocarcinoma in patients with mutant EGFR and non-smoker status in patients with wild-type EGFR were associated with better overall survival after TK inhibitor treatment. In both patient groups with mutant EGFR or wild-type EGFR, the effectiveness of gefitinib and erlotinib, including drug response or overall survival, were not different.

Conclusions

Our study revealed the obvious disparity in drug selection between erlotinib and gefitinib in clinical practice. Type of TK inhibitors did not influence treatment outcomes in patients with EGFR mutation or wild-type EGFR.     

T790M mutation is associated with better efficacy of treatment beyond progression with EGFR-TKI in advanced NSCLC patients

Background and purpose

Continuous EGFR-TKI treatment beyond progression has shown promising benefit for some patients with acquired resistance to EGFR-TKIs. The aim of this study was to investigate the association of secondary T790M mutation at the time of progression with the efficacy of EGFR-TKI treatment beyond progression.

Methods

From March 2011 to March 2013, patients with advanced NSCLC who developed acquired resistance to EGFR-TKI and where a re-biopsy was performed at Tongji University Cancer Institute were included into this study. Scorpion ARMS was used to detect EGFR mutation status.

Results

A total of 54 patients were enrolled in this study with a median progression-free survival time (PFS1) of 10.9 months according to RECIST criteria. In all, 53.7% (29/54) had T790M mutation after the failure of EGFR-TKIs; PFS1 was not statistically significantly different between patients with T790M mutation and without (13.0 vs. 10.5 months, p = 0.894). In all, 41 patients received TKI treatment beyond progression, including 22 with local progression to receive additional local therapy and 19 with gradual progression to receive additional chemotherapy. The median progression-free survival time (PFS2) of patients who received EGFR-TKI beyond progression treatment was 3.5 months (95% CI, 2.689–4.311). Patients with T790M mutation had significantly longer PFS2 (6.3 vs. 2.6 months, p = 0.002) and overall survival (39.8 vs. 23.2 months, p = 0.044) than those without.

Conclusion

Patients with secondary T790M mutation at the time of progression having gradual or local progression after acquired resistance to EGFR-TKI benefit more from EGFR-TKI treatment beyond progression compared to those without T790M mutation.     

Clinical implications of T790M mutation in patients with acquired resistance to EGFR tyrosine kinase inhibitors

Objectives

Although T790M mutation is considered to be the major mechanism of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC), its clinical implication remains undetermined.

Methods

Post-progression tumor specimens were prospectively collected for T790M mutation analysis in NSCLC patients with acquired resistance to initial EGFR TKIs. Clinical features were compared between patients with and without T790M.

Results

Out of 70 cases, 36 (51%) were identified to have T790M mutation in the rebiopsy specimen. There was no difference in the pattern of disease progression, progression-free survival for initial TKIs (12.8 and 11.3 months), post-progression survival (14.7 and 14.1 months), or overall survival (43.5 and 36.8 months) in patients with and without T790M. In total, 34 patients received afatinib after post-progression biopsy as a subsequent treatment, and the response rate was 18%. The median progression-free survival for afatinib was 3.7 months for the entire group, and 3.2 and 4.6 months for the groups with and without T790M, respectively (P = 0.33).

Conclusions

The identification of T790M as acquired resistance mechanism was clinically feasible. Although T790M had no prognostic or predictive role in the present study, further research is necessary to identify patients with T790M-mutant tumors who might benefit from newly developed T790M-specific TKIs.     

Audit of Molecular Mechanisms of Primary and Secondary Resistance to Various Generations of Tyrosine Kinase Inhibitors in Known Epidermal Growth Factor Receptor-Mutant Non-small Cell Lung Cancer Patients in a Tertiary Centre

AimsPresently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary).Materials and methodsThis was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent? Oncomine? Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit.ResultsThe results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation.ConclusionThis study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.     

ASCL1-coexpression profiling but not single gene expression profiling defines lung adenocarcinomas of neuroendocrine nature with poor prognosis

Background

Lung adenocarcinoma is heterogeneous regarding histology, etiology and prognosis. Although there have been several attempts to find a subgroup with poor prognosis, it is unclear whether or not adenocarcinoma with neuroendocrine (NE) nature has unfavorable prognosis.

Materials and methods

To elucidate whether a subtype of adenocarcinoma with NE nature has poor prognosis, we performed gene expression profiling by cDNA microarray for 262 Japanese lung cancer and 30 normal lung samples, including 171 adenocarcinomas, 56 squamous cell carcinomas and 35 NE tumors. A co-expression gene set with ASCL1, an NE master gene, was utilized to classify tumors by non-negative matrix factorization, followed by validation using an ASCL1 knock-down gene set in DMS79 cells as well as an independent cohort (n = 139) derived from public microarray databases as a test set.

