[Buccal ulcerations in an Ecuadorian patient]. / Ulcérations buccales chez une patiente équatorienne.

The goal of this text is to remind the place of histoplasmosis in the differential diagnosis of chronical buccal lesions, even if this pathology is extremely rare in our countries. The diagnosis is easy and fast because we can do biopsy in a place easily accessible.

Steroid-free medium discloses oestrogenic effects of the bisphosphonate clodronate on breast cancer cells.

Tamoxifen is the standard first-line endocrine therapy for breast cancer, but recent data indicate that it is likely to be replaced by the effective aromatase inhibitors (AIs), in both the metastatic and adjuvant settings. Aromatase inhibitors induce complete oestrogen deprivation that leads to clinically significant bone loss. Several ongoing or planned trials combine AIs with bisphosphonates, even more so that recent data reveal that clodronate may reduce the incidence of bone metastases and prolong survival in the adjuvant setting. Bisphosphonates can inhibit breast cancer cell growth in vitro, but they have never been studied in steroid-free medium (SFM), an in vitro environment that mimics the effects of AIs in vivo. Quite surprisingly, in SFM, clodronate stimulated MCF-7 cell growth in a time- and dose-dependent manner by up to two-fold (crystal violet staining assay), whereas it had no mitogenic activity in complete medium. The bisphosphonate similarly increased the proliferation of IBEP-2 cells, which also express a functional oestrogen receptor (ER), while it weakly inhibited the growth of the ER-negative MDA-MB-231 cells. Expectedly, 17beta-oestradiol stimulated the growth of MCF-7 and IBEP-2 cells cultured in SFM, and had no effect on MDA-MB-231 cells. Moreover, partial (4-OH-tamoxifen) and pure antioestrogens (fulvestrant, ICI 182,780), in combination with clodronate, completely suppressed the mitogenic effect of the bisphosphonate, suggesting that it was mediated by an activation of ER. In accordance with this view, clodronate induced ER downregulation, weakly increased progesterone receptor expression, and stimulated the transcription of an oestrogen-responsive reporter gene. In conclusion, we report a previously unknown stimulatory effect of clodronate on MCF-7 cells grown in SFM, in vitro conditions that are potentially relevant to the use of AIs for breast cancer. Moreover, our data suggest that ER is involved in these effects of clodronate on cancer cell growth.

Extracellular calcium downregulates estrogen receptor alpha and increases its transcriptional activity through calcium-sensing receptor in breast cancer cells.

Skeleton is the most common organ targeted by breast cancer cells, especially from estrogen receptor alpha (ER)-positive neoplasms. Metastatic cells can stimulate directly or indirectly osteoclast-mediated bone resorption. Tumor-induced osteolysis is often extensive and leads to the release of large quantities of calcium. Metastatic cancer cells can be thus exposed to high calcium concentrations (40 mM has been reported at the resorption site). However, the effects of Ca2+ on breast cancer cells have been minimally examined. We showed that 20-mM extracellular Ca2+ induced a downregulation of ER protein in MCF-7 cells and caused ER-mediated transactivation of a reporter gene by 55 +/- 10% (mean +/- SD) in MVLN cells (MCF-7 cells stably transfected with ERE and luciferase reporter gene). Moreover, 3 mM Ca2+ increased progesterone receptor (PgR) expression by 45 +/- 8%. Mg2+ tested at up to 20 mM did not exert any effects, while 17beta-estradiol downregulated ER, transactivated the reporter gene, and enhanced PgR expression. The pure antiestrogen ICI 182,780 was able to abrogate the transactivation of the reporter gene and the increase in PgR levels induced by Ca2+, indicating that Ca2+ may exert a weak and specific estrogenic effect in MCF-7 cells. Ca2+ effects on ER probably start at the cell membrane level since a large Ca2+ influx caused by the ionophore A23187 failed to activate ER. We have thus studied the involvement of the membrane calcium-sensing receptor (CaR) that is known to be expressed notably in MCF-7 cells. We first tested the effects of a specific activator of CaR. Exposure to 10(-4) M calcimimetic NPS R-467 mirrored the changes observed with extracellular Ca2+ by inducing a marked decrease in ER protein levels, increasing the transcriptional activity of ER (67 +/- 12%) and stimulating PgR expression (41 +/- 4%). As expected, the NPS S-467 isomer was less effective. Furthermore, a highly selective CaR antagonist partly suppressed the downregulation of ER as well as transactivation of the reporter gene induced by Ca(2+). Our results suggest that the effects of extracellular Ca2+ on ER expression and activity are mediated, at least in part, by the CaR. In summary, calcium released during the process of metastatic bone destruction could modulate the functions of the estrogen receptor, a key receptor involved in breast cancer cells growth and function, and thus participate in the pathogenesis of tumor-induced osteolysis.

