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Myxofibrosarcoma (MFS) is a well-established nosologic entity different from the myxoid variant of malignant fibrous histiocytoma. In an attempt to better define the representative cytologic criteria of MFS, we undertook a review and a reanalysis of a series of 14 cytology samples in 12 patients whose tumors were diagnosed as MFS.Using FNA technique and reviewing the original diagnoses, 11 cases were diagnosed as malignant and three as benign tumors. The cytologic diagnosis of MFS was accurate in seven cases (2 primary tumors, 4 recurrences, and 1 metastasis). Four cases were classified malignant myxoid sarcoma (1 primary and 3 recurrences), whereas three cases (2 primary and 1 recurrence) were false-negative.The smears were cell-rich in 12 cases and cell-poor in two cases. They were constantly composed of isolated and regular small spindle-shaped and stellated cells with elongated nuclei containing small inconspicuous nucleoli. Cytoplasm was pale with elongated processes. Clusters of wavy spindle-shaped cells, round cells without specific pattern, moderate cytonuclear atypia, and abundant myxoid background as well as curvilinear vascular structures were always seen. In the vast majority of cases, the cytologic distinction of MFS from other low-grade myxoid lesions is difficult. Entities such as myxoid MFH, myxoid liposarcoma (MLP), myxoid DFSP, and myxoma should be considered in the differential diagnosis. The cytological misdiagnosis is of limited clinical consequence because FNA findings suggestive of a myxoid tumor will be indicative for a surgical removal followed by the histopathological analysis.

To determine diagnostic cytomorphologic features of rhabdomyosarcoma (RMS) on fine-needle aspiration (FNA) material, the cytologic material and corresponding histologic slides of 180 tumors obtained from 109 patients were reviewed. Fifty eight (32.2%) tumors were primary, 34 (18.9%) recurrent, and 88 (48.9%) metastatic. A review of original cytology reports revealed that 176 of 180 (97.8%) tumors were either diagnosed accurately or as round cell sarcoma, while 3 (1.7%) were reported as suspicious. In one case (0.5%), the material was unsatisfactory. No false negative samples were seen. When FNA morphology was correlated with different histological subtypes, the alveolar subtype RMSs were more cellular than the nonalveolar ones (91.4% vs. 64.9%). Similarly, alveolar subtype RMSs compared with nonalveolar ones exhibited more rhabdomyoblastic cells (77.1% vs. 52.7%), alveolar structures (67.6% vs. 10.8%), giant, multinucleated cells (22.9% vs. 6.7%), mitotic figures (57.1% vs. 18.9%), and cyto-nuclear atypia (77.1% vs. 43.2%). Inversely, spindle-shaped cells were more frequently seen in nonalveolar versus alveolar RMSs (37.8% vs. 20.9%)., (Copyright 2007 Wiley-Liss, Inc.)

To determine diagnostic cytomorphologic features of osteosarcoma on fine-needle aspiration materials, we reviewed the cytologic material and corresponding histologic slides of 126 tumors in 107 patients. Fifty-five (43.6%) tumors were primary, 31 (24.6%) were recurrent, and 40 (31.8%) were metastatic. Review of original cytology reports revealed that 120 (95.3%) tumors were diagnosed as malignant. Six (4.7%) cases were reported as suspicious, false-negative, or unsatisfactory samples. Our findings showed that osteoblastic roundish cells, spindle-shaped cells, reactive giant cells, and osteoid were the most consistent features representative of osteosarcoma. Periosteal reactions, fractures with callous formation, giant cells of osteoclastic type in various conditions, chondrosarcoma with enchondral ossification are entities to consider in the differential diagnosis., ((c) 2007 Wiley-Liss, Inc.)

Schwannoma accounts for one of the most common benign mesenchymal neoplasms of soft tissues. Although it is well defined in the cytology literature, particular histologic subtypes such as "ancient," cellular and epithelioid variants could be a source of diagnostic difficulties. We have reviewed cytology aspirates and corresponding histologic sections from 34 schwannomas diagnosed at Institut Curie. Histologically, 24 cases were classic, 5 were "ancient," 4 were cellular, and 1 was epithelioid schwannomas. No example of melanotic schwannoma was recorded. Original cytologic diagnosis was schwannoma in 13 (38.2%) cases, benign soft tissue tumor in 11 (32.4%), pleomorphic adenoma in 2 (6%) cases, angioma in 1 (2.9%) case, nodular fasciitis in 1 (2.9%) case, suspicious in 3 (8.8%) cases, and not satisfactory in 3 (8.8%) cases. There were no major differences between classical, "ancient," cellular, and epithelioid variants on cytology smears. Myxoid stroma, mast cells, and intranuclear inclusions were limited to classical subtype. Similarly, cyto-nuclear atypia was more frequent in classical subtype than in other subtypes. Schwannoma should be differentiated from well-differentiated malignant peripheral nerve sheath tumor, neurofibroma, and pleomorphic adenoma, in the last instance particularly for head and neck lesions., ((c) 2006 Wiley-Liss, Inc.)

There is a limited number of correlative cytopathological studies of fibrous histiocytoma (FHC). To better define cytopathological criteria of diagnosis, we have reviewed fine-needle aspirates (FNA) from 36 FHCs (32 classical, 1 myxoid, and 3 aneurysmal variants on corresponding histological sections). Original cytological diagnoses were benign in 33 (91.7%) cases (22 accurate) and false positive in 3 (8.3%) cases. All smears were surprisingly homogenous and composed of histiocytic cells with finely vacuolated cytoplasm in 27 (75%) cases, small regular spindle cells in 25 (69%) cases, and giant cells in 17 (47%) cases. Histiocytic cells were attached to vascular structures in 9 (25%) cases. Slight cytonuclear atypia was seen in five (14%) cases. Three (8.3%) cases showed numerous siderophages. In two (5.6%) cases, there were abundant inflammatory backgrounds and in one (3%) case there was a scant myxoid background. Storiform patterns, round cells, prominent atypia, necroses, or mitotic figures were not seen. FHC should be differentiated from other benign, low- and intermediate-grade spindle-cell neoplasms such as low-grade fibrosarcoma, dermatofibrosarcoma protuberans, nodular fasciitis, spindle-cell malignant melanoma, and monophasic synovial sarcoma. Some cases may be misinterpreted as malignant, especially in cases of recurrence or in patients with a cancer history., (copyright (c) 2004 Wiley-Liss, Inc.)

