Marianne Oulhen

Circulating Tumor Cells in Lung Cancer

Circulating tumor cells (CTCs) have emerged as potential biomarkers in several cancers such as colon, prostate, and breast carcinomas, with a correlation between CTC number and patient prognosis being established by independent research groups. The detection and enumeration of CTCs, however, is still a developing field, with no universal method of detection suitable for all types of cancer. CTC detection in lung cancer in particular has proven difficult to perform, as CTCs in this type of cancer often present with nonepithelial characteristics. Moreover, as many detection methods rely on the use of epithelial markers to identify CTCs, the loss of these markers during epithelial-to-mesenchymal transition in certain metastatic cancers can render these methods ineffective. The development of personalized medicine has led to an increase in the advancement of molecular characterization of CTCs. The application of techniques such as FISH and RT-PCR to detect EGFR, HER2, and KRAS abnormalities in lung, breast, and colon cancer, for example, could be used to characterize CTCs in real time. The use of CTCs as a ‘liquid biopsy’ is therefore an exciting possibility providing information on patient prognosis and treatment efficacy. This review summarizes the state of CTC detection today, with particular emphasis on lung cancer, and discusses the future applications of CTCs in helping the clinician to develop new strategies in patient treatment.

Phenotypic and genetic heterogeneity of tumor tissue and circulating tumor cells in patients with metastatic castration-resistant prostate cancer: a report from the PETRUS prospective study

Molecular characterization of cancer samples is hampered by tumor tissue availability in metastatic castration-resistant prostate cancer (mCRPC) patients. We reported the results of prospective PETRUS study of biomarker assessment in paired primary prostatic tumors, metastatic biopsies and circulating tumor cells (CTCs). Among 54 mCRPC patients enrolled, 38 (70%) had biopsies containing more than 50% tumour cells. 28 (52%) patients were analyzed for both tissue samples and CTCs. FISH for AR-amplification and TMPRSS2-ERG translocation were successful in 54% and 32% in metastatic biopsies and primary tumors, respectively. By comparing CellSearch and filtration (ISET)-enrichment combined to four color immunofluorescent staining, we showed that CellSearch and ISET isolated distinct subpopulations of CTCs: CTCs undergoing epithelial-to-mesenchymal transition, CTC clusters and large CTCs with cytomorphological characteristics but no detectable markers were isolated using ISET. Epithelial CTCs detected by the CellSearch were mostly lost during the ISET-filtration. AR-amplification was detected in CellSearch-captured CTCs, but not in ISET-enriched CTCs which harbor exclusively AR gain of copies. Eighty-eight percent concordance for ERG-rearrangement was observed between metastatic biopsies and CTCs even if additional ERG-alteration patterns were detected in ISET-enriched CTCs indicating a higher heterogeneity in CTCs. Molecular screening of metastatic biopsies is achievable in a multicenter context. Our data indicate that CTCs detected by the CellSearch and the ISET-filtration systems are not only phenotypically but also genetically different. Close attention must be paid to CTC characterization since neither approach tested here fully reflects the tremendous phenotypic and genetic heterogeneity present in CTCs from mCRPC patients.

Circulating Tumor Cells with Aberrant ALK Copy Number Predict Progression-Free Survival during Crizotinib Treatment in ALK-Rearranged Non–Small Cell Lung Cancer Patients

The duration and magnitude of clinical response are unpredictable in ALK-rearranged non-small cell lung cancer (NSCLC) patients treated with crizotinib, although all patients invariably develop resistance. Here, we evaluated whether circulating tumor cells (CTC) with aberrant ALK-FISH patterns [ALK-rearrangement, ALK-copy number gain (ALK-CNG)] monitored on crizotinib could predict progression-free survival (PFS) in a cohort of ALK-rearranged patients. Thirty-nine ALK-rearranged NSCLC patients treated with crizotinib as first ALK inhibitor were recruited prospectively. Blood samples were collected at baseline and at an early time-point (2 months) on crizotinib. Aberrant ALK-FISH patterns were examined in CTCs using immunofluorescence staining combined with filter-adapted FISH after filtration enrichment. CTCs were classified into distinct subsets according to the presence of ALK-rearrangement and/or ALK-CNG signals. No significant association between baseline numbers of ALK-rearranged or ALK-CNG CTCs and PFS was observed. However, we observed a significant association between the decrease in CTC number with ALK-CNG on crizotinib and a longer PFS (likelihood ratio test, P = 0.025). In multivariate analysis, the dynamic change of CTC with ALK-CNG was the strongest factor associated with PFS (HR, 4.485; 95% confidence interval, 1.543-13.030, P = 0.006). Although not dominant, ALK-CNG has been reported to be one of the mechanisms of acquired resistance to crizotinib in tumor biopsies. Our results suggest that the dynamic change in the numbers of CTCs with ALK-CNG may be a predictive biomarker for crizotinib efficacy in ALK-rearranged NSCLC patients. Serial molecular analysis of CTC shows promise for real-time patient monitoring and clinical outcome prediction in this population. Cancer Res; 77(9); 2222-30. ©2017 AACR.

