Giant phyllodes tumour of the breast.

Phyllodes tumours are fibroepithelial lesions and count for 0.4% of breast tumours. Telling the difference between phyllodes tumours and fibroadenomas is sometimes difficult but of importance because wide resection is the mainstay of treatment for phyllodes tumours. We present a female patient, 55 years old with a giant phyllodes tumour (38 x 31 x 23 cm) of the breast. The breast reconstruction was done using a pedicled transverse rectus abdominis myocutaneous (TRAM) flap.

[Vertical reduction mammaplasty for gigantomastia with massive fibroadenomatosis: a case report]. / Réduction mammaire selon la technique verticale pour gigantomastie avec fibroadénomatose massive: à propos d'un cas clinique.

Vertical reduction mammaplasty is one of the most debated << short-scar >> breast reduction technique. Advantages and drawbacks of the technique are discussed; most of the authors do not accept it as the technique of choice for high glandular resection weights. In our case report we achieve it for a resection weight up to two kilograms with an areolar transposition distance of more than ten centimetres. We show that it is reasonable to realize it dealing with gigantomastia. The massive fibroadenomatosis is observed following immunosuppressive treatment for kidney transplantation. Cyclosporine intake, even sporadic, is at the origin of the growth of these multiple, bilateral and large fibroadenomas. Drug-induced cytokines stimulate their development.

[Breast reconstruction by DIEP free flap: about 100 cases]. / Reconstruction mammaire par lambeau DIEP: à propos de 100 cas.

Autologous breast reconstruction was stigmatised because the muscular sacrifice of the rectus abdominis muscle. This problem could be avoided by the DIEP flap as much for immediate as delayed reconstruction with the creation of an aesthetic and natural shaped reconstructed breast. This retrospective study about 100 cases performed between January 1997 and June 2002 concern 94 patients, 88 unilateral reconstructions and six bilateral. The reconstruction was delayed in 83%, immediate in 8% or realised after failed attempt to reconstruct the breast with implant in 9%. Risk factors were also present: smokers (66%), one or more abdominal scars (40%), obesity (30%). The recipient vessels were the internal mammary vessels (86%), the circumflex scapular vessels (10%) and the subscapular vessels (4%). We noted four total flap loss, 5% of partial loss and 2% localized liponecrosis. Mean operating time was 6 hours 28 minutes for unilateral reconstruction and 9 hours 30 minutes for bilateral reconstruction. Mean hospital stay was 7,3 days. Two moderated abdominal bulging were noted. The tedious dissection of small vessels of the DIEP flap allowed for a similar rate of complication as the free TRAM flap, by respecting of the integrity of the rectus abdominis muscle, to reduce morbidity of harvest with less postoperative pain, shorter hospital stay and faster recovery.

[Report of 243 vertical mammoplasties for very large, heavy breasts and/or severe ptosis. Analysis of the result and technical]. / A propos de 243 plasties mammaires verticales pour hypertrophie et/ou ptôse importantes. Etude rétrospective, analyse des résultats et description des modifications techniques.

Vertical mammaplasties for very large breasts and/or severe ptosis were evaluated in 124 patients who underwent operation in our unit between September 1993 and June 2001. In 119 cases it was reduction mammaplasty and in 5 cases unilateral symmetrization after contralateral reconstruction. The mean age was 36 years (13-62 years). Among inclusion criteriae, we choose the resection weight > or = 700 g and/or a ptosis > or = 30 cm. We report a few technical modifications of the initial Lejour vertical technique. About hypertrophic breasts (179 breasts): mean resection weight was 905 g (710-1750 g), mean ptosis was 33.5 cm (30-42 cm). The following complications were noted: 3 haematomas with only one evacuation on the same day, no seroma, 4 infections controlled by local treatment, 44 wound dehiscence or delayed skin healing (> 30 d), 8 partial necrosis, 12 secondary correction under local anaesthesia, 1 cheloïd scar of the areolar complex. About breasts with severe ptosis (64 breasts): the mean value was 31.8 cm (30 cm). Complications were as follow: 2 haematoma, 0 seroma, 16 healing delay, 3 partial necrosis, 6 secondary correction under local anaesthesia. The mean procedure time was 94 mn. The mean stay in hospital was 2.8 days, same as the suction drains. The vertical mammaplasty is a fast technique, giving good results. Scarring amount is reduced. Breasts are well projected, well shaped and remain stable in time. Their base can be modified. We think this technique can safely be applied to large breasts and/or severe ptosis.

