International study on inter-reader variability for circulating tumor cells in breast cancer.

INTRODUCTION: Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. METHODS: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. RESULTS: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). CONCLUSIONS: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.

Comparison of two next-generation sequencing-based approaches for liquid biopsy analysis in patients with non-small cell lung cancer: a multicentre study.

In the era of personalised medicine, testing for an increasing number of predictive biomarkers is becoming a priority. However, tissue biopsies from these patients are oftentimes insufficient for conventional approaches, a common issue that deprives them of the clinical benefits of biomarker-directed treatments. To tackle this problem, many clinical laboratories are resorting to circulating tumour DNA (ctDNA), which is becoming increasingly appreciated as a valuable source for biomarker testing. In this context, next-generation sequencing (NGS) has become essential. Indeed, different NGS systems are able to detect several clinically relevant low-frequency hot-spot mutations simultaneously in a single run. However, their reproducibility in the analysis of ctDNA has not yet been investigated. The purpose of this study was to evaluate the reproducibility of using Illumina MiSeq and Thermo Fisher Ion S5 Plus platforms to assess pathogenic alterations in non-small cell lung cancer (NSCLC) liquid biopsy specimens. Using the in vitro diagnostic (IVD) NGS panel Myriapod NGS Cancer panel DNA (Diatech Pharmacogenetics) on MiSeq platform (Illumina), we reanalysed ctDNA extracted from a retrospective series of n=40 patients with advanced NSCLC previously tested with a custom NGS panel (SiRe) on Thermo Fisher Ion S5 Plus system. Overall, 13 out of 40 (32.5%) ctDNA samples displayed pathogenic alterations in at least two genes, namely, EGFR and KRAS A concordance rate of 100% was identified between the two methodologies in terms of sample mutational status and total number of detected variables. All NGS platforms featured a high degree of concordance.

An Italian Multicenter Perspective Harmonization Trial for the Assessment of MET Exon 14 Skipping Mutations in Standard Reference Samples.

Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation (Seracare Life Sciences, Milford, MA, USA), which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice.

Final results of a multicenter phase II clinical trial evaluating the activity of single-agent lapatinib in patients with HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells. A proof-of-concept study.

This multicenter phase II trial was designed to evaluate the activity of lapatinib in metastatic breast cancer patients with HER2-negative primary tumors and HER2-positive circulating tumor cells (CTCs). In this study MBC patients with HER2-negative primary tumors and HER2-positive CTCs previously treated with at least a first-line therapy for metastatic disease received lapatinib 1500 mg/day. The CellSearch System® was used for CTCs isolation and bio-characterization. HER2 status was assessed on CTCs by immunofluorescence. A case was defined as CTCs positive if ≥2 CTC/7.5 ml of blood were isolated and HER2-positive if ≥50% of CTCs were HER2-positive. 139 HER2-negative patients were screened, 96 patients were positive for CTCs (mean number of CTCs: 85; median number of CTCs: 19; range 2-1637). Seven of the 96 patients (7%) had ≥50% HER2-positive CTCs and were eligible for treatment with lapatinib. No objective tumor responses occurred in this population. In one patient, disease stabilization lasting 254 days (8.5 months) was observed. From the findings of this study, we concluded that a subset of patients with a HER2-negative primary tumor presents HER2-positive CTCs during disease progression, although the HER2 shift rate seems to be lower than previously reported. Despite the lack of objective response, the durable disease stabilization observed in one patient cannot rule out the hypothesis that lapatinib may have some activity in this patient population. However, considering that only 1/139 screened patients may potentially have derived benefit from this approach, future trials designed according to the presented strategy cannot be recommended.

Comparison between two different next generation sequencing platforms for clinical relevant gene mutation test in solid tumours.

In the present study, we analysed 44 formalin fixed paraffin embedded (FFPE) from different solid tumours by adopting two different next generation sequencing platforms: GeneReader (QIAGEN, Hilden, Germany) and Ion Torrent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). We highlighted a 100% concordance between the platforms. In addition, focusing on variant detection, we evaluated a very good agreement between the two tests (Cohen's kappa=0.84) and, when taking into account variant allele fraction value for each variant, a very high concordance was obtained (Pearson's r=0.94). Our results underlined the high performance rate of GeneReader on FFPE samples and its suitability in routine molecular predictive practice.

