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M. Macmanus
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MINI 26 - Circulating Tumor Markers (ID 148)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 14
- Moderators:M. Macmanus, C. Aggarwal
- Coordinates: 9/09/2015, 16:45 - 18:15, 205+207
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- Abstract
- Presentation
Background:
Plasma cell-free DNA (cfDNA) contains genetic information from primary and metastatic cancer foci. We utilized multiplex deep sequencing technology to investigate the clinical significance of cfDNA concentration and mutational burden in advanced non-small-cell lung cancer patients treated with EGFR tyrosine kinase inhibitors (TKIs).
Methods:
Between January 2012 and February 2014, seventy-one eligible patients from Sun Yat-sen University Cancer Center were enrolled. All the patients provided written informed consent and donated 2 ml plasma before taking EGFR-TKIs. Plasma DNA was isolated and purified using QIAamp Circulating Nucleic Acid Kit. CfDNA concentration was determined by Qubit Fluorometer. A set of 234 primer pairs were designed to amplify sequences covering hotspots of 35 genes. The amplicon libraries were prepared and entered into deep sequencing on Ion Torrent PGM chip. Variants were called by established bioinformatics methods. Circulating DNA mutational burden was defined as the number of somatic variants other than EGFR mutations. Objective response rate (ORR) and disease control rate (DCR) between different groups were compared using Fisher’s exact test while progression-free survival (PFS) and overall survival (OS) between different groups were compared by Kaplan-Meier curves and log-rank tests. Multivariate stepwise Cox regression analyses were performed to identify independent prognostic factors.
Results:
Forty-nine out of 71 patients were observed to harbor at least one variant and at most 7 variants, involving 10 genes (totally 124 variants). Higher cfDNA concentration was associated with impaired DCR (18.6% vs 81.4%; p=0.008), PFS (median PFS, 3.5 vs 15.2 months; HR=3.03; p=0.001) and OS (median OS, 27.3 vs not-reached; HR=2.38; p=0.042) compared with low cfDNA concentration group. Higher mutational burden was associated with unfavorable ORR (31.6% vs 73.7%; p=0.004) and PFS (median PFS; 8.6 vs 17.8 months; HR=1.61; p=0.050) compared with low mutational burden group. EGFR mutation conferred better ORR, DCR and PFS compared with EGFR wild-type (Figure 1). Multivariate analyses revealed that apart from EGFR mutation status, cfDNA concentration and mutational burden were also associated with the efficacy and/or the prognosis of EGFR-TKIs.Figure 1
Conclusion:
We for the first time showed that cfDNA concentration and mutational burden might influence the efficacy and prognosis of patients receiving EGFR-TKIs. These findings call for the need for the multiplex genetic analysis of patients’ cfDNA to tailor their treatment.
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- Abstract
Background:
To detect the consistency of the c-MET gene amplification in peripheral blood and tumor tissue of patients with non small cell lung cancer and discuss the clinical application value of c-MET gene amplification in peripheral blood.
Methods:
Real-time fluorescent quantitative PCR was used to test the tissues in 257 patients of non small cell lung cancers and the peripheral blood samples in 318 patients of non small cell lung cancer, of which 185 cases of peripheral blood specimens could match the tissue samples, and detected the c-MET gene amplification in them by comparison of amplifications consistency in blood and tissue samples, and analysed the correlation between c-MET gene amplification and clinical characteristics of patients.
Results:
The c-MET gene amplification rate was 9.75% in peripheral blood of 31 patients with non small cell lung cancer，and was 8.95% in 23 cancer tissues, the amplification consistency was 81.25% in peripheral blood-tumor tissue matched samples. The difference was statistically significant (P<0.05).
Conclusion:
The consistency of the c-MET gene amplification in peripheral blood and tissue is high. c-MET gene amplification of peripheral blood could be used for clinical diagnosis and treatment in cases when tissue specimen is hard to get．
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MINI26.04 - Discussant for MINI26.02, MINI26.03 (ID 3376)
16:55 - 17:05 | Author(s): R. Salgia
- Abstract
- Presentation
Abstract not provided
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MINI26.05 - Immunophenotyping of Circulating T Cells and TILs with Chemotherapy and Phased Ipilimumab in Non-Small Cell Lung Cancer (ID 2787)
17:05 - 17:10 | Author(s): F. Dunphy, J. Yi, M. Onaitis, R. Osborne, D. Harpole, J. Crawford, T. D'Amico, K. Weinhold, N. Ready
- Abstract
- Presentation
Background:
Ipilimumab (Ipi) is a humanized CTLA-4 antibody that blocks binding of CTLA-4 with its cognate ligands, permitting T cell activation through CD28 binding. There is evidence that phased in Ipi added to chemotherapy (C) may enhance efficacy in non-small cell lung cancer NSCLC. This trial was undertaken to gain a better understanding of the changes that occur in T cells, regulatory T cells (Tregs), and myeloid-derived suppressor (MDSC) in both the blood and tumor micro-environment with CTLA-4 blockade.
Methods:
Patients with stage T > 4 cm and/or N1, N2 NSCLC were offered neoadjuvant carboplatin AUC6 plus paclitaxel 200 mg/m[2] every 21 days 3 cycles with ipilimumab 10 mg/kg day 1 cycles 2 and 3. Blood for immune profiling of circulating T cells was collected prior to cycle 1, after cycle 1 chemotherapy alone, and after cycle 3 chemotherapy plus Ipi. If patients underwent tumor resection and excess tumor was available, viable tumor infiltrating lymphocytes (TILS) were disaggregated and stored for later analysis. Phenotypic and functional polychromatic flow cytometry (PFC) analyses were performed on peripheral blood mononuclear cells (PBMC).
