Thijo J. N. Hiltermann

Circulating tumor cells in small-cell lung cancer: a predictive and prognostic factor

BACKGROUND Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to chemotherapy. Patients and methods In this multicenter prospective study, blood samples for CTC analysis were obtained from 59 patients with SCLC before, after one cycle, and at the end of chemotherapy. CTCs were measured using CellSearch systems. RESULTS At baseline, lower numbers of CTCs were observed for 21 patients with limited SCLC (median = 6, range 0-220) compared with 38 patients with extensive stage (median = 63, range 0-14,040). Lack of measurable CTCs (27% of patients) was associated with prolonged survival (HR 3.4; P ≤ 0.001). CTCs decreased after one cycle of chemotherapy; this decrease was not associated with tumor response after four cycles of chemotherapy. CTC count after the first cycle of chemotherapy was the strongest predictor for overall survival (HR 5.7; 95% CI 1.7-18.9; P = 0.004). CONCLUSION Absolute CTCs after one cycle of chemotherapy in patients with SCLC is the strongest predictor for response on chemotherapy and survival. Patients with low initial CTC numbers lived longer than those with higher CTCs.BACKGROUND Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to chemotherapy. PATIENTS AND METHODS In this multicenter prospective study, blood samples for CTC analysis were obtained from 59 patients with SCLC before, after one cycle, and at the end of chemotherapy. CTCs were measured using CellSearch® systems. RESULTS At baseline, lower numbers of CTCs were observed for 21 patients with limited SCLC (median = 6, range 0-220) compared with 38 patients with extensive stage (median = 63, range 0-14 040). Lack of measurable CTCs (27% of patients) was associated with prolonged survival (HR 3.4; P ≤ 0.001). CTCs decreased after one cycle of chemotherapy; this decrease was not associated with tumor response after four cycles of chemotherapy. CTC count after the first cycle of chemotherapy was the strongest predictor for overall survival (HR 5.7; 95% CI 1.7-18.9; P = 0.004). CONCLUSION Absolute CTCs after one cycle of chemotherapy in patients with SCLC is the strongest predictor for response on chemotherapy and survival. Patients with low initial CTC numbers lived longer than those with higher CTCs.

Resistance mechanisms after tyrosine kinase inhibitors afatinib and crizotinib in non-small cell lung cancer, a review of the literature

Targeted treatment of advanced non-small cell lung cancer patients with afatinib in EGFR mutation or crizotinib in ALK break positive patients results in profound tumor responses but inevitably induces resistance. In this review we present currently known resistance mechanisms for afatinib and crizotinib two recently approved drugs. Resistance mechanisms identified for afatinib include c-MET amplification and the V843I EGFR mutation. Expression of FGFR1, increased IL6R/JAK/STAT signaling, enhanced interference with aerobic glycolysis and autophagy are associated with resistance to afatinib. Most common resistance mechanisms for ALK break positive cases are gatekeeper mutations in the ALK gene. Also activation of the EGFR pathway, KRAS mutations, the autophagy pathway and epithelial mesenchymal transition (EMT), have been associated with resistance. Many of the proposed resistance mechanisms need to be functionally studied to proof a causative relationship with resistance.

Common and rare EGFR and KRAS mutations in a Dutch non-small-cell lung cancer population and their clinical outcome

Introduction In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI. Patient and Methods Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis. Results Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively. Conclusion One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival.

An instrument dedicated for modelling of pulmonary radiotherapy

BACKGROUND AND PURPOSE Radiotherapy plays a pivotal role in lung cancer treatment. Selection of patients for new (radio)therapeutic options aiming at improving outcomes requires reliable and validated prediction models. We present the implementation of a prospective platform for evaluation and development of lung radiotherapy (proPED-LUNG) as an instrument enabling multidimensional predictive modelling. MATERIALS AND METHODS ProPED-LUNG was designed to comprise relevant baseline and follow up data of patients receiving pulmonary radiotherapy with curative intent. Patient characteristics, diagnostic and staging information, treatment parameters including full dose-volume-histograms, tumour control, survival, and toxicity are scored. Besides physician-rated data, a range of patient-rated data regarding symptoms and health-related quality-of-life are collected. RESULTS After 18 months of accrual, 315 patients have been included (accrual rate, 18 per month). Of the first hundred patients included, 70 received conformal (chemo)radiotherapy and 30 underwent stereotactic radiotherapy. Compliance at 3 and 6 months follow-up was 96-100% for patient-rated, and 81-94% for physician-rated assessments. For data collection, 0.4 FTE were allocated in a 183 FTE department (0.2%). CONCLUSIONS ProPED-LUNG is feasible with high compliance rates and yields a large amount of high quality prospective disease-related, treatment-related, patient- and physician-rated data which can be used to evaluate new developments in pulmonary radiotherapy.