Hypercalcaemia of melanoma: incidence, pathogenesis and therapy with bisphosphonates.

Tumour-induced hypercalcaemia (TIH) is a frequent complication of advanced cancer but has been rarely reported in patients with malignant melanoma, and its pathogenesis remains unexplored. We studied eight patients with TIH and melanoma. We determined the incidence and pathogenesis of this complication and the effects of bisphosphonate therapy. The incidence of TIH in 751 patients with melanoma was 1.1%. All patients had liver and bone metastases at the time of hypercalcaemia. All patients had osteolytic lesions, most often multiple. The median survival was 30 days (range 4-136 days). After rehydration, the mean (+/- SEM) corrected calcium was 3.42 +/- 0.17 mmol/l. Parathyroid hormone levels were adequately suppressed and vitamin D concentrations were normal. Serum osteocalcin, a marker of bone formation, was low, except in the two patients with renal insufficiency, whereas fasting urinary calcium and hydroxyproline were increased, indicating inhibition of bone formation and stimulation of bone resorption. Increased parathyroid hormone-related protein secretion was noted in only one patient. Three of four patients became normocalcaemic after bisphosphonate therapy for a median duration of 2 weeks. In conclusion, hypercalcaemia is a rare complication of melanoma. It occurs in the context of far advanced disease and is essentially due to aggressive lytic bone metastases with an uncoupling in bone turnover. Bisphosphonates can offer short-term palliation.

A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination.

Recent work highlights the potential usefulness of MVM-based vectors as selective vehicles for cancer gene therapy (Dupont et al, Gene Therapy, 2000; 7: 790-796). To implement this strategy, however, it is necessary to develop optimized methods for producing high-titer, helper-free parvovirus stocks. Recombinants of MVMp (rMVMp) are currently generated by transiently co-transfecting permissive cell lines with a plasmid carrying the vector genome and a helper plasmid expressing the capsid genes (replaced with a foreign gene in the vector genome). The resulting stocks, however, are always heavily contaminated with replication-competent viruses (RCV), which precludes their use in vivo and particularly in gene therapy. In the present work we have developed a second-generation MVMp-based vector system specifically designed to reduce the probability of RCV generation by homologous recombination. We have constructed a new MVMp-based vector and a new helper genome with minimal sequence overlap and have used the degeneracy of the genetic code to further decrease vector-helper homology. In this system, the left homologous region was almost completely eliminated and the right sequence overlap was reduced to 74 nt with only 61% homology. We were thus able to substantially reduce ( approximately 200 x), but not completely eliminate, generation of contaminating viruses in medium-scale rMVMp preparations. Since the remaining sequence homology between the new vector and helper genomes is weak, our results suggest that contaminating viruses in this system are generated by nonhomologous recombination. It is important to note, unlike the autonomously replicating helper viruses produced from the first-generation vector/helper genomes, the contaminating viruses arising from the new packaging system cannot initiate secondary infection rounds (so they are not 'replication-competent viruses'). Our findings have important implications for the design of new MVMp-based vectors and for the construction of trans-complementing packaging cell lines.