Results:
Blood was successfully collected at all 3 time points for the first 17/18 patients who initiated trial therapy. Excess tumor (0.96-5 gms) was collected on 5 patients and ample viable CD45+ TIL cells (9.4-26x10[6]) were isolated and viably cryopreserved. Phenotypic analyses revealed that both CD4+ and CD8+ cells from all 17 patients were highly activated following two cycles of ipilimumab (cycle 3) as evidenced by greatly increased frequencies of CD28, HLA-DR, PD-1, and intracellular CTLA-4 expressing cells. The frequencies of Tregs, defined by CD4+CD25+FoxP3+ expression, were highly variable among the 17 participants, with 8 showing increased Tregs, 7 showing decreased frequencies, and 2 remaining unchanged over the course of therapy. Seven of the 17 participants had levels of MDSC cells at or above 5%, with two patients achieving MDSC levels of 13% and 26.7%. Tumor associated antigen (TAA)-specific CD4+ or CD8+ cells were detected at baseline on 4 patients (24%), but their relative frequencies were unaltered by Ipi therapy. The most commonly recognized TAA was Survivin, followed by MAGEA3 and PRAME. No patients developed detectable de novo TAA reactivities while on Ipi therapy. We will report the phenotypic and functional parameters of TILs isolated from 5 tumors of the patients enrolled on the current trial at the time of presentation.
Conclusion:
TAA-specific CD4+ or CD8+ cells were unexpectedly detected in the blood at baseline in a subset of patients. We were able to determine what common NSCLC antigens the circulating CD4+ or CD8+ T cells were activated against, and this has the potential to be a blood based biomarker for trials studying immunotherapy such as vaccines. Neoadjuvant ipilimumab therapy neither facilitated the development of anti-TAA reactivities nor enhanced the frequencies of existing TAA-reactive T cells in PBMC. Neoadjuvant ipilimumab therapy effectively enhanced the frequencies of highly activated T cells, but had no consistent effect of the frequencies of Tregs.
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MINI26.06 - Cytological Criteria Based in the Characterization of CTCs for Assessment the Response to Erlotinib (ID 2974)
17:10 - 17:15 | Author(s): M.J. Serrano, J.M. Jurado, J. Valdivia, M. Mellado, C. Bayarri, J. Exposito-Fernandez, J.L. Garcia-Puche
- Abstract
- Presentation
Background:
During the past decade, circulating tumor cells (CTCs) have been accepted as new prognostic and predictive factors for some type of cancers. In non small lung cancer cells (NSCLC) their detection and characterization is especially important to identify treatment resistances that some patients develop. Here, we report the value of characterization of CTCs and established cytological criteria to assess a good response for EGFR- tyrosine-Kinase inhibitors
Methods:
From Feb 2012 to October 2014, 39 patients (median age 63 years) with metastatic lung cancer were included in this study. NSCL (26 Adenocarcinomas and 13 Squamous cell carcinoma) EGFR wild-type patients were being treated with second or higher lines therapies with erlotinib. 10 ml of blood were collected from each patient into a CellSave TM Preservatives Tubes (Veridex, LLC, Johnson & Johnson Company) blood collection tube, maintained at room temperature and processed within a maximum of 72 hr after collection according to the protocol established by our group .For CTCs enrichment from PBMCs we used “Carcinoma Cell Enrichment and Detection kit: MACS technology (Miltenyi Biotechnology), using magnetic beads labeled with a multi-CK-specific. After isolation of CTCs, samples containing CTCs CK+ were stained for EGFR in double immunofluorescence (IF) experiments, following our standard protocol.The assessment of treatment was evaluated by histological criteria (CyCaR: Cytological Criteria of assessment Response).This way, as favorable response was defined when the number of CTCS was reduced more than 50% at 6 or 12 weeks of starting erlotinib treatment
Results:
Before treatment 18/39 patients (46%) were identified as positive for CTC[CK+ ]with an average number of 3.5 cell (range 1-11); 9 of which were presented persistence of CTCs after treatment. 7/ 18 patients positive for the presence of CTC[CK + ]were positive to presence EGFR marker (CTC[CK+ /EGFR +).]. More important was, that we found A positive correlation between CTCs and survival: patients with CTC(CK+ /EGFR-) presented a shorter OS (34 vs 53 weeks) and PFS (13 vs 17 weeks) compared with those patients with CTC(CK+ /EGFR +) which responded favorably to erlotinib. Patients without cytological response criteria had a worse survival OS (28 vs 53 weeks, p=0.057) and PFS (11,5 vs 21,3 weeks, p=0.06).
Conclusion:
Our study suggests that the characterization of CTC based in the EGFR expression might be useful as a marker for the therapeutic selection and monitoring of lung cancer patients sensitive to treatment with inhibitors of tyrosin-kinase.
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MINI26.07 - Circulating Tumor Cells (CTC) Enrichment as Liquid-Biopsy for Molecular and Genomic Characterization in ALK-Rearranged (ALK+) Lung Cancer (ID 3132)
17:15 - 17:20 | Author(s): P.C. Ma, L. Yin, P. Joshi, Y. Feng, W. Zhang, M. Shapiro, M. McNamara, N. Hashemi Sadraei, N. Pennell, Q. He, N. Chen, E. Borden, M. Zborowski
- Abstract
- Presentation
Background:
Precision therapy with tyrosine kinase inhibitor (TKI) crizotinib and ceritinib against EML4-ALK (ALK+) non-small cell lung cancer (NSCLC) has advanced rapidly in recent years as a new paradigm in personalized cancer therapy. However, acquired drug resistance despite initial response still remains the rule, necessitating further investigations into mechanisms of resistance and novel therapies to overcome progressive resistant disease. Liquid-biopsy using peripheral blood circulating tumor cells (CTCs) as a minimally-invasive tool to determine patient’s disease status, tumor cells molecular-genomic make-up and evolution during therapies, is highly desirable. There is still an unmet need to develop affordable and robust technology platforms to empower such liquid-biopsy assay of CTC. There are relative advantages and pitfalls with various CTC platforms and a method to capture CTC in an unbiased fashion without pre-definition would be beneficial.
