Zofia Maciorowski

The value of pretreatment cell kinetic parameters as predictors for radiotherapy outcome in head and neck cancer: a multicenter analysis.

PURPOSE The aim of this study was to assess the potential of pre-treatment cell kinetic parameters to predict outcome in head and neck cancer patients treated by conventional radiotherapy. MATERIALS AND METHODS Data from 11 different centers were pooled. Inclusion criteria were such that the patients received radiotherapy alone, and that the radiotherapy was given in an overall time of at least 6 weeks with a dose of at least 60 Gy. All patients received a tracer dose of either iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) intravenously prior to treatment and a tumor biopsy was taken several hours later. The cell kinetic parameters labeling index (LI), DNA synthesis time (Ts) and potential doubling time (Tpot) were subsequently calculated from flow cytometry data, obtained on the biopsies using antibodies against I/BrdUrd incorporated into DNA. Each center carried out their own flow cytometry analysis. RESULTS From the 11 centers, a total of 476 patients conforming to the inclusion criteria were analyzed. Median values for overall time and total dose were 49 days and 69 Gy, respectively. Fifty one percent of patients had local recurrences and 53% patients had died, the majority from their disease. Median follow-up was 20 months; being 30 months for surviving patients. Multivariate analysis revealed that T-stage, maximum tumor diameter, differentiation grade, N-stage, tumor localization and overall time correlated with locoregional control, in decreasing order of significance. For the cell kinetic parameters, univariate analysis showed that only LI was significantly associated with local control (P=0.02), with higher values correlating with a worse outcome. Ts showed some evidence that patients with longer values did worse, but this was not significant (P=0.06). Tpot showed no trend (P=0.8). When assessing survival in a univariate analysis, neither LI nor Tpot associated with outcome (P=0.4, 0.4, respectively). Surprisingly, Ts did correlate with survival, with longer values being worse (P=0.02). In the multivariate analysis of local control, LI lost its significance (P=0.16). CONCLUSIONS The only pretreatment kinetic parameter for which some evidence was found for an association with local control (the best end-point for testing the present hypothesis) was LI, not Tpot, and this evidence disappeared in a multivariate analysis. It therefore appears that pretreatment cell kinetic measurements carried out using flow cytometry, only provide a relatively weak predictor of outcome after radiotherapy in head and neck cancer.

In vitro radiation-induced apoptosis and early response to low-dose radiotherapy in non-Hodgkin's lymphomas

PURPOSE Prospective investigation of spontaneous and in vitro radiation-induced apoptosis to predict early response to palliative radiotherapy in patients with non-Hodgkins lymphomas. PATIENTS AND METHODS Fine-needle sampling was performed in 28 tumor sites (26 patients) and yielded adequate cell numbers in 27 cases. Apoptotic cells were counted by fluorescence microscopy immediately after sampling and after 24-h culture (spontaneous apoptosis) and 24 h after 2- and 10-Gy in vitro irradiation (radiation-induced apoptosis). Early response to low-dose in vivo radiotherapy (mostly 4 Gy in two fractions over 3 days) was evaluated 15 days after treatment. RESULTS The tumor response rates at 15 days were 11 (39%) complete responses, nine (32%) responses of greater than 50% reduction in volume, six (21%) responses of less than 50% reduction in volume and two (7%) cases of no response. Tumors achieving complete or major response after in vivo irradiation had higher percentages of apoptotic cells after in vitro irradiation, while no significant differences in terms of spontaneous apoptosis were observed between responders and non-responders. CONCLUSION Spontaneous and in vitro radiation-induced apoptosis can be easily and quickly assessed on cells obtained by fine-needle sampling of non-Hodgkins lymphoma lesions. The present results suggest that in vitro radiation-induced apoptosis could be used as a predictive assay of early response to low-dose in vivo irradiation in patients with non-Hodgkins lymphomas.

Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity

Complete lack of function of the tyrosine kinase ZAP70 in humans results in a severe immunodeficiency, characterized by a lack of mature CD8+ T cells and non‐functional CD4+ T cells. We report herein an immunodeficiency with an inherited hypomorphic mutation of ZAP70 due to a single G‐to‐A substitution in a non‐coding intron. This mutation introduces a new acceptor splice site and allows low levels of normal alternative splicing and of WT ZAP70 expression. This partial deficiency results in a compromised TCR signaling that was totally restored by increased expression of ZAP70, demonstrating that defective activation of the patient T cells was indeed caused by the low level of ZAP70 expression. This partial ZAP70 deficiency was associated with an attenuated clinical and immunological phenotype as compared with complete ZAP70 deficiency. CD4+ helper T‐cell populations including, follicular helper T cells, Th1, Th17 and Treg were detected in the blood. Finally, the patient had no manifestation of autoimmunity suggesting that the T‐cell tolerogenic functions were not compromised, in contrast to what has been observed in mice carrying hypomorphic mutations of Zap70. This report extends the phenotype spectrum of ZAP70 deficiency with a residual function of ZAP70.