Decreased efficacy of bisphosphonates for recurrences of tumor-induced hypercalcemia.

Bisphosphonates are now the standard treatment for tumor-induced hypercalcemia (TIH), and pamidronate can normalize serum Ca in at least 90% of the patients treated for the first time. However, there are few data on the treatment of TIH when it recurs, and published results are contradictory. We studied 29 patients with solid tumors, 14 of whom had breast cancer and all of whom were naive to bisphosphonate therapy. They were retreated with pamidronate (median dose 1 mg/kg for both courses) for recurrence of TIH after a median interval of 78 (range 7-297) days. Fourteen of them, 7 of whom had breast cancer, were treated a third time 28 (range 5-79) days after the second course (median dose of pamidronate 1.5 mg/kg). Baseline Ca levels were not significantly different before each course, but the nadirs after each treatment progressively increased, 9.3 +/- 0.2 mg/dl, 10.5 +/- 0.3 mg/dl, and 12.3 +/- 0.4 mg/dl after the 1st, 2nd and 3rd administrations, respectively (P<0.05). The percentage of treatment failures also progressively increased: 10%, 31% and 85% (P< 0.05). This decreased hypocalcemic effect was essentially observed in patients without bone metastases or with tumors other than breast cancer. Thus, in patients without bone metastases, Ca levels did not decrease at all after the 3rd course, whereas the responses were not significantly different between the three courses in patients with bone metastases. Baseline urinary hydroxyproline, a marker of bone resorption, increased progressively from course to course, especially in patients with bone metastases or breast cancer, but this was not the case for parameters of bone formation. There was also a progressive increase in PTHrP levels accompanied by an increase in the number of patients with enhanced kidney reabsorption of Ca and a decrease in the threshold for Pi excretion, which was significant in patients without bone metastases. In conclusion, pamidronate was progressively less efficient when hypercalcemia recurred. This was observed mainly in patients with hypercalcemia of humoral origin. Tumor progression is accompanied by an enhanced release of osteolytic factors, notably PTHrP, that increase bone resorption and enhance kidney calcium reabsorption, especially in patients without bone metastases. When both phenomena occur, the response to bisphosphonates becomes minimal and the usefulness of therapy questionable.

Limb-body wall complex: 4 new cases illustrating the importance of examining placenta and umbilical cord.

Limb-body wall complex (LBWC) is a rare, sporadic, congenital defect defined as a combination of at least two of three characteristics: 1. limb defects, 2. anterior body wall defects, and 3. exencephaly or encephalocoele with/without facial clefts. Three pathogenic mechanisms have been proposed: early amnion rupture, vascular disruption and embryonic dysgenesis. In this study we carried out the pathological evaluation of four fetuses with LBWC and their placentas. None of the cases had craniofacial defects. Three fetuses showed an abdominal wall defect with eventration of abdominal organs, cloacal exstrophy, absent external genitalia, abnormal internal genitalia, scoliosis and lower limb defects. One fetus showed failure of closure of both thoracic and abdominal walls with ectopia cordis, evisceration of left lung and abdominal organs, severe reduction defect of left arm, but normal colon, anus, bladder, genitalia and lower limbs. All cases had a short, malformed umbilical cord, incompletely covered by amnion. The umbilical vessels were embedded in an amniotic sheet which connected the skin margin of the anterior body wall defect to the placenta. These anomalies suggest an abnormal body stalk development as a pathogenic mechanism for LBWC. Prenatally, the abnormal fetoplacental attachment can be detected ultrasonographically by the end of the first gestational trimester. Postnatally, the examination of placenta, umbilical cord and membranes is crucial in confirming the diagnosis of LBWC.

[Bronchial mucoepidermoid carcinoma: apropos of 3 cases]. / Carcinome muco-épidermoïde bronchique: à propos de 3 observations.