High concentrations of oxygen can induce pulmonary toxicity and cause injury to alveolar epithelial and endothelial cells. The present study was performed to determine whether the potent epithelial and endothelial fibroblast growth factor 1 (FGF-1) protected against hyperoxia-induced lung injury. Recombinant adenovirus carrying the gene encoding human secreted FGF-1 (Ad. FGF1) increased the proliferation of lung epithelial cells in vitro. Ad.FGF1 or control vector with an empty expression cassette (Ad.V152) was administered intratracheally to Wistar rats. With Ad.FGF1 (10(9), 5 x 10(9), 10(10), or 5 x 10(10) viral particles [VP]), FGF-1 protein was found in bronchoalveolar lavage fluid 4 days postinfection at levels proportional to the viral dose and was detected in plasma after doses of 10(10) VP or more were administered. Histological examination of the lungs showed intense proliferation and apoptosis of alveolar and bronchial epithelial cells, with few inflammatory cells. The alveolar architecture returned to normal within 17 days. Rats pretreated with Ad.FGF1 (10(9) or 5 x 10(9) VP) 2 days before exposure to hyperoxia (95% O2) survived, whereas rats pretreated with Ad.V152 died within 3 days. In conclusion, adenovirus-mediated FGF-1 overexpression in the lungs causes epithelial cell proliferation and has beneficial effects in hyperoxic lung injury., (Copryright Mary Ann Liebert, Inc.)

The preoperative cytological diagnosis of malignant phyllodes tumor (MPT) is challenging due to the heterogeneity of its clinical, radiological, and morphological presentation. To better define the cytopathological characteristics of MPT, we reviewed 22 examples seen at the Institut Curie. The original cytologic diagnosis was benign breast tumor in four cases (18.2%), suspicious in seven cases (31.8%) (low-grade phyllodes tumor in six cases needing histological evaluation, suspicious of sarcoma in one case), and malignant in 11 cases (50%). Smears were composed of different proportions of clusters of epithelial cells (68.2%), phyllodes fragments (31.8%), spindle cells within stromal tissue (31.8%), isolated spindle-shaped or round cells (45.5%), and bipolar naked nuclei (22.7%). Giant cells and mitotic figures were also occasionally seen. The cytological findings on smears were correlated with histopathological observations. One of the difficulties to reach an accurate cytological diagnosis for MPT is the frequent overwhelming of clearly malignant sarcomatous cell by the presence of largely predominant clusters of epithelial cell., (Copyright 2004 Wiley-Liss, Inc.)

We have reviewed cytopathology and the corresponding histopathology material of 86 liposarcomas (55 patients) seen at Institut Curie. The liposarcomas (LS) were well differentiated in 14 cases (9 pure, 2 dedifferentiated, 3 sclerosing), 64 myxoid, and 8 pleomorphic. Twenty-four tumors were primary, 34 recurrent, and 28 secondary. Smears in LS were composed in different proportions of round, spindle cells, lipoblasts, and myxoid and vascular arborizing structures. Pure well-differentiated LS were frequently composed of lipoblasts, and round or spindle cells were occasionally seen. Dedifferentiated and sclerosing liposarcomas were composed of spindle or round cells, but lipoblasts were also occasionally present. Myxoid or vascular arborizing structures were absent. Myxoid LS (including round and spindle cell LS) frequently showed a myxoid background and less frequently vascular arborizing structures. Tumor cells were round or spindle. Lipoblasts were also seen. Pleomorphic LS were composed of an admixture of all cellular and stromal elements. Well-differentiated LS should be distinguished from hibernoma and spindle cell lipoma, and myxoid LS from myxoma, myxoid chondrosarcoma, chordoma, myxoid leiomyosarcoma, and myxoid malignant fibrous histiocytoma. The demonstration of the specific translocation t(12;16)(q13;p11) of myxoid LS is very helpful to establish the diagnosis. Pleomorphic LS should be differentiated from other high-grade sarcomas, whenever possible., (Copyright 2004 Wiley-Liss, Inc.)

Dermatofibrosarcoma protuberans (DFSP) is a nodular cutaneous mesenchymal tumor of intermediate malignancy. Studies on fine-needle aspiration of DFSP are extremely rare; to our knowledge, only 33 cases have been reported. We have reviewed 14 examples of DFSP in 13 patients. Ten primary tumors were aspirated before surgical biopsy and four recurrent lesions (all from superficial lesions) were also investigated by fine-needle aspiration. All smears were surprisingly homogeneous and composed of isolated spindle cells in all cases (one unsatisfactory smear is excluded). Tissue fragments with a stroriform pattern were seen in 11 cases, fibrillary stromal fragments in 10 cases, naked nuclei in 8 cases, slight to moderate cytonuclear atypia in 5 cases. Mitotic figures, myxoid background, mast cells, and dispersed adipocytes were rare. Giant cells, necrosis, or marked cytonuclear atypia were not seen. DFSP shares morphological characteristics of some low-grade spindle-cell neoplasms. It should be differentiated from other benign low- and intermediate-grade spindle neoplasm such as low-grade fibrosarcoma, fibromyxosarcoma, low-grade malignant peripheral nerve sheath tumor, benign peripheral nerve sheath tumor, nodular fasciitis, and fibrous histiocytoma., (Copyright 2004 Wiley-Liss, Inc.)

To determine diagnostic cytomorphologic features of malignant fibrous histiocytoma (MFH) on fine-needle aspiration (FNA) materials, we reviewed the cytologic material and corresponding histologic slides of 95 tumors in 71 patients. Forty-four (46%) tumors were primary, 38 (40%) were recurrent, and 13 (14%) were metastatic. Histological variants of MFH were as follows: 52 (54.7%, 43 patients) were of the storiform/pleomorphic, seven (7.4%, five patients) were giant cells, four (4.2%, four patients) were inflammatory, and 31 (33.7%, 19 patients) were myxoid type. Review of original cytology reports showed that only 23 (24.2%) tumors were diagnosed as MFH and 68 (71.6%) as other types of malignancies. Four (4.2%) cases were reported as unsatisfactory/suspicious. Our findings showed that spindle-shaped, round, giant cells, osteoclastic-like giant, and inflammatory cells were the most consistent features that allow identification of the storiform/pleomorphic, giant cell, and inflammatory variants of MFH. The myxoid tumors had marked myxoid background matrix with spindle-shaped cells and, less frequently, round and giant cells. Pleomorphic leiomyosarcoma and dedifferentiated liposarcoma should be considered in the differential diagnosis of stroriphorm/pleomorphic, giant cells, and inflammatory variants of MFH. However, myxoid MFH may resemble their leiomyosarcoma and liposarcoma counterparts., (Copyright 2003 Wiley-Liss, Inc.)