Methods:
We conducted pilot studies with adoption of two different CTC detection and enrichment platforms. First, we used the CellSearch[®] “positive-selection” platform through EpCAM immunomagnetic separation to profile 11 pts with ALK(+) NSCLC who were treated with crizotinib prospectively. Blood samples were collected (i) pretreatment, (ii) on TKI with CR/PR/SD, and (iii) at disease progression. Second, we evaluated a novel “negative-selection” CTCs capture-isolation platform, based on unbiased immunomagnetic removal of pan-leukocyte marker CD45+ cells coupled with RBC lysis, to enable CTC isolation without predefined CTC criteria. Pilot studies utilizing ALK+ H3122 cell line and ALK+ patients’ blood samples were performed for assay optimization and comparison. Whole genome sequencing using Illumina HiSeq x TEN was performed after whole genome amplification of the CTC tumor gDNA with paired-normal germline DNA for genomic interrogation.
Results:
Using ALK+ NSCLC patients’ peripheral blood samples, we demonstrated the presence of the EML4-ALK fusion (variant 1) in QPCR assay from the enriched CTC isolated using the CellSearch® platform. Also, CellSearch[®] enumeration in our pilot ALK+ cohort revealed a trend of correlation between the CTC numbers and disease status. The spike-in experiment in “negative selection” CTC platform enriched the spiked H3122 cells by 10,000 fold from the nucleated blood cells. Applying our “negative-selection” CTC assay to a patient with known EML4-ALK variant 1 (EML4-ex13/ALK-ex20) fusion lung cancer during disease progression on crizotinib, we detected the specific EML4-ALK variant 1 fusion in QPCR assay from the CTC enriched “eluate” fraction, but not in the “feed” fraction, whether the CellSearch® platform revealed any CTCs or not. In our index case of ALK-rearrangement NSCLC, we successfully performed whole genome sequencing analysis on the pretreatment negative-selection CTCs in comparison with the germline DNA and pretreatment lymph node tumor biopsied tissue tumor DNA. Our preliminary WGS results revealed similar genomic landscapes between the CTC and the biopsied tumor tissues.
Conclusion:
Taken together, our pilot CTC study results support the high sensitivity of the unbiased “negative selection” enrichment platform and its potential to empower molecular and genomic determinations in lung cancer. We also demonstrated the feasibility of the negative-selection CTC liquid-biopsy platform to achieve whole genome sequencing analysis of the captured CTCs.
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- Abstract
Background:
The addition of bevacizumab, a monoclonal antibody against vascular endothelial growth factor, to a standard, first-line platin-based, two-agent chemotherapy regimen conferred a significant improvement in overall survival, progression-free survival, and response rate in patients with advanced non-squamous NSCLC (ns-NSCLC). The feasibility of the combination of bevacizumab and chemotherapy as the second-line or more therapy for ns-NSCLC is currently explored as well. However, no effective biomarker is validated to predict the response or clinical benefit of bevacizumab. This study aims to investigate the correlation between biomarkers and overall response to bevacizumab plus chemotherapy for patients with advanced ns-NSCLC.
Methods:
Patients with locally advanced or metastatic ns-NSCLC who were assigned to 7.5mg/kg 3-weekly doses of bevacizumab plus chemotherapy were eligible. Aaccording to investigators’ decision, chemotherapy regimens were pemetrexed (500mg/m[2]) with or without platinum (75mg/m[2]). Peripheral blood samples were collected at baseline and after 2[nd] cycle of therapy for the analysis of granulocyte colony-stimulating factor（G-CSF）, vascular endothelial growth factor A（VEGF-A） and VEGF receptor-2 (VEGFR-2). Plasma samples concomitantly with the radiological evaluation of disease progression were collected as possible. Correlation between biomarkers and overall response rate (ORR) were assessed by using a logistic regression model.
Results:
Baseline blood samples were available from 45 patients (100%, 19 patients were therapy naive, 26 patients failed of prior therapies), while samples after 2[nd] cycle were obtained from 37 patients (82.22%). For the primary analysis, there was no significant association between baseline plasma biomarkers and best overall response to the treatment. But, patients with low baseline plasma G-CSF level showed a trend toward improving ORR versus patients with high G-CSF level (odds ratio [OR], 3.846; 95%CI, 0.868 to 17.044，p＝0.076). Patients with high baseline plasma VEGF-A level showed a trend toward higher ORR versus patients with low VEGF-A levels (OR, 3.477; 95%CI, 0.857 to 14.113，p＝0.081). No significant correlations were observed between plasma biomarkers and progression-free survival (PFS). In comparison to baseline, plasma VEGF-A level increased significantly at 2[nd] cycle or radiological disease progression (p＜0.001). However, the magnitude of the difference did not correlate with ORR or PFS.
Conclusion:
Baseline or dynamic changes in plasma G-CSF, VEGF-A and VEGFR-2 did not correlate significantly with response of bevacizumab plus chemotherapy treatment for ns-NSCLC patients, while low baseline plasma G-CSF and high VEGF-A are possible candidate biomarkers for predicting response of bevacizumab plus chemotherapy for ns-NSCLC.