We report three cases of bronchial mucoepidermoid carcinoma (BMEC) of low-grade malignancy with a relafase-free follow up. BMEC are rare tumors. The microscopic findings distinguish low-grade tumors which occur in children and young adults and high-grade tumors concerning older patients; this grading is based on the study of the epidermoid component. If possible, conservative therapy is appropriate in low-grade tumors. The prognosis of high-grade tumors is poor.

[Silicone tracheobronchial endoprosthesis]. / Endoprothèses trachéobronchiques en silicone.

Silicone endoprostheses are used to replace loss of support in tracheobronchial cartilage. The main silicone stents are similar to those of the Montgomery T tube, the Westaby, Dumon and Cooper-Hood prostheses. The major indications are malignant tumours and benign stenosis after anastomotic resection or graft. All have in common a degree of narrowing greater than 50%. An initial bronchoscopy enables a precise assessment of the zone to support. The prostheses are then put in place using a rigid bronchoscope. An annual bronchoscopic review is recommended associated with clinical supervision. Their removal is simple even after being in position for a long period. A multicentre study (Marseille, Saint-Etienne, Brescia and Barcelona) report their experience of 1574 prostheses positioned in 1058 patients. The localisation was tracheal (54%), left main bronchus (21%), right main bronchus (18%). The average time in place was 1.2 years for benign tumours(maximum 6.2 years) and four months for malignant tumours (maximum 4.7 years). Complications were rare and included migrations (9.5%), granulomas (7.9%) and obstructions (3.6%). Thanks to their being well tolerated, their simplicity in handling, silicone prostheses are currently an essential choice to re-establish patency of the airways in patients presenting with benign or malignant tracheobronchial pathology.

A simple method for carbohydrate-deficient transferrin measurements in patients with alcohol abuse and hepato-gastrointestinal diseases.

Carbohydrate-deficient transferrin (CDT) is known to be increased in alcohol abuse. Several methods were developed for its measurement (e.g. isoelectric focusing with Western blotting or immunofixation, anion-exchange chromatography followed by immunoassays). We describe a greatly simplified isoelectric focusing technique which does not require immunofixation. CDT results obtained with this method were compared to other biological markers of alcohol abuse, i.e. mean corpuscular volume (MCV), aspartate aminotransferase (ASAT) and gamma-glutamyl-transferase (GGT), in 55 patients distributed in three groups (i.e. healthy control subjects, control patients suffering from various hepato-gastrointestinal diseases and alcohol abusing patients). Sensitivity and specificity were 33-89%, 61-57%, 89-49% and 83-100% for MCV, ASAT, GGT and CDT, respectively. We conclude that our method is highly suitable for routine clinical use.

The bone isoenzyme of alkaline phosphatase in hypercalcaemic cancer patients.

Alkaline phosphatase (AP) is the classic marker of bone formation, especially in cancer patients, but the interpretation of its measurement is complicated by the existence of various circulating isoenzymes, especially of liver origin. The introduction of a mass measurement of the bone isoenzyme of AP (BAP) by an immunoradiometric assay has markedly improved the sensitivity and the specificity of the determination. We measured BAP and other markers of bone turnover in 46 patients with tumour-induced hypercalcaemia (TIH), which is an interesting model for evaluating markers of bone formation because of the uncoupling between bone formation and bone resorption found by histomorphometric techniques. The extent of bone metastatic involvement was evaluated by planimetry on bone scintigraphy. Mean (+/- S.D.) BAP concentrations were slightly higher in patients with TIH than in healthy subjects, 15.5 +/- 8.5 versus 12.4 +/- 3.5 micrograms/L (P < 0.05). However, the scatter of the data in TIH patients was quite marked. Increased values (10/46 patients, 22%) occurred only in patients with bone metastases. Total AP, gamma GT and BGP levels, as well as markers of bone resorption, were not significantly different between patients with or without bone metastases. BAP levels were significantly correlated with AP (rs = 0.63; P < 0.01) but not with BGP levels nor with markers of bone resorption. BAP levels were also correlated with the extent of bone uptake at scintigraphy (rs = 0.54; P < 0.01), but this was not the case for total AP or BGP. In the 36 patients re-evaluated when normocalcemic after pamidronate therapy, BAP levels increased from 16.3 +/- 9.2 to 22.2 +/- 21.3 micrograms/L (P < 0.05) but there were no significant changes in AP or BGP concentrations. In summary, our data confirm the existence of an uncoupling in bone turnover in TIH and indicate that cancer hypercalcaemia is another pathological condition characterised by a discordance between BAP and BGP concentrations. BAP levels appear to be a better reflection of bone metastatic involvement than total AP or BGP and their short-term increase after pamidronate therapy could reflect the recently described effects of bisphosphonates on osteoblasts.