To characterize the cytological features of angiosarcomas, we reviewed the fine-needle aspiration material and corresponding histologic sections of 29 tumors in 23 patients. Histologically, 24 tumors were of the classic type, and 5 were epithelioid angiosarcomas. The original corresponding cytologic diagnoses were as follows: angiosarcoma, 17 cases; sarcoma not otherwise specified, 8 cases; and rhabdomyosarcoma, 1 case. Three samples were cell-poor and were considered suspicious of malignancy. The review of cytology samples showed that smears were cell-rich in 17 tumors and cell-poor in 12 tumors. A hemorrhagic background was present in 9 cases. Tumor cells were polymorphous, including spindle-shaped, round to oval, and polygonal epithelioid cells and giant cells in different proportions. Erythrophagocytosis was seen in 12 tumors. Smears of classic angiosarcomas were polymorphous and lacking specific characteristics, whereas smears of epithelioid tumors were morphologically similar and composed of round to oval and polygonal, epithelial cells frequently arranged in clusters, and showing erythrophagocytosis. The wide spectrum of cellular components of angiosarcomas accounts for the difficulty in establishing accurate tumor typing, particularly with cell-poor samples and low-grade classic angiosarcoma. Entities to consider in the differential diagnosis are carcinoma, epithelioid sarcoma, pleomorphic rhabdomyosarcoma, and malignant melanoma., (Copyright 2003 Wiley-Liss, Inc.)

Angiogenic factors exert protective effects on the lung. To investigate the effect of VEGF-B, a factor coexpressed in the lung with VEGF-A, we assessed chronic hypoxic pulmonary hypertension in VEGF-B knockout mice (VEGF-B-/-) and in rats with lung overexpression of VEGF-B induced by adenovirus transfer. No significant difference in pulmonary hemodynamics, right ventricular hypertrophy, distal vessel muscularization, or vascular density was found between VEGF-B-/- and control mice after 3 wk of hypoxia. When overexpressed, VEGF-B(167) or VEGF-B(186) had protective effects similar to those of human VEGF-A(165). Lung endothelial nitric oxide synthase (eNOS) expression was increased by 5 days of hypoxia or VEGF-A adenovirus vector (Ad.VEGF-A) overexpression, whereas VEGF-B(167) or VEGF-B(186) had no effect. With hypoxia or normoxia, the wet-to-dry lung weight ratio was increased 5 days after Ad.VEGF-A administration compared with control (Ad.nul), Ad.VEGF-B(167), or Ad.VEGF-B(186). Endogenous VEGF-B does not counteract the development of hypoxic pulmonary hypertension. However, when overexpressed in the lung, VEGF-B can be as potent as VEGF-A in attenuating pulmonary hypertension, although it has no effect on eNOS expression or vascular permeability.

To better define the cytological features of various leiomyosarcoma (LMS) variants, we reviewed the fine-needle aspiration material and the corresponding histologic sections of 96 tumors in 68 patients. Histological variants of LMS were as follows: 80 (83.3%) were of the classical/usual, seven (7.3%) were epithelioid, and nine (9.4%) were myxoid. Review of original cytology reports showed that 23 (24%) tumors were diagnosed as LMS and 69 (71.8%) as other types of malignancies. Two (2.1%) cases were reported as suspicious and two (2.1%) were unsatisfactory. The classical variants of LMS were characterized cytologically by various proportions of spindle-shaped, cohesive, small- or large-sized cells arranged in parallel alignment. Large spindle, round, binucleated, giant cells with intracytoplasmic granulations were frequently seen. Blunt-ended nuclei, intranuclear inclusions and mitotic figures were occasionally seen, as well as stromal fragments. The epithelioid tumors were composed of an admixture of small and large, spindle-shaped and round cells, also arranged in parallel alignment. Tumor cells with granular cytoplasm, blunt-ended nuclei, intranuclear inclusions, mitotic figures, fibrous or myxoid stroma were not observed. The myxoid tumors disclosed large amounts of background myxoid matrix containing large spindle-shaped and giant cells. Entities such as leiomyoma, malignant peripheral nerve sheath tumor, monophasic synovial sarcoma, and malignant fibrous histiocytoma should be considered in the differential diagnosis of LMS of the classical type. Epithelioid leiomyoma may share similar cytological features with epithelioid LMS. The cytological features of the myxoid variant of LMS can be easily confused with other types of benign and malignant mesenchymal tumors depicting degenerative myxoid changes and/or a myxoid matrix component., (Copyright 2003 Wiley-Liss, Inc.)

Synovial sarcoma (SS) is a high-grade malignant soft tissue tumor that manifests different phenotypic subtypes that may render their cytologic evaluation challenging. Although several cytologic studies of SS have been published, correlative studies of cytologic and corresponding histologic features are limited. To better define the cytological features of various SS forms, we reviewed the cytologic and the corresponding histologic material of 56 tumors from 36 patients. Classical patterns were defined as dispersed or small clusters of cells with bland chromatin, inconspicuous nucleoli, oval to spindle-shaped cytoplasm and branching tumor tissue fragments, vessel stalks, acinar structures in scant mucin background, seen in all 53 (94.7%) cellular cases. Epithelial, squamous, round cells, mast cells, necrosis, comma-like nuclei, marked nuclear atypia, secretory mucin, and rosette-like structures were also occasionally observed. Comparing the histological subtype we noted that epithelial cells and secretory mucin were restricted to biphasic SS, round cells to poorly differentiated SS, and comma-like nuclei to monophasic fibrous SS. We conclude that the classical pattern is highly suggestive of SS of all three monophasic, biphasic, or poorly differentiated subtypes. These characteristics, along with molecular genetic studies, may improve the cytologic diagnosis of SS., (Copyright 2002 Wiley-Liss, Inc.)

Cytomorphological patterns of malignant peripheral nerve sheath tumor (MPNST) are insufficiently documented in the literature. Cytological and histological specimens in 24 tumors in 17 patients were correlated. The review of the original cytology reports showed that four (16.6%) tumors were correctly diagnosed, eight (33.3%) were diagnosed as sarcoma not otherwise specified, four (16.7%) as fibrosarcoma, three (12.5%) as synovial sarcoma, three (12.5%) as leiomyosarcoma, and one (4.2%) case each as malignant fibrous histiocytoma and rhabdomyosarcoma. At the review tumors were histologically reclassified as well-differentiated MPNST in 11 (45.9%) cases, anaplastic MPNST in 11 (45.9%) cases, and epithelioid MPNST and malignant Triton tumor in one (4.2%) case each. Cytologically, well-differentiated MPNST were composed of polymorphous oval to round cells, small spindle-shaped cells with wavy and comma-like naked nuclei, and a fibrillary, delicate stroma. Anaplastic MPNST, moreover, were composed of anaplastic giant and polymorphous cells. The malignant Triton tumor was composed of oval to round rhabdomyoblastic cells with eccentric nuclei and the epithelioid MPNST of polymorphous and round, epithelial-like cells. The cytological diagnosis of MPNST may be difficult, especially in anaplastic tumors. The correlation between the cytological features and the clinical information--origin of the tumor from a nerve trunk, a preexisting neurofibroma, patients with known history of neurofibromatosis 1--could be indicative of an MPNST diagnosis., (Copyright 2002 Wiley-Liss, Inc.)