Laminin-1-induced migration of multiple myeloma cells involves the high-affinity 67 kD laminin receptor.

The 67 kD laminin receptor (67LR) binds laminin-1 (LN), major component of the basement membrane, with high affinity. In this study, we demonstrated that human multiple myeloma cell lines (HMCL) and murine 5T2MM cells express 67LR. CD38(bright+) plasma cells in fresh multiple myeloma (MM) bone marrow (BM) samples showed weaker 67LR expression, but expression increased after direct exposure to a BM endothelial cell line (4LHBMEC). LN stimulated the in vitro migration of 3 HMCL (MM5.1, U266 and MMS.1), primary MM cells and the murine 5T2MM cells. 67LR has been shown to mediate the actions of LN through binding to CDPGYIGSR, a 9 amino acid sequence from the B1 chain of LN. MM cell migration was partially blocked by peptide 11, a synthetic nonapeptide derived from this amino sequence and also by a blocking antiserum against 67LR. Co-injection of peptide 11 with 5T2MM cells in a murine in vivo model of MM resulted in a decreased homing of 5T2MM cells to the BM compartment. In conclusion, LN acts as a chemoattractant for MM cells by interaction with 67LR. This interaction might be important during extravasation of circulating MM cells.

Selective initial in vivo homing pattern of 5T2 multiple myeloma cells in the C57BL/KalwRij mouse.

One of the main characteristics of multiple myeloma cells is their predominant localization in the bone marrow. It is, however, unclear whether this is due to a selective initial entry, or whether this entry is more random and other processes like survival and/or growth stimulation, only present in the medullar microenvironment, are unique. To investigate this, in vivo homing kinetics of murine 5T2MM cells shortly after injection were assessed in bone marrow, liver, spleen, lungs, heart, intestines, kidney and testis by tracing of radiolabelled cells, by immunostaining of isolated cells and by polymerase chain reaction analysis. We demonstrated the presence of 5T2MM cells in bone marrow, spleen and liver with all other organs being negative. Adhesion assays of 5T2MM cells to different types of endothelial cells demonstrated a selective adhesion of 5T2MM cells to bone marrow and liver and not to lung endothelial cells. We here demonstrate that the specific in vivo localization of the 5T2MM cells is a result of the combination of a selective entry/adhesion of the 5T2MM cells in the bone marrow, spleen and liver, and a selective survival and growth of these tumour cells in the bone marrow and spleen but not in the liver.

Bone sialoprotein mRNA and protein expression in human multiple myeloma cell lines and patients.

Bone sialoprotein (BSP) is a glycoprotein essentially found in mineralizing connective tissues. We have recently demonstrated that BSP is ectopically expressed by carcinomas that metastasize to bone with high frequency. Multiple myeloma (MM) is characterized by the localization of tumour plasma cells in the bone marrow. In this study, BSP expression was evaluated in human myeloma cell lines and in bone marrow aspirates and one ascites fluid from MM patients. BSP was detectable in conditioned media of MM cell lines. Using FACS analysis and in situ hybridization, we demonstrated that tumour cells from all MM patients and cell lines analysed express BSP at both the protein and the mRNA level.

Homing of the myeloma cell clone.