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MINI26.09 - Correlation between Circulating Tumor Biomarkers and Positron-Emission Tomography in Advanced Non-Small Cell Lung Cancer (ID 2940)
17:25 - 17:30 | Author(s): C. Genova, E. Rijavec, F. Biello, G. Barletta, C. Maggioni, S. Coco, I. Vanni, A. Truini, A. Alama, G. Buzzatti, S. Morbelli, G. Ferrarazzo, F. Bongioanni, G. Sambuceti, M. Massollo, F. Grossi
- Abstract
- Presentation
Background:
Circulating tumor cells (CTCs) and plasma circulating-free DNA (cfDNA) are promising candidates as non-invasive prognostic markers in malignant diseases. 18-fluorodeoxyglucose positron emission tomography integrated with computed tomography (18FDG-PET/TC) has a well-recognized diagnostic and prognostic value in non-small cell lung cancer (NSCLC). Very little is known about the mutual relationship between circulating biomarkers (CTCs and cfDNA) and 18FDG-PET/CT indicators in NSCLC.
Methods:
Peripheral blood samples from 28 patients affected by advanced/metastatic NSCLC were collected before starting first-line chemotherapy. CTCs were isolated by size using a filtration-based device (ScreenCell) and then identified and enumerated; cfDNA was isolated from plasma (QIAamp DNA Blood Mini Kit, Qiagen) and quantified by qPCR method using human telomerase reverse transcriptase (hTERT). All patients underwent 18FDG-PET/TC (Biograph 16 Siemens) at baseline. Maximum diameter (dmax) of the primary lesion (T), dmax of the greater lymph nodal (N), and metastatic (M) lesions were measured. Similarly, maximum and mean standardized uptake value (SUVmax, SUVmean) and size-incorporated SUVmax (SIMaxSUV) were computed for T, N and M, respectively; SIMaxSUV was calculated with the following formula for T, N, and M: SIMaxSUV= SUVMax*dmax. Presence (B+) and absence (B-) of metabolically active bone lesions (bone mets) were recorded. The association among CTCs, cfDNA and 18FDG-PET/CT-derived parameters was evaluated through multivariate analysis. T-test was performed to evaluate the difference in CTCs and cfDNA in B+ and B- groups, respectively.
Results:
Twenty-eight patients were evaluated; median age was 66 years (range: 51-80); male/female ratio was 18/10; 15 patients were current smokers, while 11 were former-smokers and 2 were never-smokers. Histo-types were grouped as it follows: adenocarcinoma= 22; squamous cell carcinoma= 5; not otherwise specified NSCLC= 1. Nine patients out of 28 had metabolically active bone lesions. Median CTC count was 7 CTCs/3ml (range: 0-47 CTCs/3ml), while median HTERT copy number was 109.0 (range: 16.7-1405-5).
At multivariate analysis, SUVmax of T was the only variable independently associated with cfDNA (p=0.036). No correlations were highlighted between CTCs and all PET-derived parameters. A trend towards significance between high HTERT and the presence of metabolically active bone lesions was observed (p=0.058).18FDG-PET/CT PARAMETERS MEAN STANDARD DEVIATION P T Size 44.93 20.25 0.175 SUV max 10.16 4.48 0.036 SUV mean 10.6 3.4 0.994 SIMaxSuv 487.7 333.5 0.472 N Size 22.2 10.9 0.313 SUV max 7.4 4.0 0.318 SUV mean 5.8 3.0 0.294 SIMaxSuv 172.8 158.1 0.231 M Size 23.9 15.0 0.083 SUV max 7.5 4.1 0.318 SUV mean 7.4 1.2 0.307 SIMaxSuv 216.4 206.5 0.463
Conclusion:
Our data demonstrate that the expression of cfDNA is correlated with the metabolic activity of the primary tumor lesion. Since SIMaxSUV was not correlated with HTERT, it appears that the expression of cfDNA depends from tumor metabolism rather than its burden. Further analyses on 18FDG-PET/TC-derived metabolic tumor volume are ongoing.
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MINI26.10 - Discussant for MINI26.05, MINI26.06, MINI26.07, MINI26.08, MINI26.09 (ID 3377)
17:30 - 17:40 | Author(s): B. Halmos
- Abstract
- Presentation
Abstract not provided
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MINI26.11 - Longitudinal Monitoring of EGFR Mutations in Plasma of EGFR Mutant NSCLC Patients Treated with EGFR TKIs: Korean Lung Cancer Consortium (ID 1130)
17:40 - 17:45 | Author(s): J.Y. Lee, B. Yali, W. Xiumin, B.S. Hyeon, H.Y. Lee, J. Sun, S. Lee, J.S. Ahn, E.K. Cho, H.R. Kim, Y.J. Min, D. Kim, K. Park, X. Qing, M. Mao, M. Ahn
- Abstract
- Presentation
Background:
Detection of epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancer (NSCLC) patients is mainly based on tissue biopsy, which is invasive and time consuming. Furthermore, there is still a need for serial monitoring of EGFR mutations and detection of EGFR tyrosine kinase inhibitors (TKIs) resistance. We hypothesized that plasma-based EGFR mutation analysis may be feasible for monitoring response to EGFR TKIs and could be used to predict the resistance.
Methods:
From January 2012 to October 2014, 200 EGFR mutant NSCLC patients were enrolled and treated with EGFR TKIs (141 patients for gefitinib, 46 patients for erlotinib, and 13 patients for afatinib). Plasma samples were prospectively obtained every 2 months from baseline until disease progression. The longitudinally collected plasma samples (n = 368) from 81 patients who progressed were analyzed using droplet digital PCR (ddPCR). We identified an association between serial EGFR mutant titers in plasma cell-free DNA (cfDNA) samples and the patient’s clinical response to EGFR TKIs.