Mice expressing human apolipoprotein A-IV (apoA-IV) mainly in the intestine were obtained in an apolipoprotein E-deficient (apoE(0)) background (apoA-IV/E(0) mice). Quantification of aortic lesions and plasma lipid determination showed that compared with their control apoE(0) counterparts, the apoA-IV/E(0) mice are protected against atherosclerosis without an increase in HDL cholesterol. Because oxidized lipoproteins play an important role in atherogenesis, we tested whether the protection observed in these animals is accompanied by an in vivo reduction of the oxidation parameters. The lag time in the formation of conjugated dienes during copper-mediated oxidation, the aggregation state of LDL, and the presence of anti-oxidized LDL antibodies were measured. The presence of oxidized proteins in tissues and the presence of oxidation-specific epitopes in heart sections of atherosclerotic lesions were also analyzed. Except for lag time, the results showed that the oxidation parameters were reduced in the apoA-IV/E(0) mice compared with the apoE(0) mice. This suggests that human apoA-IV acts in vivo as an antioxidant. In addition, human apoA-IV accumulation was detected in the atherosclerotic lesions of apoA-IV/E(0) mice, suggesting that apoA-IV may inhibit oxidative damage to local tissues, thus decreasing the progression of atherosclerosis.

High levels of high-density lipoprotein (HDL) cholesterol have been reported to protect against the development of atherosclerosis in humans by increasing reverse cholesterol transport and inhibiting the oxidation of low-density lipoprotein (LDL) due to the paraoxonase content of HDL. The purpose of the present study was to assess if there are any relationships between in vivo increases in serum levels of immunological LDL oxidation markers [autoantibodies against oxidized LDL, autoantibodies against malondialdehyde-modified LDL, LDL immune complexes and anti-cardiolipin autoantibodies], paraoxonase activity and the development of atherosclerosis in control rabbits and in transgenic rabbits expressing human apolipoprotein (apo) A-I. A total of 13 apo A-I transgenic rabbits and 18 non-transgenic littermates were fed on a cholesterol-rich diet (0.4%, w/w) for 14 weeks, and were monitored at weeks 0, 2, 6, 10 and 14. Aortic atherosclerotic lesions were measured at the end of this period. Human apo A-I transgenic rabbits with high HDL cholesterol levels were not protected against the development of atherosclerosis when they were fed on a cholesterol-rich diet which induced dramatic hypercholesterolaemia. Immunological markers of LDL oxidation increased and serum paraoxonase activity decreased similarly in control and transgenic rabbits. In conclusion, the present study demonstrates that high HDL cholesterol levels are ineffective in inhibiting increases in immunological markers of LDL oxidation and the development of atherosclerosis in a mammal with severe hypercholesterolaemia.

Studies performed in vivo have been controversial regarding the implication of human apolipoprotein (apo)A-II in the atherogenic process. Expression of human apoA-II in transgenic mice fed a chow diet leads to (1) a bimodal distribution of high density lipoprotein (HDL) size as in humans, (2) a reduction in total cholesterol concentration that is mainly due to a reduction in non-HDL cholesterol level, and (3) a dramatic reduction in mouse endogenous apoA-I and apoA-II. After 20 weeks on an atherogenic diet, transgenic mice had reduced total cholesterol concentrations because of a reduction in cholesterol associated with all lipoprotein classes. Endogenous apoA-I and apoA-II were also dramatically decreased in transgenic mice. The mean area of atherosclerotic lesions was drastically decreased in transgenic mice (-44%, P=0.0027) compared with control mice. The amount of aortic surface covered by lesions was positively correlated with very low density lipoprotein cholesterol (P<0.01) and intermediate density lipoprotein cholesterol levels (P<0.05). Transgenic mice were protected against the development of atherosclerosis despite a marked decrease in HDL cholesterol and apoA-I concentrations. This protection may be related to the marked reduction in circulating low density lipoprotein (very low density and intermediate density lipoprotein) levels in transgenic mice.

Numerous diseases are linked to the absence or insufficient concentration of a specific plasma protein. Gene transfer is an appealing strategy for correction of such diseases. We report high and sustained plasma secretion of human secreted alkaline phosphatase and of human Factor IX by skeletal muscle of mice. This was obtained by delivering square-wave unipolar electric pulses of low field strength (200 V/cm) and long duration (20 ms) to skeletal muscle previously injected with plasmid DNA encoding for the secreted protein. This intramuscular electrotransfer method allows 30- to 150-fold increase in reporter protein secretion, compared to simple plasmid DNA injection. This increase allows one to obtain values of up to 2200 ng/ml of a reporter circulating protein. Moreover, this high level of secretion remains stable for several months.

Serum paraoxonase (PON1) is believed to protect against the development of atherosclerosis because of its ability to retard the oxidation of low-density lipoprotein (LDL) by hydrolysing LDL-associated phospholipid and cholesteryl-ester hydroperoxides. We have examined the relationship between PON1 and atherosclerosis development in transgenic rabbits overexpressing human apolipoprotein (apo) A-I and nontransgenic littermates fed a pro-atherogenic diet. PON1 activity was higher in transgenic (4006.1 +/- 716.7 nmol/min/ml) compared to control (3078.5 +/- 623.3 nmol/min/ml) rabbits (P < 0.01) while high-density lipoprotein (HDL) cholesterol was 1.84 +/- 0.54 mmol/L in transgenic rabbits and 0.57 +/- 0.21 mmol/L in control rabbits (P = 0.0001). After feeding rabbits a high-cholesterol diet for 14 weeks HDL-cholesterol fell by 70% in both transgenic and control rabbits (P < 0.001 compared to week 0) PON1 activity fell by 50% in both groups of rabbits (P < 0. 01 compared to week 0). The amount of thoracic aortic surface area covered by lesions was 29 +/- 16% in the control group and 26 +/- 15% in the transgenic group (P = NS). A pro-atherosclerotic diet reduces PON1 which may exaggerate the effects of the diet on the development of atherosclerosis., (Copyright 2000 Academic Press.)