The potential diagnostic power of circulating tumor cell analysis for non-small-cell lung cancer.

In non-small-cell lung cancer (NSCLC), genotyping tumor biopsies for targetable somatic alterations has become routine practice. However, serial biopsies have limitations: they may be technically difficult or impossible and could incur serious risks to patients. Circulating tumor cells (CTCs) offer an alternative source for tumor analysis that is easily accessible and presents the potential to identify predictive biomarkers to tailor therapies on a personalized basis. Examined here is our current knowledge of CTC detection and characterization in NSCLC and their potential role in EGFR-mutant, ALK-rearranged and ROS1-rearranged patients. This is followed by discussion of the ongoing issues such as the question of CTC partnership as diagnostic tools in NSCLC.

Detection of Gene Rearrangements in Circulating Tumor Cells: Examples of ALK-, ROS1-, RET-Rearrangements in Non-Small-Cell Lung Cancer and ERG-Rearrangements in Prostate Cancer

Circulating tumor cells (CTCs) hold promise as biomarkers to aid in patient treatment stratification and disease monitoring. Because the number of cells is a critical parameter for exploiting CTCs for predictive biomarkers detection, we developed a FISH (fluorescent in situ hybridization) method for CTCs enriched on filters (filter-adapted FISH [FA-FISH]) that was optimized for high cell recovery. To increase the feasibility and reliability of the analyses, we combined fluorescent staining and FA-FISH and developed a semi-automated microscopy method for optimal FISH signal identification in filtration-enriched CTCs . Here we present these methods and their use for the detection and characterization of ALK-, ROS1-, RET-rearrangement in CTCs from non-small-cell lung cancer and ERG-rearrangements in CTCs from prostate cancer patients.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap): Toward a real liquid biopsy in metastatic breast and prostate cancer

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a “liquid biopsy”. In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as “gold standard” reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0–32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1–4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Routine clinical use of circulating tumor cells for diagnosis of mutations and chromosomal rearrangements in non-small cell lung cancer—ready for prime-time?

In non-small cell lung cancer (NSCLC), diagnosis of predictive biomarkers for targeted therapies is currently done in small tumor biopsies. However, tumor biopsies can be invasive, in some cases associated with risk, and tissue adequacy, both in terms of quantity and quality is often insufficient. The development of efficient and non-invasive methods to identify genetic alterations is a key challenge which circulating tumor cells (CTCs) have the potential to be exploited for. CTCs are extremely rare and phenotypically diverse, two characteristics that impose technical challenges and impact the success of robust molecular analysis. Here we introduce the clinical needs in this disease that mainly consist of the diagnosis of epidermal growth factor receptor (EGFR) activating alterations and anaplastic lymphoma kinase (ALK) rearrangement. We present the proof-of-concept studies that explore the detection of these genetic alterations in CTCs from NSCLC patients. Finally, we discuss steps that are still required before CTCs are routinely used for diagnosis of EGFR-mutations and ALK-rearrangements in this disease.

Filter-Adapted Fluorescent In Situ Hybridization (FA-FISH) for Filtration-Enriched Circulating Tumor Cells

Circulating tumor cells (CTCs) may represent an easily accessible source of tumor material to assess genetic aberrations such as gene-rearrangements or gene-amplifications and screen cancer patients eligible for targeted therapies. As the number of CTCs is a critical parameter to identify such biomarkers, we developed fluorescent in situ hybridization (FISH) for CTCs enriched on filters (filter-adapted-FISH, FA-FISH). Here, we describe the FA-FISH protocol, the combination of immunofluorescent staining (DAPI/CD45) and FA-FISH techniques, as well as the semi-automated microscopy method that we developed to improve the feasibility and reliability of FISH analyses in filtration-enriched CTC.