Results:
Of a total 58 baseline cfDNA samples available for ddPCR, 43 (74%) samples demonstrated same mutation in the matched tumors (i.e. sensitivity: 70.8% (17/24) for L858R vs 76.5% (26/34) for exon 19 deletions). The concordance rate of plasma with tissue results of EGFR mutation was 88% for L858R and 86% for exon 19 deletion, respectively. Of the 54 patients with both before and after treatment plasma samples, 40 patients showed a dramatic decrease of mutant copies (greater than 50%) in blood in the first 2 months after treatment. We also found the secondary mutation (T790M) emerged in 28 patients around 3~13 months after treatment and in 4 patients before the treatment. Elevated circulating mutations (L858R/ex19/T790M) can be detected in 5 patients before disease progression as determined by CT scan.
Conclusion:
These results suggest that ddPCR is an appropriate method for determining plasma-based EGFR mutation status and may aid in monitoring response to EGFR TKIs and early detection of EGFR TKIs resistance.
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MINI26.12 - Circulating Tumor DNA for Noninvasive Monitoring of Non-Small Cell Lung Cancer Patients Receiving EGFR-Targeted Therapies (ID 372)
17:45 - 17:50 | Author(s): D. Tsui, M. Muhammed, A.S. Wong, O.M. Rueda, T. Forshew, R. Soo, H. Wong, B.C. Goh, T. Eisen, F. Marass, D. Gale, W. Liu, T.M. Chin, N. Rosenfeld
- Abstract
Background:
Analysis of circulating tumor DNA (ctDNA) in plasma offers an opportunity to noninvasively monitor tumor burden and identify alternative drivers of disease progression in real-time. However, cancer progression during targeted therapy, such as EGFR-targeted therapies in non-small cell lung cancer (NSCLC), is driven by clonal evolution, and how this impacts the levels of targeted mutations in circulating tumor DNA (ctDNA) for monitoring disease burden is unclear.
Methods:
We collected serial plasma samples from 47 NSCLC patients receiving EGFR-targeted therapy (gefitinib) and hydroxychloroquine, and analysed mutations in EGFR, TP53, PTEN and PIK3CA in plasma by digital PCR and tagged-amplicon deep sequencing (TAm-Seq) of ctDNA.
Results:
We identified the same EGFR mutations in tumor and plasma samples in over 97% of patients, and found that patients with high pre-treatment levels of ctDNA are associated with worse progression-free survival and overall survival. Serial plasma analysis of 32 patients reveals clonal dynamics in ctDNA in response to treatment. In >72% of patients (23/32), EGFR mutations levels increased preceding clinical progression, with the resistant mutation T790M detected in around 50% of these patients (13/23) a median of 6 months before progression became clinically evident. In the remaining 9 of the 32 patients, EGFR-mutant ctDNA levels became uninformative during treatment, and in two patients we identified alternative driver mutations in ctDNA that correlated with progression. In one patient we also showed that the analysis of relative representations of resistant and sensitizing mutations may provide insight to the response to sequential treatment.
Conclusion:
Our results demonstrate the potential of ctDNA for noninvasive stratification and monitoring disease progression in NSCLC patients, and highlight that targeted therapy may drive the selection of alterative mutations. This may impact the representation of the targeted mutations in plasma for assessing disease burden. We therefore propose that effective ctDNA-based monitoring of targeted therapies in oncogene-addicted cancers requires tracking of multiple mutations beyond the targeted genes.
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MINI26.13 - Serial ctDNA Assessment of Response and Resistance to EGFR-TKI for Patients with EGFR-L858R Mutant Lung Cancer from a Prospective Trial (ID 3107)
17:50 - 17:55 | Author(s): Q. Zhou, J.-. Yang, Z.-. Chen, X.-. Zhang, H.-. Yan, J. Su, H.-. Chen, C.-. Xu, H.-. Tu, W.-. Zhong, X.-. Yang, Y.-. Wu
- Abstract
- Presentation
Background:
Plasma circulating tumor DNA (ctDNA) has been widely accepted as a form of liquid biopsy to detect EGFR mutations in NSCLC for its high concordance rate with tumor tissues. There are some retrospective studies about the ctDNA quantitative changes of EGFR mutations in EGFR-TKI treatment, but there is no report about serial ctDNA assessment of response and resistance to EGFR-TKI by detecting the dynamic changes of EGFR mutations during the whole course of EGFR-TKI treatment based on prospective clinical trial.
Methods:
Based on a randomized trial initiated to compare erlotinib with gefitinib in advanced NSCLC harboring EGFR exon 21 L858R mutation in tumor tissues (CTONG0901, NCT01024413), we prospectively collected serial plasma samples as preplanned schedule (baseline, one week after treatment, one month after treatment and then every 8 weeks until disease progression) and quantitatively detected EGFR L858R mutation in ctDNA by using fluorescence quantitative polymerase chain reaction. We made a serial ctDNA assessment of response and resistance to EGFR-TKI and its correlation with survival outcomes. Four patients’ serial plasma samples were selected to undergo next generation sequencing (NGS).
Results:
From 108 patients enrolled in the trial, serial plasma of 80 patients were collected as schedule and tested the quantity of L858R. As a whole, the quantity of L858R decreased to the lowest level when patients achieved best response to EGFR-TKI and increased to the highest level when disease progressed. Further analysis by Ward's Hierarchical Clustering Method showed that the dynamic changes of quantity of L858R could be categorized into two groups, Ascend Group and Stable Group (Figure 1A). Median progression-free survival (PFS) was 11.1 months (95%CI=6.6-15.6) and 7.5 months (95%CI=1.4-13.6) in two groups, respectively (HR=0.57, 95%CI=0.34-0.97, P=0.035) (Figure 1B). Median overall survival was 20.1 months (95%CI=15.7~24.5) vs. 16.4 months (95%CI=13.3~19.6) (HR=0.73, 95% CI =0.38~1.38, P=0.322). In multivariate Cox proportional hazards regression analysis, changing group was independent predictive factor for PFS. In plasma samples of 4 patients underwent NGS, similar dynamic changing characteristics were confirmed and more genetic mutations were found. Detailed data will be presented on site.Figure 1
Conclusion:
This is the first report about serial ctDNA assessment of response and resistance to EGFR-TKI by detecting the dynamic changes of EGFR mutation based on a prospective clinical trial. The quantity of plasma L858R has different changing patterns during EGFR-TKI treatment and higher L858R mutation abundance on EGFR-TKI resistance is correlated with longer PFS.