Comparative evaluation of markers of bone resorption in patients with breast cancer-induced osteolysis before and after bisphosphonate therapy.

The understanding of the pathophysiology and the monitoring of metastatic bone disease remains unsatisfactory. We compared several new markers of bone turnover in normocalcaemic patients with breast cancer-induced osteolysis before and after a single infusion of the bisphosphonate pamidronate. We studied 19 ambulatory patients with advanced breast cancer and extensive bone metastases who did not receive any systemic antineoplastic therapy. Pamidronate was administered at doses of 30, 60, 90 or 120 mg and the patients were followed weekly during a mean of 8 (range 4-10) weeks. Compared with healthy premenopausal women, the percentage of elevated values at baseline was 47% for fasting urinary calcium (uCa), 74% for hydroxyproline, 83% for CrossLaps (a new marker of type I collagen degradation) and 100% for the collagen cross-links (measured by high performance liquid chromatography), namely pyridinoline (Pyr) and deoxyPyr (D-Pyr). Pretreatment levels of uCa did not correlate significantly with any of the four markers of bone matrix resorption, whereas the correlations between these four markers were generally significant (r(s)=0.43-0.71). Alkaline phosphatase correlated significantly with markers of bone matrix resorption (r(s)=0.54-0.74). All parameters, except phosphaturia (uPi) and the bone formation markers (osteocalcin and alkaline phosphatase), fell significantly after pamidronate therapy, up to day 42 for hydroxyproline, D-Pyr and CrossLaps and day 56 for uCa. This longer lasting effect was probably due to the parathyroid hormone (PTH) surge following the decrease in serum calcium, implying that the decrease in uCa can overestimate the effects of bisphophonates on bone resorption. The decrease in bone turnover parameters was most marked for CrossLaps, indicating the potential of this new marker for monitoring therapy. Sequential determinations of markers of bone matrix resorption should be useful in delineating the optimal therapeutic schemes of bisphosphonates and for evaluating treatment effects on bone in cancer patients.

Retrobulbar optic neuritis after pamidronate administration in a patient with a history of cutaneous porphyria.

We describe the case of a patient who developed reversible retrobulbar optic neuritis after intravenous pamidronate therapy for established osteoporosis. This possible complication has never been previously reported and, since our patient had a history of porphyria, it suggests that bisphosphonates should be administered cautiously in patients with this disease.

Tumor-induced hypercalcemia in a patient with extensive soft tissue sarcoma: effects of bisphosphonate therapy and surgery.

Tumor-induced hypercalcemia (TIH) is a frequent complication of advanced cancer, but it has been rarely reported in patients with sarcoma. We describe the case of a young female patient with TIH and with an extensive synoviosarcoma of the left lower limb destroying the bony structures. Hypercalcemia was severe (18.3 mg/dl) and accompanied by low serum Pi and suppressed parathyroid hormone (PTH) and 1,25(OH)2 vit D3 serum concentrations. Hypercalcemia was successfully treated with ibandronate, a new third-generation bisphosphonate, and radical surgery was performed when the patient was normocalcemic. Circulating levels of PTH-related protein (PTHrP) were elevated at 22.5 pmol/L (NI < 9). PTHrP levels did not change after successful therapy of TIH, in contrast with PTH, which increased sharply. PTHrP levels were normalized after radical surgery. Moreover, low serum Pi with reduced threshold for phosphate excretion and increased tubular calcium reabsorption supported the notion that PTHrP was indeed the essential mediator of paraneoplastic hypercalcemia in this case despite the extensive bone destruction.