The apolipoprotein E (apoE)-deficient mouse is a relevant animal model of human atherosclerosis. Although the prevention of atherosclerosis development has been documented after somatic gene transfer into animal models, regression of lesions remains to be demonstrated. Thus, we used this genetically defined mouse model nn the nude background to show atherosclerosis regression. ApoE-deficient nude mice were infected with 5 x 10(8) or 10(9) plaque-forming units of a first-generation adenovirus encoding human apoE cDNA. The secretion of human apoE resulted in a rapid decrease of total cholesterol, which normalized the hypercholesterolemic phenotype within 14 days (from 600+/-100 to <100 microg/mL). Transgene expression was observed during a period of >4 months, with a normalization of cholesterol and triglyceride levels during 5 months. At that time, we successfully reinjected the recombinant adenovirus and observed the appearance of the human protein as well as the correction of lipoprotein phenotype. In mice killed 6 months-after the first infection, we observed a dose-dependent regression of fatty streak lesions in the aorta. We showed sustained expression of a transgene with a first-generation adenoviral vector and a correction of dyslipoproteinemia phenotype leading to lesion regression. These data demonstrate that somatic gene transfer can induce plaque regression.

Background: To define the role of metalloproteinases (MMPs) in the development of lipid-rich atherosclerotic lesions in relation to the balance between proteolytic and antiproteolytic activities, we investigated the impact of adenovirus-mediated elevation in the circulating levels of human tissue inhibitor of MMP (TIMP-1) in atherosclerosis-susceptible apolipoprotein E-deficient (apoE(-/-)) mice., Methods and Results: Infusion of apoE(-/-) mice fed a lipid-rich diet with rAd.RSV.TIMP-1 (1x10(11) viral particles) resulted in high hepatic expression of TIMP-1. At 2 weeks after injection, plasma TIMP-1 levels ranged from 7 to 24 micrograms/mL (mean 14.8+/-6.8). Marked overexpression of TIMP-1 was transient, with levels of TIMP-1 decreasing to 2.5 to 8 micrograms/mL (mean 4.3+/-2.1) at 4 weeks. Plasma lipid and lipoprotein levels in mice treated with rAd.RSV.TIMP-1 were similar to those treated with rAd.RSV.betaGal. However, rAd.RSV.TIMP-1-infused mice displayed a marked reduction (approximately 32%; P<0.05) in mean lesion area per section (512+/-121 micrometers(2)x10(3); n=12 sections from 4 animals) as compared with rAd.RSV.betaGal-infused mice (750+/-182 micrometers(2)x10(3); n=12 sections from 4 animals). Similarly, marked reduction in macrophage deposition as well as MMP-2, MMP-3, and MMP-13 antigens was observed., Conclusions: Histological and immunohistologic analyses of atherosclerotic lesions revealed increases in collagen, elastin, and smooth muscle alpha-actin content in mice treated with rAd.RSV.TIMP-1. These qualitative and quantitative features were the consequence of TIMP-1 infiltration from plasma to arterial intima, as immunohistochemical analyses revealed an abundance of TIMP-1 specifically in lesions of rAd.RSV. TIMP-1-treated mice.

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.

Background: Apolipoprotein (apo) A-I is the major component of HDL, and it displays antiatherogenic properties., Methods and Results: The human apoA-I gene has been transferred into different mouse models by use of a recombinant adenovirus under the control of an RSV-LTR promoter (AV RSV apoA-I). Administration of AV RSV apoA-I to C57BL/6 mice resulted in moderate expression of human apoA-I for 3 weeks, leading to a transient elevation (40% at day 11 after injection) of HDL cholesterol concentration. In contrast, administration of AV RSV apoA-I to human apoA-I-transgenic mice induced a large increase of human apoA-I and HDL cholesterol concentrations (300% and 360%, respectively, at day 14 after injection) for 10 weeks, indicating that an immune response to the transgene was one major hurdle for long-term duration of expression. Recombinant adenovirus expressing human apolipoprotein A-I (AV RSV apoA-I) was also injected into human apoA-I-transgenic/apoE-deficient mice, which are prone to develop atherosclerosis. Over a 6-week period, overexpression of human apoA-I inhibited fatty streak lesion formation by 56% in comparison with control., Conclusions: Somatic gene transfer of human apoA-I prevents the development of atherosclerosis in the mouse model.

Background: Gene delivery of the thymidine kinase (tk) gene combined with ganciclovir (GCV) limits intimal hyperplasia after abrasion of normal arteries. However, the low efficiency of adenoviral-mediated gene transfer to atherosclerotic arteries has raised concerns about the applicability of this strategy to the prevention of restenosis., Methods and Results: A replication-defective adenoviral vector expressing tk (Ad-RSVtk) demonstrated selective toxicity toward GCV-treated arterial smooth muscle cells, with oligonucleolytic cleavage suggesting apoptosis. In vivo, after demonstration of tk expression after Ad-RSVtk delivery, the combination of Ad-RSVtk followed by GCV was tested in a rabbit model of angioplasty of atheromatous iliac arteries. Angioplasty (8 atm, 20 minutes) was performed by use of a hydrogel balloon coated with Ad-RSVtk (4x10(9) plaque forming units). GCV was infused (25 mg.kg(-1) I.V. BID) from days 2 through 7 after angioplasty in 8 of 12 rabbits. Four weeks later, morphometric analysis demonstrated a reduced intima-to-media ratio in the group receiving combination therapy compared with Ad-RSVtk alone (3.0+/-1.2 versus 5.2+/-0.5, P<.018). GCV per se had no effect on intimal hyperplasia after arterial injury., Conclusions: In vitro, Ad-RSVtk demonstrates selective toxicity toward GCV-treated arterial smooth muscle cells involving apoptosis. In vivo, GCV conditions reduction of neointimal formation after percutaneous delivery of Ad-RSVtk during angioplasty of atheromatous arteries.

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.

Apolipoproteins are protein constituents of plasma lipid transport particles. Human apolipoprotein A-IV (apoA-IV) was expressed in the liver of C57BL/6 mice and mice deficient in apoE, both of which are prone to atherosclerosis, to investigate whether apoA-IV protects against this disease. In transgenic C57BL/6 mice on an atherogenic diet, the serum concentration of high density lipoprotein (HDL) cholesterol increased by 35 percent, whereas the concentration of endogenous apoA-I decreased by 29 percent, relative to those in transgenic mice on a normal diet. Expression of human apoA-IV in apoE-deficient mice on a normal diet resulted in an even more severe atherogenic lipoprotein profile, without affecting the concentration of HDL cholesterol, than that in nontransgenic apoE-deficient mice. However, transgenic mice of both backgrounds showed a substantial reduction in the size of atherosclerotic lesions. Thus, apoA-IV appears to protect against atherosclerosis by a mechanism that does not involve an increase in HDL cholesterol concentration.

