Label-free enrichment of rare unconventional circulating neoplastic cells using a microfluidic dielectrophoretic sorting device

Cellular circulating biomarkers from the primary tumor such as circulating tumor cells (CTCs) and circulating hybrid cells (CHCs) have been described to harbor tumor-like phenotype and genotype. CHCs are present in higher numbers than CTCs supporting their translational potential. Methods for isolation of CHCs do not exist and are restricted to low-throughput, time consuming, and biased methodologies. We report the development of a label-free dielectrophoretic microfluidic platform facilitating enrichment of CHCs in a high-throughput and rapid fashion by depleting healthy peripheral blood mononuclear cells (PBMCs). We demonstrated up to 96.5% depletion of PBMCs resulting in 18.6-fold enrichment of cancer cells. In PBMCs from pancreatic adenocarcinoma patients, the platform enriched neoplastic cells identified by their KRAS mutant status using droplet digital PCR with one hour of processing. Enrichment was achieved in 75% of the clinical samples analyzed, establishing this approach as a promising way to non-invasively analyze tumor cells from patients.

B-Cell Lymphopenia and Circulating Neoplastic B-Cell Populations Are Associated with High Risk Disease in DLBCL: Results from the UK Remodl-B Trial

Background: Preliminary results from the UK REMoDL-B trial have shown that circulating neoplastic B-cells and B-lymphopenia are frequently detected by flow cytometry in presenting DLBCL patients. Uncertainty remains about the significance of these findings and how they relate to the course of clinical disease. Aim: The aim of this study was to assess whether numerical and phenotypic peripheral B-cell abnormalities are related to advanced disease and adverse prognosis. Methods: The study was carried out using peripheral blood samples collected from patients prior to first treatment. Peripheral blood was analysed using a panel of antibodies comprising of a CD19 and CD20 backbone, with Kappa, Lambda, CD5, CD45, CD49d, LAIR-1, CXCR5, CD31, CD95, CD38 and CD10, supplemented in some cases by CD81, CD79b, and CD43. Analysis allowed B-cell enumeration, identification of monoclonal populations and phenotypic classification as CLL, germinal centre (GC), non-GC or not otherwise specified (NOS) where the phenotype was indeterminate. In addition, gene expression profiling (GEP) was performed on the diagnostic tissue biopsy (FFPE) using the Illumina WG-DASL assay to classify tumours as GCB-type, ABC-type or unclassified. Results: A total of 845 patients with an average age of 62.7 years (range 20.9-87.8) were included. A monoclonal population was detected in 174/845 (20.5%) of cases; CLL 3%, GC 7.8%, non-GC 7.7% and NOS 2.1%. Numerical abnormalities were also detected; 33% of patients had B-lymphopenia (<0.06 x 109/l) and 2.8% had B-lymphocytosis (>1 x 109/l). There was minimal overlap between numerical abnormalities and the presence of neoplastic B-cells, with 86% of cases with an abnormal population having a normal B-cell count. Circulating neoplastic cells were present in a small proportion of cases with B-lymphopenia (17/264) and a higher percentage of cases with B-lymphocytosis (8/19), as would be expected. Neoplastic B-cells were detected in 33% of cases an International Prognostic Index (IPI) score of 4-5, compared to 14% of cases with an IPI of 0-2. B-lymphopenia was present in 39% of individuals with an IPI of 4-5 compared to 24% of individuals with an IPI of 0-2. 51% (58/113) of cases with BM involvement had a circulating monoclonal population, compared to 15% (116/732) of cases with no overt marrow disease. Conversely, 66% of cases with circulating neoplastic cells did not have marrow involvement but this may, in part, be a sensitivity issue as staging was based on morphological assessment only. Tumour GEP results were available for 690 individuals; 362 (52%) GCB, 184 (26%) ABC and 144 (21%) unclassified. 86% of populations identified in the ABC subgroup were phenotyped non-GC or NOS. Presence of a GC-population by flow was highly predictive of GCB GEP (88% GC-type populations detected were GCB cases). The number of discordant cases and CLL clones detected approximated to the numbers that would have been expected in a normal age-matched population. Conclusion: Both the presence of circulating monoclonal B-cells and B-lymphopenia showed an association with higher risk disease. In addition, there was strong concordance between clonal phenotypes and the GEP of the underlying tumours. It is likely that in most cases the peripheral clonal populations are circulating tumour cells or a closely related precursor clone. Absolute B-cell counts are known to decline with age but there was no correlation with age in this patient group. Immuosuppression has a role in the pathogenesis of DLBCL in the elderly and the observed B-lymphopenia may be as a result of underlying immune dysfunction. Alternatively it may reflect suppression of normal B-cells by the neoplastic clone. Either way this has implications for the efficacy of immunomodulatory drugs, especially if the T-cell subsets are also affected. Multiparameter flow is highly applicable for diagnostic screening of both numerical and phenotypic B-cell abnormalities and may have a role in the prognostic assessment of DLBCL. Outcome data will be available shortly allowing the impact on progression free overall survival to be assessed. Disclosures Rawstron: Celgene: Honoraria; Abbvie: Honoraria; Pharmacyclics: Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria; BD Biosciences: Patents & Royalties. Johnson:Takeda: Honoraria; Pfizer: Honoraria; Janssen: Research Funding. Davies:Seattle Genetics: Research Funding; Takeda: Honoraria. Jack:Jannsen: Research Funding.