The presence of myeloma cells in the blood circulation. implicates that these cells must have the potential to extravasate and home to the bone marrow environment. Using the 5T2 MM mouse model, we could demonstrate that the restricted localization of myeloma cells in the bone marrow is the result of selective migration of myeloma cells in the bone marrow combined with a selective growth of the tumour cells in the bone marrow microenvironment. Moreover, we showed that 5T2 MM cells bind in vitro selectively to bone marrow-derived endothelial cells (EC) and not to lung-derived EC. In order to identify which chemotactic molecules mediate the transendothelial migration of myeloma cells, we examined the motility-inducing effect of different extracellular matrix proteins on myeloma cell lines. We found that laminin-1 a major component of the basement membrane, triggers the motility of both human myeloma cells and 5T2 MM cells, through the 67 kD laminin receptor. Because of the broad distribution of laminin in extracellular matrices throughout the body, it is clear that this molecule on itself can not be the only factor that determines the specificity of myeloma cell homing. In the 5T2 MM model we identified IGF-1 as a more specific bone marrow derived chemoattractant for myeloma cells. In addition we demonstrated that the marrow microenvironment can upregulate the expression of the IGF-1 receptor on 5T mouse myeloma cells. In the end phase of the disease, increasing numbers of myeloma cells are detectable in the peripheral blood and extramedullary tumour growth can occur. We found that the stroma-independent variant of the human MM5 myeloma cell line showed an increased in vitro motility as compared to the stroma-dependent variant. By representational difference analysis we demonstrated that the stroma-dependent MM5 cells show a downregulation of the motility-related protein (MRP-I CD9) which might reflect the involvement of this molecule in the regulation of myeloma cell extravasation.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) DNA sequences are absent in leukapheresis products and ex vivo expanded CD34+ cells from multiple myeloma patients.

Recently it was reported that Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) infects bone marrow (BM) dendritic cells (DC) in multiple myeloma (MM) patients and therefore might play a role in MM development. Because of the use of myeloid growth factors like GM-CSF and G-CSF for the mobilization of peripheral blood progenitor cells (PBPC), the subsequent increase of DC precursors might imply a risk for KSHV contamination in PBPC grafts. Therefore, in this study leukapheresis products and ex vivo cultured CD34+ cell suspensions were analysed. KSHV DNA could not be amplified in any of them.

Homing behaviour of the malignant cell clone in multiple myeloma.

Multiple myeloma (MM) represents a B cell malignancy characterised by the presence of a monoclonal population of end-stage B cells in the bone marrow. Although fully matured bone marrow plasma cells are the predominant cell type in MM, there is much evidence that also more immature B cells are included in the malignant cell clone which are considered to be the myeloma precursor cells. The fact that these cells are detectable in the blood circulation and that their number increases with disease progression, makes it very likely that they represent the component of the tumour clone that mediates disease dissemination. This implies that these cells must have the potential to extravasate and home to the bone marrow environment. Like the migration mechanisms used by normal leukocytes and/or metastatic tumour cells of non-haematopoietic origin, it can be assumed that this bone marrow homing process is mediated by adhesive interactions and chemotactic signals provided by the microenvironment of the tumour. Once in the bone marrow compartment, myeloma cells will receive the appropriate signals to grow and survive. This aspect of tumour-homing is found to be the result of a functional interplay between the myeloma cells and the surrounding microenvironment, involving the action of several cytokines and adhesion molecules. In the end phase of the disease, myeloma cells can lose their stroma-dependency resulting in extramedullary tumour growth. We review normal B cell homing and discuss molecular mechanisms that determine the homing behaviour of the malignant cell clone in MM.

Structure of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene. Promoter analysis and alternative splicing of the SERCA3 pre-mRNA.

Human chromosome 17-specific genomic clones extending over 90 kilobases (kb) of DNA and coding for sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The presence of the D17S1828 genetic marker in the cosmid contig enabled us to map the SERCA3 gene (ATP2A3) 11 centimorgans from the top of the short arm p of chromosome 17, in the vicinity of the cystinosis gene locus. The SERCA3 gene contains 22 exons spread over 50 kb of genomic DNA. The exon/intron boundaries are well conserved between human SERCA3 and SERCA1 genes, except for the junction between exons 8 and 9 which is found in the SERCA1 gene but not in SERCA3 and SERCA2 genes. The transcription start site (+1) is located 152 nucleotides (nt) upstream of the AUG codon. The 5'-flanking region, including exon 1, is embedded in a 1.5-kb CpG island and is characterized by the absence of a TATA box and by the presence of 14 putative Sp1 sites, 11 CACCC boxes, 5 AP-2-binding motifs, 3 GGCTGGGG motifs, 3 CANNTG boxes, a GATA motif, as well as single sites for Ets-1, c-Myc, and TFIIIc. Functional promoter analysis indicated that the GC-rich region (87% G + C) from -135 to -31 is of critical importance in initiating SERCA3 gene transcription in Jurkat cells. Exon 21 (human, 101 base pairs; mouse, 86 base pairs) can be alternatively excluded, partially included, or totally included, thus generating, respectively, SERCA3a (human and mouse, 999 amino acids (aa)), SERCA3b (human, 1043 aa; mouse, 1038 aa), or SERCA3c (human, 1024 aa; mouse, 1021 aa) isoforms with different C termini. Expression of the mouse SERCA3 isoforms in COS-1 cells demonstrated their ability to function as active pumps, although with different apparent affinities for Ca2+.