Novel features of boundary cap cells revealed by the analysis of newly identified molecular markers.

Neural crest (NC) cells are a multipotent, highly migratory cell population that generates most of the components of the peripheral nervous system (PNS), including the glial Schwann cells (SC) and boundary cap (BC) cells. These latter cells are located at the interface between the central nervous system and PNS, at the exit/entry points of ventral motor/dorsal sensory axons and give rise to all SC in the nerve roots and to a subset of nociceptive neurons and satellite cells in the dorsal root ganglia. In the present study we have compared BC cells with two closely related cell types, NC and Schwann cell precursors (SCpr), by RNA profiling. This led to the definition of a set of 10 genes that show specific expression in BC cells and/or in their derivatives along the nerve roots. Analysis of the expression of these genes during mouse development revealed novel features, of those most important are: (i) dorsal and ventral nerve root BC cell derivatives express different sets of genes, suggesting that they have distinct properties; (ii) these cells undergo major modifications in their gene expression pattern between embryonic days 14.5 and 17.5, possibly linked to the SCpr‐immature Schwann cell transition; (iii) nerve roots SC differ from more distal SC not only in their origins and locations, but also in their gene expression patterns. In conclusion, the identification of these novel makers opens the way for a detailed characterization of BC cells in both mouse and man.

Preferential Occurrence of Chromosome Breakpoints within Early Replicating Regions in Neuroblastoma

Neuroblastoma (NB) is a frequent paediatric extra cranial solid tumour characterized by the occurrence of unbalanced chromosome translocations, frequently, but not exclusively, involving chromosomes 1 and 17. We have used a 1 Mb resolution BAC array to further refine the mapping of breakpoints in NB cell lines. Replication timing profiles were evaluated in 7 NB cell lines, using DNAs from G1 and S phases flow sorted nuclei hybridised on the same array. Strikingly, these replication timing profiles were highly similar between the different NB cell lines. Furthermore, a significant level of similarity was also observed between NB cell lines and lymphoblastoid cells. A segmentation analysis using the Adaptative Weights Smoothing procedure was performed to determine regions of coordinate replication. More than 50% of the breakpoints mapped to early replicating regions, which account for 23.7% of the total genome. The breakpoints frequency per 108 bases was therefore 10.84 for early replicating regions, whereas it was only 2.94 for late replicating regions, these difference being highly significant (p

Comparative analysis of apoptosis measured by Hoechst and flow cytometry in non-Hodgkin's lymphomas

Fine-needle samples of 75 non-Hodgkins lymphomas were investigated for apoptosis immediately and after 24 h of culture after in vitro irradiation (2 Gy, 10 Gy, and nonirradiated controls). Apoptotic cells were simultaneously quantified by fluorescence microscopic enumeration of apoptotic cells using Hoechst 33342 staining, and by flow cytometric detection of sub-G1 peak cells. The nonirradiated controls showed a similar mean percent apoptotic cells using both methods, analyzed immediately (9% by morphology vs. 10% by flow) or after 24 h of culture (40% by morphology vs. 41% by flow). In the irradiated samples, the mean percent apoptotic cells quantified by morphology was higher than by flow cytometry (64% by morphology vs. 55% by flow after 2 Gy irradiation, and 71% vs. 58% after 10 Gy). The results of the two methods were correlated, although large differences were seen between the techniques in individual tumors. In our system, flow cytometric sub-G1 peak analysis appears to underestimate apoptosis. Of these two methods, we find the Hoechst morphology method to be more reliable for quantitation of apoptosis utilizing fresh fine-needle sample material, in that discrimination of apoptotic cells from debris is easier and that both early and late apoptotic cells are detectable.

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells’ metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.

Signal amplification of FISH for automated detection using image cytometry

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification systems and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Diagnostic and prognostic information obtained on fine-needle aspirates of primary neuroblastic tumors: Proposal for a cytology prognostic score.

The use of cytological material for diagnosis and prognosis in patients with neuroblastic tumors is poorly described in the literature.

Basic Multicolor Flow Cytometry

Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest, allowing rapid analysis on tens of thousands of cells per second, with the possibility of isolating pure, viable populations by cell sorting for further experimentation. This unit covers the tools needed by the beginning immunologist to plan and run multicolor experiments, with information on fluorochromes and their characteristics, spectral spillover, compensation and spread, instrument and reagent variables, and the basic elements of multicolor panel design. Protocols to quantify and maximize sensitivity by titration of reagents and optimization of instrument settings, as well as basic surface and intracellular cell staining, are included.