Abstract 856: Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib

The duration of clinical response is unpredictable in ALK-rearranged NSCLC patients treated by crizotinib but all patients invariably develop resistance. Using filter-adapted fluorescence in situ hybridization (FA-FISH) we previously reported ALK-rearrangement detection in circulating tumor cells (CTCs) from ALK-rearranged patients. Here we report the monitoring of ALK-rearranged (≥1×3’/5’, ≥1x(3’ and 5’)) and ALK-amplified (>2×3’/5’) CTCs at baseline and at an early time point under crizotinib therapy in an extended cohort of ALK-rearranged patients. The correlation between CTC subsets harboring distinct FISH patterns and clinical parameters including progression-free survival (PFS) and overall survival (OS) is presented. Forty ALK-rearranged patients were recruited. Blood samples were collected at baseline to crizotinib and at 1-3 months. Abnormal ALK-FISH patterns were examined in CTCs using immunofluorescence staining (DAPI/CD45) combined with FA-FISH after isolation by size of epithelial tumor cells (ISET) filtration. CTCs were defined into five distinct subsets according to the number of FISH ‘break-apart’ signals (3’ and 5’) or isolated red signals (3’) and/or native copies (3’/5’). All ALK-abnormal cells were validated by a cytogenetician. Clinical data was collected retrospectively. Confirming our previous data, ALK rearrangement was detected in CTCs: 32/39 patients (82%) had ≥4 ALK-rearranged CTCs per 1 ml of blood. Age >60 years and smoking status >15 pack years were associated with a higher mean number of ALK-amplified CTCs (p = 0.0423 and p = 0.0407 respectively). No statistically significant correlation was observed between the different CTC subsets with abnormal FISH patterns at baseline and PFS or OS. However we observed a statistically significant correlation (p = 0.0196) between the evolution under crizotinib of the numbers of ALK-amplified CTCs and PFS. An increase in the numbers of ALK-amplified CTCs during treatment is associated with a PFS of 6.5 months, while a decrease in this subset is associated with a PFS of 25 months. There was no corresponding correlation with OS. The evolution under crizotinib therapy of numbers of ALK-amplified CTCs is significantly correlated with PFS. Although not dominant, amplification of the ALK gene has been reported to be one mechanism of acquired resistance to crizotinib therapy in tumor biopsies. CTCs with an amplification of ALK do not express the oncogenic ALK fusion protein and are therefore not targeted by crizotinib. FISH patterns suggested these CTCs may have a high degree of ploidy and chromosomal instability which may contribute to promote the emergence of CTC subclones with a high metastatic potential. Our data suggest that number of ALK-amplified CTCs may be a predictive biomarker allowing prediction of ALK-rearranged NSCLC patients who are at risk of early resistance to crizotinib. Citation Format: Emma Pailler, Marianne Oulhen, Kirsty Ross, Fanny Billiot, Nathalie Auger, Isabelle Borger, Maud NgoCamus, Jordi Remon-Masip, David Planchard, Jean-Charles Soria, Benjamin Besse, Francoise Farace. Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 856.

Abstract 1470: Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small cell lung cancer.

Purpose: The diagnostic test of ALK rearrangement in non-small-cell lung cancer (NSCLC) for crizotinib treatment is currently done on tumor biopsies or fine needle aspirations. The present study was designed to evaluate -1) whether ALK-rearrangement diagnosis could be performed using circulating tumor cells (CTCs), -2) whether CTCs harboring ALK-rearrangement could be monitored in ALK-positive patients treated by crizotinib. Patients and Methods: CTCs were isolated in 18 ALK-positive and 14 ALK-negative patients by blood filtration using ISET (Isolation by Size Epithelial Tumor cells) and tested by filter adapted-fluorescence in situ hybridization (FA-FISH), a FISH method optimized for filters. Numbers of ALK-rearranged cells and patterns of ALK-rearrangement were determined in CTCs and compared to those present in tumor biopsies. ALK-rearranged CTCs and tumor specimens were characterized for epithelial (cytokeratin, E-cadherin) and mesenchymal (vimentin, N-cadherin) markers expression. ALK-rearranged CTCs were monitored in ALK-positive patients treated by crizotinib. Results: ALK-rearranged CTCs [ranging from 4 to 34 CTCs /1mL] were detected in all ALK-positive patients, while no or only one ALK-rearranged CTCs was detected in blood samples obtained from ALK-negative patients. ALK-rearranged CTCs harboured a unique (3’ 5’) split pattern while heterogeneous patterns (3’ 5’, only 3’) of splits were present in tumors. ALK-rearranged CTCs exclusively expressed a mesenchymal phenotype and contrasted with heterogeneous epithelial and mesenchymal marker expressions in tumors. Variations in levels of ALK-rearranged CTCs and in CTCs harboring a gain of ALK native copies were detected under crizotinib treatment. Conclusion: We report that ALK-rearrangement can be detected in CTCs of ALK-positive NSCLC patients using ISET and FA-FISH, enabling diagnostic testing for crizotinib treatment. Our results suggest that CTCs harboring a unique ALK-rearrangement and mesenchymal phenotype may arise from the clonal selection of tumor cells that have acquired the potential to drive metastatic progression of ALK-positive NSCLC. The molecular characterization of CTCs in patients undergoing crizotinib treatment might provide new insights into mechanism of resistance to ALK tyrosine kinase inhibitors, and possible strategies to overcome this resistance. Citation Format: Emma Pailler, Julien Adam, Amelie Barthelemy, Marianne Oulhen, Nathalie Auger, Alexander Valent, David Planchard, Melissa Taylor, Isabelle Borget, Fabrice Andre, Jean-Charles Soria, Philippe Vielh, Benjamin Besse, Francoise Farace. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1470. doi:10.1158/1538-7445.AM2013-1470