Results

The co-expression gene set classified the adenocarcinomas into alveolar cell (AL), squamoid, and NE subtypes. The NE subtype, which clustered together almost all the NE tumors, had significantly poorer prognosis than the AL subtype that clustered with normal lung samples (p = 0.0075). The knock-down gene set also classified the 171 adenocarcinomas into three subtypes and this NE subtype also had the poorest prognosis. The co-expression gene set classified the independent database-derived American cohort into two subtypes, with the NE subtype having poorer prognosis. None of the single NE gene expression was found to be linked to survival difference.

Conclusion

Co-expression gene set with ASCL1, rather than single NE gene expression, successfully identifies an NE subtype of lung adenocarcinoma with poor prognosis.     

Osimertinib With Ramucirumab in EGFR-mutated,T790M-positive Patients With Progression During EGFR-TKI Therapy: Phase Ib Study

Background

Epidermal growth factor receptor (EGFR) T790M mutation is the most frequent mechanism of resistance among patients with progression during EGFR-tyrosine kinase inhibitor (TKI) therapy. A third-generation EGFR-TKI, osimertinib, demonstrated durable efficacy with mild adverse events in a phase III trial and is considered a novel standard regimen. The tolerability of osimertinib monotherapy has allowed for the development of a more efficacious combination regimen. Preclinical and clinical study data have suggested that vascular endothelial growth factor inhibition can enhance EGFR-TKI activity with tolerable toxicity.

Low EGFR/MET ratio is associated with resistance to EGFR inhibitors in non-small cell lung cancer

Purpose

Although activating mutations in the epidermal growth factor receptor (EGFR) gene are predictive markers for response to EGFR inhibitors, 30–40% of EGFR-mutant non-small cell lung cancer (NSCLC) patients are de novo non-responders. Hence, we sought to explore additional biomarkers of response.

Methods

We conducted a prospective pilot study to characterize the expression and/or activation of key receptor tyrosine kinases (RTKs) in stage IIIB-IV NSCLC tumors. A total of 37 patients were enrolled and 34 underwent EGFR inhibitor treatment.

Results

As expected, patients bearing activating EGFR mutations showed increased progression free survival (PFS) compared to patients with wild-type EGFR status (9.3 vs 1.4 months, p = 0.0629). Analysis of baseline tumor RTK profiles revealed that, regardless of EGFR mutation status, higher levels of EGFR relative to MET correlated with longer PFS. At multiple EGFR/MET ratio cut-offs, including 1, 2 and 3, median PFS according to below vs. above cut-offs were 0.4 vs. 6.1 (p = 0.0001), 0.5 vs. 9.3 (p = 0.0006) and 1.0 vs. 11.2 months (p = 0.0008), respectively.

Conclusion

The EGFR/MET ratio measured in tumors at baseline may help identify NSCLC patients most likely to benefit from prolonged PFS when treated with EGFR inhibitors.     

MET phosphorylation predicts poor outcome in small cell lung carcinoma and its inhibition blocks HGF-induced effects in MET mutant cell lines

Background:

Small cell lung carcinoma (SCLC) has poor prognosis and remains orphan from targeted therapy. MET is activated in several tumour types and may be a promising therapeutic target.

Methods:

To evaluate the role of MET in SCLC, MET gene status and protein expression were evaluated in a panel of SCLC cell lines. The MET inhibitor PHA-665752 was used to study effects of pathway inhibition in basal and hepatocyte growth factor (HGF)-stimulated conditions. Immunohistochemistry for MET and p-MET was performed in human SCLC samples and association with outcome was assessed.

Results:

In MET mutant SCLC cells, HGF induced MET phosphorylation, increased proliferation, invasiveness and clonogenic growth. PHA-665752 blocked MET phosphorylation and counteracted HGF-induced effects. In clinical samples, total MET and p-MET overexpression were detected in 54% and 43% SCLC tumours (n=77), respectively. MET phosphorylation was associated with poor median overall survival (132 days) vs p-MET negative cases (287 days)(P<0.001). Phospho-MET retained its prognostic value in a multivariate analysis.

Conclusions:

MET activation resulted in a more aggressive phenotype in MET mutant SCLC cells and its inhibition by PHA-665752 reversed this phenotype. In patients with SCLC, MET activation was associated with worse prognosis, suggesting a role in the adverse clinical behaviour in this disease.     

MET Expression in Advanced Non–Small-Cell Lung Cancer: Effect on Clinical Outcomes of Chemotherapy,Targeted Therapy,and Immunotherapy

Background

The receptor tyrosine kinase MET is implicated in malignant transformation, tumor progression, metastasis, and acquired treatment resistance. We conducted an analysis of the effect of MET expression and MET genomic aberrations on the outcome of patients with advanced or metastatic pulmonary adenocarcinomas prospectively enrolled in an institutional precision oncology program.