Immunoglobulin VH gene sequence analysis of spontaneous murine immunoglobulin-secreting B-cell tumours with clinical features of human disease.

The 5T series of multiple myelomas (MM) and Waldenstrsöm's macroglobulinaemia-like lymphomas (WM), which developed spontaneously in ageing mice of the C57BL/KaLwRij strain, shows clinical and biological features that closely resemble their corresponding human diseases. In order to compare the patterns of somatic mutation in VH genes of mouse tumours with those of human counterparts, we have determined and analysed sequences of immunoglobulin VH genes in five cases of murine MM, two of WM and one of biclonal benign monoclonal gammopathy (BMG). Four of five MM and 2/2 WM cases used VH genes of the large J558 family; one MM used a gene of the VGAM3.8 family, and both clones of the BMG used genes of the 36-60 family. N-region insertions were observed in all cases, but D-segment genes were only identified in 6/9 cases, which were all from the D-SP family and translated in reading frame 3. Compared with human MM, in which the VH genes have been found to be consistently hypermutated (mean% +/- SD = 8.8 +/- 3.2), the degree of somatic mutation in the murine tumours was significantly lower (mean% +/- SD = 2.9 +/- 2.3). There was no significant evidence of clustering of replacement mutations in complementarity determining regions (CDR), a feature considered to be characteristic of antigen-selected sequences. However, one clone of the biclonal BMG case showed intraclonal variation, a feature described in some cases of human BMG. These results indicate that murine VH genes in mature tumours differ from human counterparts in the level and distribution of somatic mutations, but support the concept that BMG may be distinct from MM.

Differential gene expression of the neural cell adhesion molecule (N-CAM) in a panel of multiple myeloma cell lines.

A striking feature of myeloma plasma cells concerns their expression of the neural cell adhesion molecule (N-CAM). The regulation of this particular expression is, however, not known. In this study, the N-CAM (CD56) gene regulation was examined in a panel of multiple myeloma (MM) cell lines. In this panel, both N-CAM-positive and -negative cells were analysed, reflecting the in vivo situation where a minority of MM patients have CD56-negative plasma cells at diagnosis or where in cases of extramedullary involvement CD56 expression decreases. At least two N-CAM mRNAs were found in the cell lines expressing the 140 kDa isoform. With one exception, no N-CAM transcripts could be detected in the N-CAM-negative cell lines. No structural differences could be found in the genomic organization of the N-CAM gene, or in the regulatory promoter region when CD56-positive and -negative cell lines were compared. In transfection studies, however, transcriptional activity of the N-CAM promoter was observed in N-CAM-negative cells, leading us to conclude that the up-regulation of N-CAM in MM cannot be explained by a simple transcriptional gene activation.

Establishment and characterization of a human stroma-dependent myeloma cell line (MM5.1) and its stroma-independent variant (MM5.2).