Recent advances in systemic therapy: new diagnostics and biological predictors of outcome in early breast cancer.

The key to optimising our approach in early breast cancer is to individualise care. Each patient has a tumour with innate features that dictate their chance of relapse and their responsiveness to treatment. Often patients with similar clinical and pathological tumours will have markedly different outcomes and responses to adjuvant intervention. These differences are encoded in the tumour genetic profile. Effective biomarkers may replace or complement traditional clinical and histopathological markers in assessing tumour behaviour and risk. Development of high-throughput genomic technologies is enabling the study of gene expression profiles of tumours. Genomic fingerprints may refine prediction of the course of disease and response to adjuvant interventions. This review will focus on the role of multiparameter gene expression analyses in early breast cancer, with regards to prognosis and prediction. The prognostic role of genomic signatures, particularly the Mammaprint and Rotterdam signatures, is evolving. With regard to prediction of outcome, the Oncotype Dx multigene assay is in clinical use in tamoxifen treated patients. Extensive research continues on predictive gene identification for specific chemotherapeutic agents, particularly the anthracyclines, taxanes and alkylating agents.

Correlation of HER2 status between primary tumors and corresponding circulating tumor cells in advanced breast cancer patients.

Biocharacterization of circulating tumor cells (CTCs) in the peripheral blood of advanced breast cancer (ABC) patients may represent a real-time tumor biopsy. We assessed HER2 status on CTCs from blood samples of ABC patients. CTCs were separated and stained using the CellSearch System((R)). HER2 status was assessed by immunofluorescence and, when technically feasible, by fluorescence in situ hybridization. Blood samples were obtained from 66 ABC patients. Forty patients had a positive CTC sample (61%) and of these, 15 (37%) had HER2 + CTCs. We found non-concordant results in 32% of cases: 29% (8/28) of HER2-negative primary tumors had HER2-positive CTCs and 42% (5/12) of HER2-positive primary tumors had HER2-negative CTCs (k = 0.278). Our study suggests that a subset of patients with HER2-negative primary tumors develops HER2-positive CTCs during disease progression.

Inter- and intra-tumoral heterogeneity in DNA damage evaluated by comet assay in early breast cancer patients.

There are no clinical tools to functionally assess degree of DNA damage in breast cancer. The comet assay is an accepted research tool for assessing DNA damage, however, most cancer studies have assessed lymphocytes as surrogate cells. The aim of this pilot study was to use the comet assay in early breast cancer directly in tumor tissue to compare DNA damage between and within traditionally defined subgroups, and to explore intra-tumoral heterogeneity. Scrapings of tumor and healthy breast tissue were obtained at primary surgery from 104 women. Comet assay was applied to quantitatively assess DNA damage, revealing substantial inter- and intra-subgroup variation. Marked intra-tumoral heterogeneity was evident across all subgroups. The degree of DNA damage for an individual could not be predicted by breast cancer subgroup. Comet assay warrants further study as a potential clinical tool for identification of tumoral DNA damage and ultimately, individualised use of DNA damaging therapy.

HER2-positive circulating tumor cells in breast cancer.

PURPOSE: Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging. METHODS: Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®. RESULTS: Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluorescence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0-8.4%) whereas 4.1% (95%CI 1.4-11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1-14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1-3 cells) and 35.9% (95%CI 22.7-51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1-42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03). CONCLUSION: HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.

Significance of micrometastases: circulating tumor cells and disseminated tumor cells in early breast cancer.

Adjuvant systemic therapy targets minimal residual disease. Our current clinical approach in the adjuvant setting is to presume, rather than confirm, the presence of minimal residual disease. Based on assessment of the primary tumor, we estimate an individual's recurrence risk. Subsequent treatment decisions are based on characteristics of the primary tumor, with the presumption of consistent biology and treatment sensitivity between micrometastases and the primary lesion. An alternative approach is to identify micrometastatic disease. Detection of disseminated tumor cells (DTC) in the bone marrow and circulating tumor cells (CTC) from peripheral blood collection may offer quantification and biocharacterization of residual disease. This paper will review the prognostic and predictive potential of micrometastatic disease in early breast cancer.