Heterogeneous response of individual multicellular tumour spheroids to immunotoxins and ricin toxin.

The cytoreductive effects of anti-transferrin receptor (anti-TfnR) immunotoxins (ITs) and of ricin toxin against tumour micromasses have been evaluated in a multicellular tumour spheroid (MTS) model. More than 600 (656) MTSs obtained with human breast carcinoma (MCF7) or rat glioblastoma (9L) cell lines were treated individually with ITs or toxin and the effects induced by the treatment were measured for each MTS as volume variation vs time by applying the Gompertz growth model. Two dose-dependent patterns of MTS growth were observed in MTSs of both cell lines in response to IT or toxin treatment: (1) complete inhibition of MTS growth (''sterilisation''); and (2) partial/complete inhibition (''heterogeneous response''). Within the range of IT or toxin concentrations resulting in partial inhibition of MTS growth, the sensitivity of treated MTSs was extremely heterogeneous (the cytoreductive effects varying between 0.1 and 4 logs of cells killed for a given IT or toxin concentration). Analysis of the post-treatment regrowth kinetics indicated that treated non-sterilised and control MTSs reached the same final limiting volumes. However, the doubling time estimated for the surviving cells of treated MCF7 and 9L MTSs ranged between 15 and 50 h, indicating that each MTS had individual growing potential. In conclusion, our results indicate that at substerilising IT concentrations individual heterogenicity of MTSs may greatly influence the cytoreductive potential of ITs. An implication of our study is that the efficacy of an IT treatment in eradicating disseminated micrometastases may not be predictable a priori. The MTS model that we describe in this paper may help in dissecting out factors limiting the effect of ITs in three-dimensional tumours.     

Ultrastructural characteristics of human mesenchymal stromal (stem) cells derived from bone marrow and term placenta

Human mesenchymal stromal (stem) cells (hMSCs) isolated from adult bone marrow (BM-hMSCs) as well as amnion (AM-hMSCs) and chorion (CM-hMSCs) term placenta leaves were studied by transmission electron microscopy (TEM) to investigate their ultrastructural basic phenotype. At flow cytometry, the isolated cells showed a homogeneous expression of markers commonly used to identify hMSCs, i.e., CD105, CD44, CD90, CD166, HLA-ABC positivities, and CD45, AC133, and HLA-DR negativities. However, TEM revealed subtle yet significant differences. BM-hMSCs had mesenchymal features with dilated cisternae of rough endoplasmic reticulum (rER) and peripheral collections of multiloculated clear blisters; this latter finding mostly representing complex foldings of the plasma membrane could be revelatory of the in situ cell arrangement in the niche microenvironment. Unlike BM-hMSCs, CM-hMSCs were more primitive and metabolically quiescent, their major features being the presence of rER stacks and large peripheral collections of unbound glycogen. AM-hMSCs showed a hybrid epithelial-mesenchymal ultrastructural phenotype; epithelial characters included non-intestinal-type surface microvilli, intracytoplasmic lumina lined with microvilli, and intercellular junctions; mesenchymal features included rER profiles, lipid droplets, and well-developed foci of contractile filaments with dense bodies. These features are consistent with the view that AM-hMSCs have a pluripotent potential. In conclusion, this study documents that ultrastructural differences exist among phenotypically similar hMSCs derived from human bone marrow and term placenta leaves; such differences could be revelatory of the hMSCs in vitro differentiation potential and may provide useful clues to attempt their in situ identification.     

Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.     

Multimodality stereotactic approach to the treatment of cystic craniopharyngiomas.

OBJECTIVES: To present the therapeutic reliability of a multimodality stereotactic approach (MSA) to cystic craniopharyngiomas (CPs), combining neuroendoscopy, intracavitary bleomycin and gamma knife (GK) radiosurgery. METHODS: 8 patients with mono- or multicystic CP (7/8 regrowths/recurrence) underwent stereotactic neuroendoscopy and subsequently treatment with intracavitary bleomycin and GK. They were clinically characterized by hypopituitarism (7 cases), visual impairment (7), endocranial hypertension (7), cognitive and behavioral disturbances (3), and cranial nerve deficits or focal signs (3). Concomitant hydrocephalus was observed in 3/8 patients. According to Backlund's classification, the treated CPs were classified as type A (3 cases), type Cc (4 cases) and type Dc (1 case). In all 8 patients, neuroendoscopy allowed evacuation of the cyst and, in multi-cystic CPs, fenestration of the interposed septa so as to create a single communicating cavity. Thus, a single catheter and Ommaya reservoir system was sufficient both for subsequent aspirations and for bleomycin injection (doses of 1.5-3 mg, usually repeated every 7-8 days, with total doses ranging from 3-35 mg). GK radiosurgery was carried out at a later stage on the collapsed cyst in type A forms, while in types Cc and Dc, it was used on the solid nodule on the same day as the neuroendoscopy. RESULTS: The median follow-up period was 42.5 months. Neurological improvement was observed in 8/8 patients. A complete response (reduction of the entire tumor volume > 90 %) was observed in 3/8 cases (type A), a subtotal response (reduction > 50 %) in 4/8 cases (types Cc and Dc), and a partial response (reduction < 50 %) in 1/8 cases (type Cc). Treatment of CP alone led to normalization of the ventricular morphology in the 3 patients with associated hydrocephalus. Transient GK-related visual worsening was recorded in one case only. One patient died because of ventriculitis after repeated shunt replacements. CONCLUSIONS: This MSA seems to be an effective and safe treatment alternative to microsurgery, above all in patients with regrowing/recurrent cystic CPs.