Background: Prospective epidemiological studies support the hypothesis that high levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I limit atherosclerosis development. However, more data from studies with animal models of atherosclerosis that resemble the human disease are required to demonstrate the effect of apo A-I in the inhibition of atherogenesis. The rabbit is a good animal model for human atherosclerosis., Methods and Results: Human apo A-I-transgenic rabbits have been produced, and we have evaluated the effect of apo A-I on the development of atherosclerosis in transgenic rabbits fed a cholesterol-rich diet for 14 weeks. Plasma cholesterol levels of atherogenic apo B-containing lipoproteins were similar for transgenic and control rabbits (> 1000 mg/dL), while plasma levels of HDL cholesterol in the transgenic group were always about twice that of the control group (68 +/- 11 versus 37 +/- 3 mg/dL at 14 weeks; P < .001). At the end of the experiment, the amount of aortic surface area covered by lesions as well as the amount of lipid accumulation in the aorta were significantly less in transgenic rabbits compared with the control group (15 +/- 12% versus 30 +/- 8%, P < .0027 for the surface area of the thoracic aorta; 116 +/- 31 versus 247 +/- 39 mumol/g aorta, P < .0068 for cholesterol content in total aorta)., Conclusions: Overexpression of human apo A-I in rabbits inhibits the development of atherosclerosis in this animal model that resembles, in many respects, human atherosclerosis.

Inhibition of chondrogenesis in limb bud cell micromass cultures has been proposed as a short-term teratogen detection test. Validation studies were performed by testing large series of reference compounds and comparing their teratogenic potential with their ability to inhibit chondrogenesis; however, there are few reports describing the histological and ultrastructural changes associated with inhibition of chondrogenesis in vitro. The objective of this study was to provide a qualitative description of the histological and ultrastructural alterations induced by three chondrogenesis inhibitors: retinoic acid (RA) and 6-aminonicotinamide (6AN), two teratogens, and doxylamine succinate (DS), a nonteratogen compound. In addition, in order to have a basis for the interpretation of the morphological alterations induced by the test compounds, the histological and ultrastructural changes which occur during the time course of chondrogenesis in control cultures were described and compared with those in rat embryo limb buds. We found that RA at 0.5 micrograms/ml led to a marked decrease in the number and size of cartilaginous foci; most cells lacked morphological signs of differentiation but their ability to proliferate was unaffected. At concentrations of 2 micrograms/ml and more, 6AN delayed cell proliferation, reduced staining of the extracellular matrix, and induced the formation of endoplasmic cisternae. DS at 50 micrograms/ml affected both differentiation and proliferation; pigment deposits were observed in chondrocytes, suggesting phospholipid metabolism disorders. In conclusion, this study showed that inhibition of chondrogenesis in this simple cell culture system can be associated with different types of histological and ultrastructural alterations. Examination of these alterations can provide useful information about the teratogenic potential of tested compounds and their mechanism of action.

Basaloid squamous carcinoma (BSC) is a rare distinct variant of squamous cell carcinoma of the head and neck region. This entity may commonly pose diagnostic difficulties, especially on small biopsy material. We report the clinicopathological characteristics of 12 new cases and compare their immunohistochemical features with those of solid adenoid cystic carcinomas and conventional squamous cell carcinomas. Our results show that neoplastic BSCs and squamous cell carcinomas do not react to vimentin and S100 protein, while adenoid cystic carcinomas manifest both. The BSCs, however, are S100 protein-positive in intratumoral dendritic Langerhans' cells that are lacking in squamous cell and adenoid cystic carcinomas. Our findings indicate that the immunohistochemical differences between BSC and adenoid cystic carcinoma could assist in their differential diagnosis.

The lipopeptide lauroyl-L-Ala-gamma-D-Glu-L,L-A2pm (LtriP) increased the resistance of mice to the lethal effect of gamma-ray irradiation. The radioprotective effect was dependent on the doses of LtriP and of radiation. Maximum survival was observed when the lipopeptide was injected on two successive days before irradiation. This activity seems to be related to immunostimulating functions, since the non-immunostimulating analog lauroyl-L-Ala-gamma-D-Glu-D,D-A2pm-Gly, containing D,D-diaminopimelic acid, was not radioprotective. The protective activity might result from an induction of cytokines, such as IL-1, TNF and M-CSF, since LtriP induced the mRNA expression and the secretion of these immunomodulators.

From May 1981 to June 1989, 84 children with non-B-cell lymphoma (82 lymphoblastic, 1 T-cell immunoblastic, 1 unclassified diffuse lymphoma) were treated in the pediatric department of the Institut Gustave Roussy according to a protocol called LMT81, which was derived from the LSA2L2 protocol of Wollner and modified by the adjunction of 10 courses of high dose methotrexate to improve the CNS prophylaxis. No planned irradiation was performed except in cases of initial tests (2 patients) or CNS (5 patients) involvement and residual mass (2 patients). Sixty patients had mediastinal involvement; for the others, primaries were in the head and neck (7), nodes (2), (sub)cutaneous (4), bone (7), and elsewhere (2). According to Murphy's staging system, there were 2 stage I, 6 stage II, 33 stage III, and 43 stage IV. Among the stage IV patients, 41 had bone marrow involvement, 24 of them with more than 25% blast cells in bone marrow and 19 with blast cells in blood; 7 had CNS involvement. Three patients did not achieve complete remission, 4 died in remission (two measles, one post-transfusion AIDS, one unexplained definitive aplasia) and 13 relapsed at 2 to 29 months (median-13 months). Among the 77 patients without initial CNS involvement, there was only one isolated CNS relapse. With a median follow-up of 57 months (10-106 months), the event-free survival is 75% (SE 2.5) for the 84 patients with a plateau at 29 months, 73% (SE 8) for stage I and II patients, 79% (SE 4) for stage III, and 72% (SE 4) for stage IV patients. Survival was similar in each stage group. Reasons for failure of treatment, however, were different, being toxic deaths in stage II; initial therapy resistance, early relapses, and toxic deaths in stage III; and tumor failures in stage IV. In conclusion, this protocol is efficacious on T and non-T, non-B childhood lymphoma with a low incidence of CNS relapse. A future study will seek to diminish toxicity and long-term sequellae while at least maintaining the same cure rate of patients.

