Background:Circulating tumour cells (CTCs) can provide information on patient prognosis and treatment efficacy. However, there is no universal method to detect CTC currently available. Here, we compared the performance of two CTC detection systems based on the expression of the EpCAM antigen (CellSearch assay) or on cell size (ISET assay).Methods:Circulating tumour cells were enumerated in 60 patients with metastatic carcinomas of breast, prostate and lung origins using CellSearch according to the manufacturer's protocol and ISET by studying cytomorphology and immunolabelling with anti-cytokeratin or lineage-specific antibodies.Results:Concordant results were obtained in 55% (11 out of 20) of the patients with breast cancer, in 60% (12 out of 20) of the patients with prostate cancer and in only 20% (4 out of 20) of lung cancer patients.Conclusion:Our results highlight important discrepancies between the numbers of CTC enumerated by both techniques. These differences depend mostly on the tumour type. These results suggest that technologies limiting CTC capture to EpCAM-positive cells, may present important limitations, especially in patients with metastatic lung carcinoma.

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The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors.

The activation of T- and NK-cell sub-populations in vivo was evaluated in a phase-I study (18 patients) with a 3-month course of low-dose s.c. IL-2, 1, 3 and 6 x 10(6) IU/day once daily, 6 days a week. At the higher doses, we observed early on (day 15) an increase in CD3+ CD56-, CD3- CD56+ and CD56+ DR+ cell counts, as well as an increase in circulating sIL-2R and non-MHC-restricted cytotoxicity against K562 and Daudi cells. In contrast, at the lowest dose, T- and NK-cell counts were not appreciably altered, while a substantial increase in NK cytotoxic activity was still observed. In addition, thyroid dysfunction resembling that described in auto-immune thyroiditis, was documented in 6 out of the 14 patients studied. Using a high-resolution method analyzing CDR3 sizes of TCR beta transcripts, we observed the appearance of dominant T-cell clonotypes in 1 patient out of 2 analyzed, corresponding to the clonal expansion of T cells primed in vivo. Overall, these results show that long courses of low-dose s.c. IL-2 treatment lead to the activation of discrete T- and NK-cell sub-populations.

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We have attempted to improve negative selection procedures for the large scale purification of human CD-3 CD56+ NK cells. In a series of experiments, purifications of NK cells from 10(8) PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3+ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10(8) cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10(9) PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior to in vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.

We investigated the biological response of 73 patients with metastatic renal-cell carcinoma (MRCC) treated by repetitive weekly cycles of high-dose interleukin 2 (IL-2) (protocol 1, 40 patients) or IL-2 plus interferon-gamma (IFN-gamma) (protocol 2, 33 patients). The objectives of this study were (i) to evaluate the effects of this IL-2 administration schedule on biological response, (ii) to compare the effects of IL-2 alone with those of IL-2 plus IFN-gamma, (iii) to search for any correlation between certain biological marker values and the clinical response to treatment. Mean CD56+ lymphocyte counts (i.e., NK cells) were significantly higher than those of CD3+ cells in the 2 protocols and a subpopulation of CD56bright cells in protocol 1 was found to be preferentially expanded in vivo. Cytotoxic activity against K562 and Daudi cell lines as well as TNF-alpha and sTNF-alpha R (but not IL-6) significantly increased following treatment. Comparison of the data obtained from patients treated with IL-2 alone vs. IL-2 plus IFN-gamma did not show any significant changes except for eosinophilia (higher in protocol 1). Therefore, addition of IFN-gamma did not affect either lymphocyte distribution or non-MHC-restricted cytotoxicity in vivo. No difference in cell subpopulation or cytotoxicity was detected between responders and non-responders. Pre-treatment sTNF-alpha R concentration, in contrast to IL-6 and TNF-alpha, was significantly higher in progressive than in stable and responder groups, suggesting that this parameter may be predictive of the clinical response.

SUMMARY Alterations of imniunological parameters were analysed in patients with advanced malignancies during a phase I trial with rIL‐2. Five‐day infusions of rIL‐2 at doses from 1 × 106 to 24 × 106 biological response modifiers program (BRMP) U/m2 per day were given to 29 patients, with a minimum of three patients per dose. The dose of 24 × 106 U/m2 per day was the maximal tolerated dose (MTD). Immunological parameters were analysed at days 0, 8 and 11 of the rIL‐2 courses. Following a leucopenia during rlL‐2 infusion, a lymphocytosis was found in all patients except one. The lymphocylosis peaked at day 8 and was detected at doses of rIL‐2 as low as 1 × 106 U/m2 per day, reaching a plateau at a dose of 16 × 106 U/m2 per day. Although all lymphocyte subsets were increased in patients receiving rIL‐2. some patients had predominant T cells (CD3+, NKH1(CD56)‐), others had predominant natural killer (NK) cells (CD3‐. NKH1(CD56)+), and yet others showed a mixed profile. A strong induction of cells cytotoxic for K.562 targets was found in all patients at days 8 and 11. Eighteen patients received, 1 month later, a second treatment in which infusion of rIL‐2 was preceded by a course of 5 days infusion of 2 × 106 U/m2 per day recombinant interferon‐gamma (rIFN‐γ)‐ The infusion of rIFN‐γ prior to rIL‐2 had no effect on the rIL‐2‐induced alterations of immunological parameters. Taken together, our results suggest that immune stimulation by rIL‐2 occurs even at low doses and is maximal at a dose below the MTD; and that pretreatment with low‐dose rlFN‐γ does not modify the immune stimulation by rIL‐2.