Technical and clinical validation of a new immunoradiometric assay for human osteocalcin.

The measurement of circulating osteocalcin or bone GLA protein (BGP) constitutes a well established and non-invasive means for evaluating preferentially the bone formation rate, but most available commercial assays suffer from several technical constraints, notably a rapid degradation of BGP at room temperature or after thawing and the inability to measure subnormal values. We evaluated, from a technical and a clinical viewpoint, a newly available two-site sandwich immunoradiometric assay (IRMA) using standard of human origin and two different monoclonal antibodies. The theoretical and functional assay detection limit was 0.3 ng/ml. Concentrations of BGP progressively decreased when the serum was left at 4 degrees C or at room temperature (mean apparent loss of 15% after 24 h). Two cycles of freezing-thawing only lightly reduced the BGP concentrations. The mean (+/- SD) BGP concentration was 19.6 +/- 7.9 ng/ml in healthy subjects (NI, N = 61); the normal range was 8.1-35.6 ng/ml. There was a marked difference between pre- and postmenopausal women: 15.1 +/- 4.4 vs 22.3 +/- 8.4 ng/ml, respectively (p < 0.05). The mean BGP concentration in patients with tumor-induced hypercalcemia (N = 29) was not significantly different from NI, but nine patients (31%) had subnormal levels and five (17%) had elevated BGP levels. Concentrations of BGP were significantly increased in patients with hyperparathyroidism (N = 14) (45.1 +/- 21.0 ng/ml) and significantly lower than NI in patients with hypoparathyroidism (N = 18) (7.3 +/- 4.6 ng/ml). Concentrations of BGP were also measured by a classical radioimmunoassay using bovine standards and tracer; the correlations between both sets of measurements were significant in all groups, except in patients with hypoparathyroidism. In summary, this newly available IRMA for measuring circulating human BGP appears to be quite sensitive, reproducible and robust. It should be especially useful for investigating clinical conditions characterized by a low bone formation rate.

Cyclical pamidronate infusions in postmenopausal osteoporosis.

OBJECTIVES: Until recently, two bisphosphonates, pamidronate (APD) and etidronate were available for clinical purposes. Contrary to etidronate, pamidronate was not extensively studied in osteoporosis. Therefore, we investigated the effect of cyclic intravenous APD treatment in postmenopausal osteoporosis. METHODS: Parameters of bone remodelling and lumbar spine bone mineral density (BMDL) were assessed in 36 postmenopausal women with osteoporosis (BMDL t-score < -2.5). They received five courses of APD. Intervals between courses were defined according to the fasting urinary calcium excretion (UCa/Cr, mg/mg creatinine) which was measured before each APD course and every 2 weeks after the first treatment. The patients were retreated when UCa/Cr had reached baseline levels. Serum biochemical parameters and urinary hydroxyproline (UOHPro/Cr, mg/mg) were measured before each APD. RESULTS: UCa/Cr decreased during 21-28 days after each course but UCa/Cr measured before APD infusion remained unchanged. UOHPro/Cr significantly fell after the third APD (P = 0.02). Serum calcium was however not modified. Parameters of bone remodelling decreased with time: bone-GLA protein (BGP) started to fall after the first APD (P = 0.0001) and continued to decrease until the fourth APD course, alkaline phosphatase (ALP) significantly decreased after the first APD (P = 0.005); intact PTH significantly increased at the fifth APD (P = 0.02). BMDL significantly increased after 1 year treatment: +2.9% of baseline value. CONCLUSIONS: Cyclical pamidronate treatment of postmenopausal osteoprosis appeared to be effective in reducing bone turnover assessed by BGP, ALP and OHPro/Cr. This effect is followed by an increase in vertebral BMD.