In vivo studies have shown that the testis is a target organ for drugs and chemicals. In order to evaluate the testicular toxicity of compounds and to identify the mechanisms of their toxicity, we have developed a miniaturized primary culture of immature pig Leydig cells. Five well-known drugs with differing mechanisms of toxicity on testicular functions were tested to validate the model. Testosterone and progesterone secretion were measured to evaluate testicular function. Cell viability was assessed quantitatively using a colorimetric assay based on the reduction of a tetrazolium salt (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) which stains viable cells only, thus allowing discrimination between specific inhibitors of Leydig cell function and nonspecific cytotoxic drugs. Ketoconazole and aminoglutethimide inhibited both testosterone and progesterone secretion, but without modifying cell viability. Spironolactone specifically blocked testosterone secretion and increased progesterone concentration without inducing cell mortality. Cycloheximide altered testicular steroid secretion by another mechanism of action. Chlorpromazine, which interferes with the secretion of gonadotropins in vivo, produced a significant inhibition of progesterone and testosterone secretion as a result of the cytotoxic effects of the drug. In conclusion, this in vitro test enables one to discriminate accurately between specific and nonspecific inhibitors of steroidogenesis and could reduce the number of false positives when screening for potential testicular toxins.

One hundred and two bone marrow samples were analysed by histological and immunohistochemical methods for neurone specific enolase (NSE). The biopsies were performed to determine the extent of bone marrow disease in 84 neuroblastomas, nine embryonal rhabdomyosarcomas, five Ewing's sarcomas, two cases of Hodgkin's disease and two lymphoblastic lymphomas. Twenty seven (32%) of neuroblastoma bone marrows showed metastases by conventional histological techniques and 33 (39%) after immunohistochemical staining with NSE. Five embryonal rhabdomyosarcomas, five Ewing's sarcomas, and two lymphoblastic lymphomas showed bone marrow metastases. Only one of these cases was reactive for NSE. NSE represents a very sensitive immunomarker for the follow up of neuroblastoma and improves detection of bone marrow invasion by neuroblastoma.

From 1974 to 1987, 450 children with non-Hodgkins' lymphoma (NHL) were seen at the Institut Gustave Roussy (IGR); 14 children had malignant lymphoma of bone (MLB). Eleven of the 14 were newly diagnosed, whereas three presented in relapse. Nine patients presented with multifocal bone involvement. The median age of these eight girls and six boys was 9.5 years (range 1.25-15 years). Bone pain was present in all patients as the initial symptom. Evaluation included physical examination, routine serum chemistries, complete blood count, chest roentgenography, skeletal survey, radionuclide bone scan, lumbar puncture, bone marrow aspiration, and intravenous pyelography, and/or abdominal ultrasonography. Hypercalcemia was found in six patients. Biopsy was performed in 12 patients, revealing high-grade lymphoblastic lymphomas in all. In two patients diagnosis was made on cytological examination of bone marrow aspirate. Immunophenotyping in four cases, demonstrated non-B, non-T cell origin in three and pre-B cell origin in one. Three patients were treated prior to 1982 with Cyclophosphamide/Oncovin/Prednisone/ADriamycin (COPAD) and seven patients, seen after 1982, were treated with a modified LSA2L2 protocol (LMT). None of the previously untreated patients received radiotherapy. All patients treated with COPAD have died, whereas four out of seven treated with LMT are alive with a median follow up of 51 months (range 36-82 months). One child treated on a pilot study died. One of the three children seen at relapse is disease-free with a follow-up of 98 months after high-dose chemotherapy followed by autologous bone marrow transplantation (ABMT). Five out of six patients presenting with hypercalcemia have died. Results with LMT are encouraging and together with published results suggest that sufficiently intensive chemotherapy can result in complete remission and cure in MLB. Radiotherapy does not seem to be necessary, avoiding possible serious long-term effects. Hypercalcemia is a bad prognostic feature.

Two cases of pancreatoblastoma in children are reported here. Only biopsies were made at laparotomy as surgical resection by duodeno-pancreatectomy was not possible. In both children a dramatic response was observed with chemotherapy: doxorubicin plus cisplatin for one, cyclophosphamide, actinomycin D, bleomycin, vinblastine sulfate, and cisplatin for the other. After completion of the chemotherapy the first patient had a local resection; then he had radiotherapy. He is alive in first remission 40 months after the end of the treatment. In the second patient, regional recurrence occurred 8 months after chemotherapy was ended. A transient second remission was obtained with ifosfamide plus etoposide alternating with epirubicin plus vincristine. The patient died 36 months after the diagnosis. Therefore, these two cases suggest that chemotherapy may be proposed before any attempt at surgical excision. Nevertheless, early consolidation by radical resection or irradiation must be considered.

The authors describe the case of a patient with stage II non seminomatous germinal cell tumor of the testis with a generalized sarcoidosis. They review the existing literature and discuss the problem of diagnosis.

Thirty-four cases of soft tissue tumors (STT) other than rhabdomyosarcoma in infants under 1 year of age were seen in our institution between 1955 and 1985. All were diagnosed initially as malignant tumors except for three cases of fibromatosis, and, thus, they received therapy appropriate at that time. During a recent pathologic review, four were seen to have had hemangioma, six hemangiopericytoma, one hamartoma, seven fibromatosis, eight fibrosarcoma, and eight unclassified sarcomas. Of these 34 cases, the initial histological diagnosis was confirmed in only 17. Sixteen patients were believed to have received inappropriately aggressive therapy; indeed, four of these cases diagnosed prior to 1975 had been benign. Major long-term side effects were seen in 7/16 treated patients (six radiotherapy, one surgery), who, in retrospect, should have received less aggressive treatment. The reasons for these initial errors in diagnosis included doubtful histologic features associated in some cases with alarming clinical presentation. We believe that difficulties still remain in distinguishing benign from malignant STT in this age group by histological examination. Therefore, we urge extreme caution in using aggressive therapy before the diagnosis is certain.

In a retrospective study of infants under 1 year of age treated at our institution over a 30-year period for soft tissue tumors, eight fibrosarcomas (FS) were seen, six of which were congenital. Therapy consisted of local excision (n = 3), radiotherapy (n = 1), surgery + radiotherapy (n = 1), surgery + chemotherapy (n = 1), and surgery + chemotherapy + radiotherapy (n = 2). Among these eight patients, four are alive in first complete remission (CR) with 13, 17, 23, and 27 years of follow-up. Of the remaining four patients, two had local recurrences and are still alive in CR after re-excision of the tumor, while the other two had both local and distant relapses and died. Interestingly, in the two patients who developed distant metastases, the pathologic pattern was that of malignant fibrous histiocytoma (MFH) at the time of local recurrence. To our knowledge, no similar cases of transitions between infantile FS known for its favorable outcome and MFH have been reported in this age group. The relevance of such transitions is difficult to assess. However, given the known metastatic potential of MFH, we believe that chemotherapy regimens currently used in the management of childhood soft tissue sarcomas should be used in similar cases.