Detection of circulating neoplastic cells by reverse-transcriptase polymerase chain reaction in malignant melanoma: association with clinical stage and prognosis.

PURPOSE Circulating melanoma cells can be detected in peripheral blood by means of tyrosinase mRNA amplification by reverse-transcriptase polymerase chain reaction (RT-PCR). We conducted a prospective study to evaluate the clinical significance of the presence of circulating neoplastic cells in the blood of patients with malignant melanoma (MM). METHODS A sensitive RT-PCR assay was used to detect tyrosinase mRNA in the peripheral blood of patients with stages I to IV melanoma. Healthy subjects or patients with other malignancies were used as negative controls. RESULTS Ninety-one assessable patients were included in the study. There was a statistically significant association between RT-PCR positivity and clinical stage. Circulating melanoma cells were detected in 36% of patients with localized disease (stages I and II), in 45% of patients with regional nodal involvement (stage III), and in 94% of patients with metastatic disease (stage IV) (P < .001). In stage II-III patients who were RT-PCR-positive for mRNA tyrosinase in blood, the recurrence rate and disease-free survival were significantly worse than patients who were RT-PCR-negative. In multivariate analysis, RT-PCR was an independent prognostic factor for recurrence in patients with nonmetastatic disease (P = .002). CONCLUSION The detection of circulating melanoma cells in peripheral blood by RT-PCR correlated with the clinical stage of patients with melanoma and was an independent prognostic factor for recurrence. Further studies are warranted to better assess the significance of this test in the evaluation of prognosis, early detection of relapse, and in monitoring the effectiveness of systemic therapy.

Chimeric fusion protein toxin DAB486IL-2 in advanced mycosis fungoides and the Sezary syndrome: correlation of activity and interleukin-2 receptor expression in a phase II study

DAB486IL-2 is a recombinant toxin with the cell surface-binding domain of diphtheria toxin (DT) replaced by interleukin-2 (IL-2). To correlate clinical response with expression of components of the IL-2 receptor (IL-2R), 14 patients with cutaneous T-cell lymphoma (CTCL) received five daily 90-minute infusions every 21 days. There were no complete responses, 1 partial response (PR), 2 major biologic effects (major cutaneous improvement without change in circulating neoplastic cells), 3 stable disease (SD), and 8 progressive disease (PD). Responders had easily detected expression of CD25 (Tac; alpha-chain of IL-2R) in skin, and in two responders expression of the beta chain of the IL-2 receptor (beta-IL-2R) was detectable by reverse transcriptase-polymerase chain reaction. CD25 was also detected in 8 of 11 SD or PD patients, with beta-IL-2R in 3 of 8 SD or PD patients. Two of the three responders had anti-DT antibodies before treatment. Reversible increased hepatic transaminases occurred in 13 of 14 patients during the first course, with decreased frequency in repeated courses. The maximal serum concentration after the first infusion of DAB486IL-2 varied (1,369 +/- 1,155 ng/mL [mean +/- SD]; n = 14; range, 55 to 3,999 ng/mL) with a short half-life (T1/2 beta = 0.21 +/- 0.12 h [mean +/- SD]; range, 0.099 to 0.57 h). The area under the concentration curve varied inversely with anti-DT antibody titer. We conclude that DA-B486IL-2 has valuable activity in certain patients with CTCL. Expression of the IL- 2R may be necessary but is not sufficient to predict response.