Although IL-6 has been identified as a major growth factor in multiple myeloma (MM), it is believed that maintenance of tumor growth in vivo depends on one or more additional stroma-derived factors. We describe a new human myeloma cell line (MM5.1) that can be maintained in the presence of bone marrow-derived stromal cell layers, and not only when cultured with exogeneous IL-6. This cell line expresses the same immunoglobulin kappa light chain RNA sequence as the patient's original tumor cells, has a plasma cell morphology and expresses plasma cell antigens (cytoplasmic kappa light chain, CD38, BB4). Without the presence of stromal factors, MM5.1 cells become apoptotic. A low proliferative effect was observed in the presence of oncostatin M (OSM) but other cytokines (IL-10, IL-11, stem cell factor (SCF) and leukemia inhibitory factor (LIF)) had no effect at all. We observed that MM5.1 cells also grow when physically separated from stromal cell layers by a 0.45 microm microporous membrane or when cultured in conditioned medium from stromal marrow cells. Unexpectedly, the growth in stromal supernatants was markedly inhibited by an anti-IL-6 antiserum and an anti-IL-6 receptor transducer chain (gp130) mAb in a dose-dependent manner. This implies that MM5.1 cells are IL-6 responsive only when exposed to one or more additional soluble factor(s) derived from bone marrow stroma. Coculturing MM5.1 cells with IL-6 and cytokines that were described to increase the IL-6 responsiveness of myeloma cells (G-CSF, GM-CSF and IL-3) had no effect on the growth or survival. A strong proliferative effect was observed when MM5.1 cells were cultured with IL-6 and soluble IL-6 receptor (sgp80). However no sgp80 could be detected in stromal supernatants using a sensitive immunoassay. This indicates that sustained proliferation of the MM5.1 cell line depends on a combination of IL6 and at least one, thus far unidentified, stroma-derived factor. After more than 1 year in continuous culture, we could obtain a variant of the line (MM5.2) that shows an improved growth rate and grows stroma independently. Molecular analysis revealed clonal identity with the early passage form and Epstein-Barr virus antigen expression was negative. The two variants of this cell line offer a useful model to identify molecular mechanisms involved in clonal evolution towards stroma-independent growth of myeloma cells.

Expression of human pICln and ClC-6 in Xenopus oocytes induces an identical endogenous chloride conductance.

pICln is a protein that induces an outwardly rectifying, nucleotide-sensitive chloride current (ICln) when expressed in Xenopus oocytes, but its precise function (plasma-membrane anion channel versus cytosolic regulator of a channel) remains controversial. We now report that a chloride current identical to ICln is induced when Xenopus oocytes are injected with human ClC-6 RNA. Indeed, both the pICln and the ClC-6 induced current are outwardly rectifying, they inactivate slowly at positive potentials and have an anion permeability sequence NO3- > I- > Br- > Cl- > gluconate. Cyclamate, NPPB, and extracellular cAMP block the induced currents. The success rate of current expression is significantly increased when the injected Xenopus oocytes are incubated at a higher temperature (24 or 37 degrees C) prior to the analysis. In addition, the ICln current was detected in 6.2% of noninjected control Xenopus oocytes. We therefore conclude that the ICln current in Xenopus oocytes corresponds to an endogenous conductance that can be activated by expression of structurally unrelated proteins. Furthermore, functional, biochemical, and morphological observations did not support the notion that pICln resides in the plasma membrane either permanently or transiently after cell swelling. Thus, it is unlikely that pICln forms the channel that is responsible for the ICln current in Xenopus oocytes.

Persistence of residual tumour cells after cytokine-mediated ex vivo expansion of mobilized CD34+ blood cells in multiple myeloma.

Mobilized CD34+ blood cells were immunomagnetically enriched from leukapheresis products in five multiple myeloma (MM) patients. Thawed samples of selected CD34+ cells were cultured for up to 21 d in a liquid and stroma-free culture system with different combinations of recombinant cytokines. The most successful cell expansion was obtained when a combination of rh-IL-1beta, rh-IL-3, rh-IL-6, rh-SCF, rh-G-CSF and rh-GM-CSF was used. After 14 d this mixture gave a 120-187-fold overall increase of total nuclear cells and a 4-8-fold overall increase of early CFU-GM numbers. In four patients a very sensitive patient-specific PCR analysis showed the presence of monoclonal cells in the initial leukapheresis products. After immunomagnetic separation a tumour cell depletion of 2-4 logs was observed, although all samples still contained malignant cells. Cell suspensions that were cultured with the most potent cytokine combination showed tumour contamination in two-thirds of evaluable cases at the moment of maximal CFU-GM output. Serial cDNA dilution experiments indicated that the positive PCR results at day 14 reflected the persistence of pre-culture tumour cells rather than in vitro expansion of tumour cells in two cases. This study demonstrates that ex vivo expansion of myeloid precursor cells from mobilized CD34+ cells in MM patients does not always result in an effective purging of residual tumour cells. On the other hand, our culture conditions do not seem to favour in vitro expansion of malignant cells, despite the use of a cytokine cocktail that includes potential myeloma growth factors.