Picoliter‐Droplet Digital Polymerase Chain Reaction‐Based Analysis of Cell‐Free Plasma DNA to Assess EGFR Mutations in Lung Adenocarcinoma That Confer Resistance to Tyrosine‐Kinase Inhibitors

Purpose.

The objective of this study was to evaluate the utility of analyzing cell-free plasma DNA (cfDNA) by picoliter-droplet digital polymerase chain reaction (ddPCR) to detect EGFR mutations that confer resistance to tyrosine-kinase inhibitors (TKIs) used for treatment of lung adenocarcinoma (LADC).

Experimental design.

Thirty-five LADC patients who received epidermal growth factor receptor (EGFR)-TKI therapy, including ten who received tumor rebiopsy after development of resistance, were subjected to picoliter-ddPCR-cfDNA analysis to determine the fraction of cfDNA with TKI-sensitive (L858R and inflame exon 19 deletions) and -resistant (i.e., T790M) mutations, as well as their concordance with mutation status in rebiopsied tumor tissues.

Results.

cfDNA samples from 15 (94%) of 16 patients who acquired resistance were positive for TKI-sensitive mutations. Also, 7 (44%) were positive for the T790M mutation, with fractions of T790M (+) cfDNA ranging from 7.4% to 97%. T790M positivity in cfDNA was consistent in eight of ten patients for whom rebiopsied tumor tissues were analyzed, whereas the remaining cases were negative in cfDNA and positive in rebiopsied tumors. Prior to EGFR-TKI therapy, cfDNAs from 9 (38%) and 0 of 24 patients were positive for TKI-sensitive and T790M mutations, respectively. Next-generation sequencing of cfDNA from one patient who exhibited innate resistance to TKI despite a high fraction of TKI-sensitive mutations and the absence of the T790M mutation in his cfDNA revealed the presence of the L747P mutation, a known driver of TKI resistance.

Conclusion.

Picoliter-ddPCR examination of cfDNA, supported by next-generation sequencing analysis, enables noninvasive assessment of EGFR mutations that confer resistance to TKIs.

Implications for Practice:

Noninvasive monitoring of the predominance of tumors harboring the secondary T790M mutation in the activating mutation in EGFR gene is necessary for precise and effective treatment of lung adenocarcinoma. Because cells harboring the T790M mutation are resistant to epidermal growth factor receptor-tyrosine-kinase inhibitors (TKIs), the predominance of tumor cells harboring the T790M mutations influences the choice of whether to use conventional or next-generation TKIs. Digital polymerase chain reaction-based examination of cfDNA is a promising method; however, its feasibility, including its consistency with examination of rebiopsied tumor tissue, has not been fully proven. Here, picoliter-droplet digital polymerase chain reaction technology is presented as a candidate method for testing cfDNA and assessing the predominance of T790M-mutant tumors.     

ERK phosphorylation predicts synergism between gemcitabine and the epidermal growth factor receptor inhibitor AG1478

Background

Clinical trials combining epidermal growth factor receptor (EGFR) inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly RAS mutation. Furthermore, ERK, a critical growth regulator downstream of RAS, may play a role. This study aimed to explore the relationship between ERK, synergy/antagonism and cell cycle arrest in combination treatment.

Methods

A549 (mutant KRAS), H322 (wildtype KRAS) and siRNA-mediated KRAS knockdown A549 were treated with gemcitabine and/or the EGFR inhibitor AG1478 and analyzed with median effect analysis. Cell cycle distribution and ERK phosphorylation were assessed using flow cytometry and ELISA, respectively. Effect on cytotoxicity after ERK inhibition by U0126 was also assessed.

Results

Cytotoxic interaction was dose dependent with antagonism at high dose AG1478. G1 arrest was observed with both high dose AG1478 and high dose gemcitabine and therefore was inconsistently associated with antagonism. Furthermore, ERK phosphorylation was increased by gemcitabine and its suppression by AG1478 was related to antagonism particularly in H322. ERK's effect in antagonism was further confirmed by using U0126. Greater antagonism was observed in the KRAS mutant cell line and KRAS knockdown by siRNA resulted in increased sensitivity to AG1478 as well as combination treatment.

Conclusion

Our findings are consistent with a model in which ERK phosphorylation favors synergy and the outcome depends on the balance between gemcitabine-induced and AG1478-inhibited ERK phosphorylation. KRAS mutation confers resistance to AG1478 as well as combination treatment.     

Role of HGF in epithelial–stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ

Introduction

Basal-like and luminal breast cancers have distinct stromal–epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal–epithelial interactions evolve in benign and pre-invasive lesions.

Methods

To study epithelial–stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis.

Results

Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basal-like subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival.

Conclusions

Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.