Chimeric fusion protein toxin DAB486IL-2 in advanced mycosis fungoides and the Sezary syndrome: correlation of activity and interleukin-2 receptor expression in a phase II study

DAB486IL-2 is a recombinant toxin with the cell surface-binding domain of diphtheria toxin (DT) replaced by interleukin-2 (IL-2). To correlate clinical response with expression of components of the IL-2 receptor (IL-2R), 14 patients with cutaneous T-cell lymphoma (CTCL) received five daily 90-minute infusions every 21 days. There were no complete responses, 1 partial response (PR), 2 major biologic effects (major cutaneous improvement without change in circulating neoplastic cells), 3 stable disease (SD), and 8 progressive disease (PD). Responders had easily detected expression of CD25 (Tac; alpha-chain of IL-2R) in skin, and in two responders expression of the beta chain of the IL-2 receptor (beta-IL-2R) was detectable by reverse transcriptase-polymerase chain reaction. CD25 was also detected in 8 of 11 SD or PD patients, with beta-IL-2R in 3 of 8 SD or PD patients. Two of the three responders had anti-DT antibodies before treatment. Reversible increased hepatic transaminases occurred in 13 of 14 patients during the first course, with decreased frequency in repeated courses. The maximal serum concentration after the first infusion of DAB486IL-2 varied (1,369 +/- 1,155 ng/mL [mean +/- SD]; n = 14; range, 55 to 3,999 ng/mL) with a short half-life (T1/2 beta = 0.21 +/- 0.12 h [mean +/- SD]; range, 0.099 to 0.57 h). The area under the concentration curve varied inversely with anti-DT antibody titer. We conclude that DA-B486IL-2 has valuable activity in certain patients with CTCL. Expression of the IL- 2R may be necessary but is not sufficient to predict response.

Contamination of peripheral blood stem cell harvests by circulating neuroblastoma cells

Peripheral blood stem cells (PBSC) are being used as one alternative to autologous marrow rescue for patients with neuroblastoma and other solid malignancies. Some physicians prefer use of PBSC because less risk of tumor contamination is believed to exist. This hypothesis was evaluated by immunocytologic analysis of blood samples and concurrently drawn bone marrow (BM) samples and of PBSC harvests obtained from 31 patients with disseminated neuroblastoma. We found circulating neoplastic cells in 75% of specimens analyzed at diagnosis, in 36% during therapy, and in 14% of PBSC harvests. Tumor cells in blood obtained during therapy did not appear until 3 months after the time of diagnosis. Clearance of circulating neuroblastoma cells was documented after two courses of induction chemotherapy. Six of 13 patients with minimal or no BM disease had positive blood specimens. We conclude that substantial risk of tumor contamination of PB harvests exists and recommend that induction chemotherapy be administered before hematopoietic progenitor cells are collected from blood.

Contamination of peripheral blood stem cell harvests by circulating neuroblastoma cells

Peripheral blood stem cells (PBSC) are being used as one alternative to autologous marrow rescue for patients with neuroblastoma and other solid malignancies. Some physicians prefer use of PBSC because less risk of tumor contamination is believed to exist. This hypothesis was evaluated by immunocytologic analysis of blood samples and concurrently drawn bone marrow (BM) samples and of PBSC harvests obtained from 31 patients with disseminated neuroblastoma. We found circulating neoplastic cells in 75% of specimens analyzed at diagnosis, in 36% during therapy, and in 14% of PBSC harvests. Tumor cells in blood obtained during therapy did not appear until 3 months after the time of diagnosis. Clearance of circulating neuroblastoma cells was documented after two courses of induction chemotherapy. Six of 13 patients with minimal or no BM disease had positive blood specimens. We conclude that substantial risk of tumor contamination of PB harvests exists and recommend that induction chemotherapy be administered before hematopoietic progenitor cells are collected from blood.

Initial experience in treating human lymphoma with a chimeric univalent derivative of monoclonal anti-idiotype antibody

Murine monoclonal anti-idiotype antibody was raised against the surface IgM on the neoplastic cells of a patient with widespread follicular lymphoma. For therapy, a chimeric antibody derivative, FabIgG, was constructed by thioether-linking Fab'gamma, from the monoclonal antibody, to human normal IgG. FabIgG is univalent and thereby avoids rapid antigenic modulation. Its human IgG component is intended to optimize recruitment of effectors and metabolic survival while minimizing immunogenicity. Four intravenous (IV) infusions of 380 to 580 mg of anti-idiotype FabIgG were given over a period of 11 weeks. There was no significant toxicity. On each occasion, the antibody disappeared from the plasma with a half-life (t1/2) of less than 24 hours. The brief survival was evidently due to uptake by tumor, as infused control FabIgG, containing Fab'gamma from an irrelevant antibody, yielded a plasma t1/2 of greater than 10 days. With each therapeutic infusion, there was a fall in the number of circulating neoplastic cells over a 24-hour period. The numbers were largely replenished over the next week, but a net fall became discernible over the entire period of treatment. Four days after each infusion, nodal masses were swollen and tender, subsiding over approximately 8 days. At the end of the treatments, the blood lymphocyte count and nodal and splenic swellings continued to subside, so that by 6 weeks a partial remission with removal of greater than 50% of tumor was judged to have occurred. We did not detect any qualitative change in surface idiotype nor any antibody response to the infused Ig.