Surviving respiratory insufficiency with intensive care support in a pretreated, extensively metastasized patient with an EML4-ALK translocation.

Journal of Thoracic Oncology • Volume 8, Number 1, January 2013 A never-smoking 26-year-old woman presented at an outpatient clinic in February 2011 with cough, back pain, and weight loss. A computed tomography (CT) scan demonstrated a mass in the left lung, multiple bone, liver, and adrenal gland metastases. A liver biopsy revealed an adenocarcinoma originating from the lung (thyroid transcription factor-1 positive, EGFR and K-RAS wild type). She was treated with cisplatin and pemetrexed, with stable disease after two cycles. Treatment continued with two cycles carboplatin and paclitaxel with again stable disease. In the period from March till June she was radiated two times for symptomatic bone metastases. Next, she received erlotinib for 2 months, until progression occurred with a new metastasis in her right eye. In September, 1 month later, she gradually developed a paresis in both legs because of vertebral metastases that was treated with a single fraction of 8 Gy radiation. In October 2011, the tumor was tested positive for echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase translocation by fluorescence in situ hybridization. Crizotinib 250 mg twice daily was started. After 3 days of treatment, she deteriorated and developed respiratory failure because of a right-sided pneumonia and pulmonary embolism on top of her massive tumor deposition throughout her body. She was transferred to the intensive care unit for noninvasive ventilation, antibiotics, and low-molecular heparin, in addition to crizotinib. Two days later, she reported sight in her previously blind right eye. Within a week, she was discharged to our regular ward and home in the next week. After 6 weeks, she returned to our outpatient clinic in a good condition. Her preexisting paresis remained stable. CT showed a partial response (55% tumor decrease, Response Evaluation Criteria in Solid Tumors 1.1, Fig. 1B). At 12 and 18 weeks, further clinical improvement was observed and with positron emission tomography/CT imaging a near-complete response (>90% tumor decrease) was noticed (Fig. 1C). At 6 months fluorodeoxyglucose uptake increased slightly in a few liver metastases, with a continuing partial response on CT (Fig. 1D). The patient is still receiving crizotinib at 47 weeks.

Correspondence between primary and secondary care about patients with cancer: A qualitative mixed-methods analysis

Abstract Cancer care is complex and involves many different healthcare providers, especially during diagnosis and initial treatment, and it has been reported that both general practitioners and oncology specialists experience difficulties with interdisciplinary communication. The aim of this qualitative study was to explore information sharing between primary and secondary care for patients with lung, breast or colorectal cancer. A qualitative content analysis of 50 medical files (419 documents) was performed, which identified 70 correspondence‐related items. Six main topics were identified in most referral letters from primary to secondary care, but it was particularly notable that highly relevant information regarding the past medical history was often mixed with less relevant information. To lesser extents, the same held true for the medication list and presenting history. In the letters from specialists, nine topics were identified in most letters. Although information about actual treatment was always present, only limited detail, if any, was given about the intent of the treatment (curative or palliative) or the treatment alternatives. Interviews with nine healthcare providers confirmed these issues. These findings indicate that neither the initial referral nor the specialist correspondence is tailored to the needs of the recipient.

Classification of Cells in CTC-Enriched Samples by Advanced Image Analysis

In the CellSearch® system, blood is immunomagnetically enriched for epithelial cell adhesion molecule (EpCAM) expression and cells are stained with the nucleic acid dye 4′6-diamidino-2-phenylindole (DAPI), Cytokeratin-PE (CK), and CD45-APC. Only DAPI+/CK+ objects are presented to the operator to identify circulating tumor cells (CTC) and the identity of all other cells and potential undetected CTC remains unrevealed. Here, we used the open source imaging program Automatic CTC Classification, Enumeration and PhenoTyping (ACCEPT) to analyze all DAPI+ nuclei in EpCAM-enriched blood samples obtained from 192 metastatic non-small cell lung cancer (NSCLC) patients and 162 controls. Significantly larger numbers of nuclei were detected in 300 patient samples with an average and standard deviation of 73,570 ± 74,948, as compared to 359 control samples with an average and standard deviation of 4191 ± 4463 (p < 0.001). In patients, only 18% ± 21% and in controls 23% ± 15% of the nuclei were identified as leukocytes or CTC. Adding CD16-PerCP for granulocyte staining, the use of an LED as the light source for CD45-APC excitation and plasma membrane staining obtained with wheat germ agglutinin significantly improved the classification of EpCAM-enriched cells, resulting in the identification of 94% ± 5% of the cells. However, especially in patients, the origin of the unidentified cells remains unknown. Further studies are needed to determine if undetected EpCAM+/DAPI+/CK-/CD45- CTC is present among these cells.