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- Abstract
- Presentation
Background:
EGFR-T790M mutation, which is the valuable target for the next generation of EGFR-TKI, accounts for about half of the acquired resistance to current EGFR-TKI therapy in the EGFR sensitive mutation positive NSCLC patients. Due to clinical challenge in obtaining re-biopsy tumor tissues, noninvasive detection of EGFR-T790M in plasma circulating free DNA (cfDNA) has been proved to be feasible. Yet a highly sensitive assay needs to be developed to avoid false-negative detection. We here explored whether droplet digital PCR (ddPCR) of cfDNA can an alternative assay to identify the EGFR-TKI resistance mediated by EGFR-T790M in the clinical practice.
Methods:
The digital PCR method was recently developed for EGFR sensitive mutations, and its high sensitivity and specificity were validated in plasma cfDNA from EGFR-TKI-naïve NSCLC patients. In this study, we applied this method to detect EGFR-T790M in plasma cfDNA from metastatic NSCLC patients who initially responded but acquired resistance to current EGFR-TKI treatment. For the concordance analysis, the paired re-biopsy or pleural effusion cytology samples after failed EGFR-TKI were also collected for EGFR-T790M testing.
Results:
25 consecutive NSCLC patients were enrolled and analyzed in this study according to these criteria: 1. Metastatic NSCLC patients with acquired EGFR-TKI resistance. 2. The re-biopsy tissue or cytology samples and paired plasma samples were available after disease progression on EGFR-TKI. Among these 25 patients, 13 were positive and 9 were negative for EGFR-T790M mutation in both tumor tissue and plasma samples. 3 patients positive for EGFR-T790M mutation in tumor tissue were detected negative in their plasma. The overall concordance rate between plasma and tumor tissue testing was 88.00% (22/25) (Kappa=0.757, 95%CI: 0.4996-1.0). The sensitivity and specificity for plasma testing of EGFR-T790M mutation by ddPCR were 81.25% (13/16) (95%CI: 54.35%-96.00%) and 100.00% (9/9) (95%CI: 66.37%-100%), respectively. Figure 1
Conclusion:
Detection of EGFR-T790M in plasma cfDNA by ddPCR is highly sensitive and specific when compared to the pairedre-biopsy tissue or cytology samples. This noninvasive method could complement current invasive biopsy approach or provide an alternative method to identify specific mutation mediated resistance in clinic.
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MINI26.15 - Discussant for MINI26.11, MINI26.12, MINI26.13, MINI26.14 (ID 3378)
18:00 - 18:10 | Author(s): R. Perez-Soler
- Abstract
- Presentation
Abstract not provided
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MS 20 - Joint Imaging/Therapy Conference (ID 38)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Treatment of Locoregional Disease – NSCLC
- Presentations: 1
- Moderators:O.T. Brustugun, D. Grunenwald
- Coordinates: 9/09/2015, 14:15 - 15:45, 601+603
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MS20.03 - Optimal Monitoring After Combined Modality Treatment (Imaging and Markers) (ID 1939)
15:00 - 15:20 | Author(s): M. Macmanus
- Abstract
- Presentation
Abstract:
When response assessment is carried out after definitive high-dose radiation therapy (RT) or chemoRT for patients with locally-advanced non-small cell lung cancer (NSCLC), it should give an early indication of the likely prognosis of the patient. Ideally it should identify those patients most likely to experience long term freedom from progression, who require no further therapy, and it should further identify patients with persistent or progressive disease who could benefit from additional therapy or who may be candidates for clinical trials of investigational treatments designed to improve their poor prognosis. In usual clinical practice, response assessment in NSCLC involves the use of structural imaging with computed tomography (CT), to assess the effect of treatment on tumor volumes. The initial dimensions of tumor sites are compared with their dimensions after treatment, either on a single occasion or with serial images acquired over time. Potential sites of distant disease progression are also sought within the field of view of the restaging CT scan although this is a relatively insensitive test for small volume metastatic tumour. Another possible approach to response assessment is to employ a global measure of the success of therapy, typically by analysing serial blood samples for a tumor-specific biomarker. A sensitive blood-based assay could potentially detect the presence of very small amounts of persistent tumor, beyond the resolution of currently available imaging modalities. A disadvantage of a blood test compared to imaging in a locoregionally confined rather than a metastatic cancer is the absence of any indication of the likely location of persistent or recurrent disease, making it impossible to implement any local salvage therapies without additional information. However, a combination of a sensitive biomarker and state of the art imaging could potentially provide detailed and clinically useful prognostic information after therapy. The use of both local and global approaches to response assessment will be discussed.Using Imaging to assess local Treatment Response in NSCLCStructural Imaging Traditionally, serial imaging with CT has been used to assess treatment response in NSCLC. Serial tumor measurements are compared with specific response assessment criteria, enshrined in systems such as the Response Evaluation Criteria In Solid Tumors (RECIST) [1]. Patients are categorized by RECIST as having either; Complete Response (CR): Disappearance of all target lesions Partial Response (PR): At least a 30% decrease in the sum of the longest diameter (LD) of target lesions, taking as reference the baseline sum LD Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started, or Progressive Disease (PD): At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions Although these categories have prognostic significance, they can be an unreliable predictor of ultimate survival in individual cases. Tumor masses are often slow to resolve after RT and their margins may be obscured by fibrotic or consolidated lung, making accurate measurements