Na(+)-Ca2+ exchange in rat dorsal root ganglion neurons.

The role of the Na(+)-Ca2+ exchanger was examined in isolated rat dorsal root ganglion (DRG) neurons. Neurons were dialyzed with the Ca2+ indicator Indo-1. Ca2+ transients were elicited by depolarizing the cells from -80 to 0 mV for 100 ms under voltage clamp conditions. In most cells (45 of 67), the decay of intracellular Ca2+ concentration ([Ca2+]i) could be fitted with a single exponential with a time constant of 2.43 s. In the remaining 22 cells, the decay of [Ca2+]i could be described with a double exponential with time constants of 0.76 and 11.84 s. In cells that displayed a biphasic [Ca2+]i relaxation, Na(+)-free medium caused resting [Ca2+]i to increase from 116 to 186 nM; the slow component of recovery to basal [Ca2+]i was nearly abolished in Na(+)-free medium or by application of 5 mM Ni2+. In 35 of 45 cells displaying a monophasic [Ca2+]i decay, omitting external Na+ increased the time constant of [Ca2+]i decay from 2.02 to 3.63 s. In the remaining 10 cells, Na(+)-free solution did not affect Ca2+ handling. The time constant of [Ca2+]i relaxation was voltage dependent. These findings demonstrate the important role of the Na(+)-Ca2+ exchanger in DRG neurons. Its presence was further confirmed both at the mRNA and the protein level.

Functional effects of expression of hslo Ca2+ activated K+ channels in cultured macrovascular endothelial cells.

The aim of the present study is to elucidate the effects of the expression of large conductance Ca2+ activated K+ channels (BK[Ca]) in an endothelial cell type normally lacking this channel. The human homologue hslo of BK(Ca) was expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which have no endogenous BK(Ca). Membrane potential, ionic currents and Ca2+ signals were investigated in non-transfected and transfected cells using a combined patch clamp and Fura-2 fluorescence technique. In non-transfected control CPAE cells, ATP evoked a Ca2+ activated Cl- current (I[Cl,Ca]). The most prominent current component during ATP stimulation in hslo expressing cells was conducted BK(Ca) which resulted in a pronounced transient hyperpolarization. This hyperpolarization, which was absent in non-transfected cells, was enhanced if I(Cl,Ca) was blocked with niflumic acid. The sustained component of the Ca2+ response during ATP stimulation was significantly larger in hslo transfected cells than in non-transfected cells. This plateau level correlated well with the corresponding effects of ATP on the membrane potential, indicating that the expression of cloned BK(Ca) exerts a positive feedback on Ca2+ signals in endothelial cells by counteracting the negative (depolarizing)effect of stimulation of Ca2+-activated Cl- channels.

Distribution of the organellar Ca2+ transport ATPase SERCA2 isoforms in the cat brain.

Of the three genes encoding the Ca2+ transport ATPases of the endoplasmic reticulum, the SERCA2 gene is the major isoform expressed in the mammalian brain. The SERCA2 transcript is alternatively processed generating two protein isoforms: SERCA2a which is expressed in cardiac and slow-skeletal muscle, and SERCA2b, the house-keeping isoform which is ubiquitously expressed. We have studied the expression of SERCA2 in the cat brain, and at a less refined level also in the rat brain, using antibodies specific for either SERCA2a or SERCA2b. The SERCA2a staining was very restricted. The SERCA2a antibody clearly labeled the cell body of the Purkinje neurons and weakly stained the giant cells of the gigantocellular reticular nuclei. In contrast, the SERCA2b isoform was found in most regions of the brain. It appeared to be largely confined to neuronal cells. Neuroglial cells were negative. The antibody stained the cell body. In heavily labeled cells such as the pyramidal cells of the hippocampus and of the cerebral cortex, it also stained the proximal portion of the dendrites. The most intense labeling was observed in the Purkinje neurons, which were stained all over the cell including the distal ramifications of the dendritic tree. Remarkably the SERCA2b labeling in neuronal cells of the hypothalamic area and the substantia nigra was very weak. The possible physiological significance of these results is discussed.