Abstract 1602: Urine cell-free DNA as early biomarker in immunotherapy response in NSCLC

Introduction: Immunotherapy shows a durable tumor response in approximately 20% of advanced NSCLC patients. Imaging by CT is used to evaluate tumor response. PD-L1 immunohistochemistry is generally used to predict survival advantages, however with low predictive value. Therefore, it is important that alternative predictive biomarkers are investigated. Minimal invasive techniques may be a solution. The role of circulating cell-free DNA (cfDNA) was found to be an early biomarker for tumor response in KRAS positive Non-Small Cell Lung Cancer (NSCLC) patients treated with immunotherapy (N=6). In this study we will determine the role of urine cell-free DNA (ucfDNA) in an extended cohort. The non-invasive method of urine collection makes it an excellent source for mutation detection and follow-up. Methods: In an ongoing study KRAS positive patients treated with immunotherapy, are being tested for cfDNA at baseline (before treatment, 1, 2, 4, 6 and thereafter every 12 weeks until disease progression) with a KRAS G12-13 or Q61H digital droplet PCR (ddPCR) screening assay (BioRad). In a pilot study, urine from these patients is collected in a tube with 5 ml of STRECK reagent at baseline and at 6 weeks. Results: In 16/27 patients with KRAS positive advanced NSCLC, the mutation was detected in plasma at baseline and subsequent evaluation time points. Urine cfDNA as a predictive marker seems promising and results will be reported and presented at the AACR meeting. Conclusion: Among patients with advanced NSCLC treated with immunotherapy plasma and urine cfDNA are collected and will be presented as non-invasive early biomarker for tumor response evaluation. Citation Format: Anthonie J. van der Wekken, Arja ter Elst, Anneke Miedema, Thijo J. Hiltermann, Harry J. Goen, Ed Schuuring. Urine cell-free DNA as early biomarker in immunotherapy response in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1602.

Abstract 2718: Molecular Tumor Board treatment predictions on rareEGFRexon 20 mutations

Purpose: To evaluate treatment response following the Multidisciplinary Molecular Tumor Board (MTB) decisions which focused on EGFR exon 20 mutations in lung adenocarcinoma. Methods: Molecular studies were routinely performed using the Ion Torrent sequencing platform for histologically or cytologically diagnosed lung adenocarcinoma. Since October 2014 patients with rare (prevalence Summary of data: Over a period of 2 years 1389 samples were tested. A total of 170 (12.2%) rare mutations were detected in lung cancer. Among these rare mutations we observed 16 EGFR exon 20 insertions and other mutations like e.g: p.T790M, p.D761N, p.D770delinsGY, p.S768_D770dup, p.V769_D770insSFL, p.N771_H773dup, p.T790S. Using an in silico modeling of protein responses with TKI were predicted to be likely in EGFR T790M mutations and p.S768_V769delinsIL, but not in p. D770delinsGY, p.S768_D770dup, p.V769_D770insSFL, and p.N771_H773dup mutations. Eight patients with an exon 20 mutation were treated with an EGFR TKI. One patient (p.D761N) had a partial response on erlotinib 300mg daily. On afatinib monotherapy 2 out of 3 pts had stable disease (PFS 3-11 months). On afatinib/cetuximab treatment 1 out of 2 pts (p.D770delinsGY) had a partial response and the other patient had stable disease (p.S768_D770dup) (PFS 11 and 4 months, resp.). Two evaluable patients were treated with osimertinib: the first patient initially progressed on afatinib (p.V769_D770insSFL). The protein model predicted no response, since we predicted hindrance of osimertinib. The model predicted no response on osimertinib in the second patient as well, and during treatment a stable disease for 4 months was observed (p.N771_H773dup). This molecular information from the weekly MTB meeting was delivered within 2 weeks to the treating physician. Feedback on treatment outcome helped to further improve treatment predictions. Conclusion: The Molecular Tumor Board is an effective multidisciplinary team to discuss rare mutations. Our pilot data show that the evaluation of the use and effectiveness of a theoretical model concerning protein structures is not possible yet, however, did provide a clear insight in protein structures in a mutated EGFR receptor supporting decision making of treatment options. Citation Format: Anthonie J. van der Wekken, Matthew R. Groves, Arja ter Elst, Nils A. 9t Hart, Lucie B. Hijmering-Kappelle, Thijo J. Hiltermann, Anke van den Berg, Wim Timens, Ed Schuuring, Harry J. Groen. Molecular Tumor Board treatment predictions on rare EGFR exon 20 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2017-2718

Investigating CTCs in NSCLC-a reaction to the study of Jia-Wei Wan: a preliminary study on the relationship between circulating tumor cells count and clinical features in patients with non-small cell lung cancer.

Liquid biopsies are emerging as a patient-friendly approach for estimating biomarkers to predict treatment outcome and overall survival. Detection and characterization of circulating tumor cells (CTCs) is a promising biomarker and its application for non-small cell lung cancer (NSCLC) is currently under investigation. The FDA approved CellSearch system is used as the standard test.