impossible. The very concept of remission is hard to define after RT in NSCLC because of the changes in the thorax that occur due to a combination of invasion and destruction of parenchyma by tumor and of the morphological changes that result from atelectasis, radiation pneumonitis and radaition induced pulmonary fibrosis. Fibrotic masses may persist indefinitely even in cured cases. Regions of dense fibrosis can harbor persistent tumor that only becomes apparent when regrowth occurs months or years after treatment is complete. The CR category, with disappearance of all lesions, may be especially hard to define on CT imaging. CT scanning, although it is the standard response assessment modality in clinical trials, has very significant limitations when used for this purpose.Functional Imaging with PET Some of the limitations of CT can be transcended by the use of molecular imaging. The advent of positron emission tomography (PET), using [18]F-fluoro-deoxyglucose (FDG) as the tracer, has provided a means of “seeing inside” areas of fibrosis and persistent mass lesions and identifying focal areas of persistent tumor. Furthermore PET imaging compensates for another major limitation of CT, that of its poor sensitivity and specificity for assessing the true status of mediastinal nodes. Unlike CT, PET can detect tumor in small (<1cm short axis) mediastinal nodes and correctly defines enlarged reactive nodes as non-malignant in the great majority of cases. Several meta-analyses have confirmed the superiority of PET-based mediastinal staging in this regard, making it a logical choice for re-staging the mediastinum after therapy. Prospective data have shown the superiority of PET-based response assessment compared to CT-based response assessment after RT in NSCLC. Our group developed FDG-PET response criteria based on visual assessment and used them prospectively in patients treated with RT/chemoRT [2]. Patients were classified into four metabolic response categories groups, namely; 1) Complete Metabolic Response (CMR): tumor FDG uptake absent or less than mediastinal blood pool. 2) Partial Metabolic Response (PMR): appreciable reduction in the intensity of tumor FDG uptake or tumor volume. 3) Stable Metabolic Disease (SMD): no appreciable change in intensity of tumor FDG uptake or volume. 4) Progressive Metabolic Disease (SMD); any new sites of disease, and/or an appreciable increase in intensity of tumour FDG uptake or volume in known tumor sites. In 73 patients, PET response was evaluated at a median of 70 days post-treatment. PET and CT responses were the same in only 40% of cases and PET response predicted survival much better than CT response. There were many more complete responders on FDG criteria (n=34) compared to CT (N=10), and no patients were inevaluable by PET on compared to 6 on CT. In this study, PET was clearly far superior to CT and in an expanded cohort it was clear that a poor PET response was strongly associated with distant metastasis [3]. Without standardization, the use of visual response criteria may be limited by interobserver variability. The Deauville criteria were developed specifically for use in lymphoma in an effort to standardise visual response assessment by comparing residual tumor FDG uptake with uptake in the liver and mediastinum [4]. Another way to reduce interobserver variability is to use a semi-quantitative method of response assessment, such as by comparing pre- and post treatment standardized uptake values (SUV). Although this is an attractive approach, accuracy may be affected by differences technique on different scanning occasions and by the fact that after treatment, uptake of FDG in radiation penumonitis is often within the range associated with the presence of tumor. This is especially so after high dose hypofractionated stereotactic body radiotherapy (SBRT). It is inappropriate therefore to consider a particular SUV cut-off as being diagnostic of persistent disease. Uptake in lung affected by radiation pneumonitis can also hamper visual response assessment but on a qualitative reading of the scan, pattern recognition can take this into account and still provide valuable prognostic information [5]. Despite the apparent superiority of PET for response assessment, no large prospective studies have yet helped refine how this information might be used. The ideal time for imaging is undecided. A longer interval between treatment and imaging is likely to be associated with greater accuracy but less clinical utility. The use of PET imaging during RT is being actively explored by several groups but remains investigational. In anecdotal cases, patients with resectable PET-detected residual disease have undergone successful salvage surgery after RT but large prospective trials are required to validate this approach.Use of circulating biomarkers to measure global treatment response in NSCLC In some cancers, the use of biomarkers in the blood to monitor disease status is a well established part of standard management. Commonly used circulating biomarkers include paraproteins in multiple myeloma, prostate specific antigen in prostate cancer and alpha-fetoprotein and human chorionic gonadotrophin in germ cell tumors. These markers can be highly specific and sensitive and can be used to guide therapy. However, in NSCLC, the search for a practical circulating biomarker with wide application has been hampered by the extreme heterogeneity of this group of diseases. Two of the most intensely investigated tumor biomarkers in NSCLC have been carcinoemryonic antigen (CEA), which is commonly detected in adenocarcinoma and CYFRA21-1 which can be detected in squamous carcinoma. In a review of the literature in 2012, Grunnet and Sorensen analysed the level of CEA as a prognostic marker in NSCLC in 23 studies of serum and two of plasma [6]. In 18 studies CEA was found to be a prognostic marker for either overall survival OS, recurrence after surgery and/or progression free survival (PFS) in NSCLC patients. The remaining 7 studies contained an excess of patients with squamous carcinoma. One study found that a tumor marker index (TMI), based on preoperative CEA and CYFRA21-1 serum levels was useful as a prognostic marker for OS. Six studies evaluated the use of CEA as a predictive marker. Four of these studies found, that serial CEA measurement had some potential as a predictive marker for recurrence and death. Although a combination of CEA and CYFRA21-1 markers have some value in a proportion of patients with NSCLC the heterogeneity of their expression limits their role in response assessment after RT [7]. Measurement of circulating tumor (ct)DNA has shown promise as a "liquid biopsy" for assessing cancer burden but ctDNA detection methods have to date been insensitive or lacked the broad coverage needed to permit clinical application in NSCLC where genetic variation is extreme. Because background circulating DNA is present in healthy individuals, tumour derived ctDNA can be detected and quantified only if it contains a tumour specific sequence. Diehn and colleagues at Stanford reported a breakthrough in ctDNA in NSCLC, which they called “Cancer Personalized Profiling by Deep Sequencing” (CAPP-Seq) [8]. This is an ultrasensitive method for quantifying ctDNA with clinical applicability. CAPP-Seq was implemented in NSCLC patients with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumours. The method detected ctDNA in 100% of patients with stage II–IV NSCLC and in 50% of patients with stage I disease, with 96% specificity for mutant allele fractions down to ~0.02%. At least one, and on average 4, mutations were covered in >95% of patients. Levels of ctDNA detected by CAPP-Seq were highly correlated with tumour volume and helped distinguish between residual disease and treatment-related imaging changes in several cases. A large clinical trial is being planned to establish the utility of ctDNA for monitoring disease status after RT in NSCLC.Conclusions Structural imaging with CT gives useful prognostic information after RT in NSCLC but is inferior to FDG-PET. Of all of the blood based methods for estimating global tumour burden, ctDNA analysis seems the most promising at present. A combination of PET and ctDNA could potentially provide prognostic information of previously unattainable accuracy and utility.References1. Therasse P, Arbuck SG, Eisenhauer EA, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000;92:205-216.2. Mac Manus MP, Hicks RJ, Matthews JP, et al. Positron emission tomography is superior to computed tomography scanning for response-assessment after radical radiotherapy or chemoradiotherapy in patients with non-small-cell lung cancer. J Clin Oncol 2003;21:1285-1292.3. Mac Manus MP, Hicks RJ, Matthews JP, Wirth A, Rischin D, Ball DL. Metabolic (FDG-PET) response after radical radiotherapy/chemoradiotherapy for non-small cell lung cancer correlates with patterns of failure. Lung Cancer 2005;49:95-108.4. Gallamini A, Barrington SF, Biggi A, et al. The predictive role of interim positron emission tomography for Hodgkin lymphoma treatment outcome is confirmed using the interpretation criteria of the Deauville five-point scale. Haematologica 2014;99:1107-1113.5. Hicks RJ, Mac Manus MP, Matthews JP, et al. Early FDG-PET imaging after radical radiotherapy for non-small-cell lung cancer: inflammatory changes in normal tissues correlate with tumor response and do not confound therapeutic response evaluation. Int J Radiat Oncol Biol Phys 2004;60:412-418.6. Grunnet M, Sorensen JB. Carcinoembryonic antigen (CEA) as tumor marker in lung cancer. Lung Cancer 2012;76:138-143.7. Okamura K, Takayama K, Izumi M, Harada T, Furuyama K, Nakanishi Y. Diagnostic value of CEA and CYFRA 21-1 tumor markers in primary lung cancer. Lung Cancer 2013;80:45-49.8. Newman AM, Bratman SV, To J, et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med 2014;20:548-554.
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ORAL 36 - Translational Science/Radiation (ID 151)
- Event: WCLC 2015
- Type: Oral Session
- Track: Treatment of Locoregional Disease – NSCLC
- Presentations: 1
- Moderators:E. Vokes, B. Kavanagh
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 2c-3c
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ORAL36.06 - 4D-VQ-PET/CT Imaging Allows Strong Correlation Between Radiotherapy Dose and Change in Lung Ventilation, Perfusion and Density (ID 211)
17:39 - 17:50 | Author(s): M. Macmanus
- Abstract
- Presentation
Background:
[68]Ga-V/Q PET/CT is a novel imaging modality for assessment of perfusion(Q), ventilation(V) and lung density changes in the context of radiotherapy (RT) for non-small cell lung cancer.
Methods:
In a prospective clinical trial, 20 patients underwent 4D-V/Q PET/CT before treatment, 4 weeks into treatment and 3 months after definitive lung RT. Eligible patients were prescribed 60 Gy in 30 fractions with or without concurrent chemotherapy. Functional images were registered to the RT planning 4D-CT and isodose volumes averaged into 10 Gy bins. Within each dose bin, relative loss in SUV was recorded for ventilation and perfusion, and loss in air-filled fraction was recorded to assess RT-induced lung fibrosis. A dose-effect relationship was described using both linear and 2-parameter logistic fit models and goodness of fit assessed using Akaike Information Criterion (AIC).
Results:
A total of 179 imaging datasets were available for analysis (1 scan unrecoverable). An almost perfectly linear dose-response relationship was observed for perfusion and air-filled fraction (r[2] = 0.99, p < 0.01), with ventilation also strongly linear (r[2] = 0.95, p < 0.01) [Figure]. Logistic models did not provide a better fit as evaluated by AIC [Table]. Perfusion, ventilation and the air-filled fraction changed by -7.5% ± 0.3%, -7.1% ± 0.6% and 4.9% ± 0.02% per 10 Gy, respectively. Within high-dose regions, higher baseline SUV was associated with greater rate of loss. At 50Gy and 60Gy the rate of loss was 1.35% (p = 0.07) and 1.73% (p = 0.05) per SUV, respectively. Of 8/20 patients with peri-tumoral reperfusion / re-ventilation during treatment, 7/8 did not sustain this effect post-treatment. Figure 1 Figure 2
Conclusion:
RT induced regional lung functional deficits occur in a dose dependent manner and can be estimated using simple linear models with 4D-V/Q PET/CT imaging. These findings may inform functional lung sparing by planning RT using this novel imaging technology.
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