Between February 1979 and May 1986, 165 children were treated with autologous bone marrow transplantation after high-dose chemotherapy without total-body irradiation for a hematologic malignancy or a solid tumor. Nonbacterial nonfungal interstitial pneumonitis was observed in 24 children and was the cause of death in 11 cases. Of the 24 cases of interstitial pneumonitis, 14 were considered to be idiopathic. Cytomegalovirus was the major pathogenic agent detected in the 10 other cases of interstitial pneumonitis (n = 5), followed by Herpes zoster (n = 2), Pneumocystis carinii (n = 1), tumor (n = 1), and adenovirus (n = 1). The only factor found to correlate significantly with the increased rate of interstitial pneumonitis was the use of high-dose 1-3 bis chloroethyl-1 nitrosourea (BCNU) (600 mg/m2), whereas BCNU at a dose of 300 mg/m2 did not affect this rate. These data, when compared with the literature, show a lower incidence of interstitial pneumonitis than in allogeneic transplantations, and an incidence similar to that observed in syngeneic transplantations, although there was no radiation toxicity in this series.

Cytochemical reactions were examined in lymph node imprints from a group of 53 previously untreated patients with histologically proven Hodgkin's disease. In 40 of 51 cases investigated, Reed-Sternberg (R-S) cells, irrespective of the cytologic appearances and the histologic types, showed moderate to strong reactions with acid phosphatase (ACP). In 12 cases ACP activity was present in more than 25% of the R-S cells. The reaction consisted of formation of small- to medium-sized granules, which were located close to the nuclei on a diffusely positive background or irregularly distributed throughout the cytoplasm. In three cases, a coarse granular reaction product with periodic acid-Schiff was present. R-S cells were positive to the naphthol-AS acetate esterase and beta-glucuronidase reactions in four and two cases, respectively. Alkaline phosphatase and naphthol-AS-D-chloroacetate esterase reactions were completely negative. Our results have revealed a pattern of staining in the diagnostic R-S cells similar to that in its morphologic variants; this supports the view that these cells may derive from a common primitive cell. Moreover, the quality and quantity of the ACP reaction product shows that R-S cells differ from both neoplastic histiocytes of malignant histiocytosis and neoplastic lymphocytes of T-cell lymphomas. This study confirms that R-S cells lack definite cytochemical characteristics of each of supposed progenitor cells: histiocytes and T-lymphocytes.

Thirty-five children with Stage III and IV nonseminomatous malignant germ cell tumors were treated, between June 1, 1977 and December 31, 1982, at Institut Gustave-Roussy, Villejuif, France, and Hospital General de Ninos, Buenos-Aires, Argentina: 11 sacrococcygeal, 12 ovarian, 6 testicular, 5 intrathoracic, and 1 intrabdominal site. All of them had yolk sac component with high level of AFP, seven had also elevated level of HCG. Thirteen patients had primary chemotherapy, 18 received chemotherapy after incomplete surgical excision, and 4 patients with testicular Stage III tumors had chemotherapy immediately following retroperitoneal lymphadenectomy after orchiectomy, because of persistently elevated AFP levels. The chemotherapy regimen for 1 year's duration, repeated every 21 days, and consisted of 6 courses of dactinomycin-Cytoxan (cyclophosphamide) D1 to D5 and vincristine, Adriamycin (doxorubicin) D21, bleomycin D23, and cisplatin D24. Only three patients received complementary radiotherapy. The toxicity of the chemotherapy regimen was severe, but only one death was due to the therapy (pulmonary fibrosis with bleomycin). The number of surviving patients without evidence of disease was 12 of 16 for Stage III and 12 of 19 for Stage IV. The 25-month survival rate was 63% with a follow-up of 11 months to 5.5 years (median, 22 months). This constitutes a dramatic improvement when compared with the survival rate of 21% of 48 similar patients treated at Institut Gustave-Roussy between 1968 and 1977 with surgery, radiotherapy, and chemotherapy (methotrexate, dactinomycin, and Cytoxan).

The testicular function of 30 adolescent or adult males having undergone polychemotherapy in childhood was assessed by means of a spermogram or testicular biopsy. At the time of examination, the patients were pubertal and had completed chemotherapy between 1 and 20 years previously (mean, 9 years). All patients who were prepubertal or intrapubertal at the time of treatment achieved normal puberty with normal growth. Twenty patients presented with azoospermia and/or severe disturbances in the germinal line on biopsy. This series confirms the toxicity of alkylating agents, in particular that of the mechlorethamine, vincristine, procarbazine, and prednisone combination (MOPP) and that of cyclophosphamide (CPM). However, dactinomycin, vinblastine, and vincristine did not appear to have a toxic effect on spermatogenesis. The prepubertal state did not protect the gonads of 19 patients who were prepubertal at diagnosis: 12 are now sterile as a result of the treatment. An increase in basal follicle-stimulating hormone (FSH) levels gives a good indication of testicular damage, although normal levels do not rule out the possibility of azoospermia.

A totally synthetic molecule (109-145 peptide) analogous to the beta-subunit carboxyl terminus was used as an antigen in the development of antibodies by the hybridoma technique. A monoclonal antibody (702 D7) specifically recognized the free native beta-human CG (beta hCG). 702 D7 was of the immunoglobulin G1 subclass and was directed against an antigenic site localized in a 10-amino acid sequence (109-118) or less. The recognition of an epitope located in the 109-118 region could explain the specific recognition of beta hCG observed with 702 D7, in contrast to monoclonal antibodies directed against a 118-145 region with a recognition of both beta hCG and whole hCG, as observed with a second monoclonal antibody (1032) to synthetic peptide. Immunohistochemical results and preliminary data obtained from the immunoradiometric assay show that 702 D7 provides a clinical tool for the detection of free beta-subunit secretion even at low concentrations, and could allow the study of this subunit or its metabolites produced by normal and tumoral cells.

It has been suggested that cytokeratin 13 is a useful marker of malignancy. We examined normal squamous cell epithelia, hyperplasia, dysplasias of various grades, intraepithelial neoplasia and invasive squamous cell carcinomas of the pyriform fossa using K13 and KL1. Positive staining for K13 was seen in all normal or hyperplastic benign epithelia, was inconstant in dysplasia, and intraepithelial neoplasia and carcinoma was negative. KL1 expression is constant and non significant. These results suggest that tumour cells are unable to synthesize keratin 13 a finding which may be valuable in surgical pathology.

Unusual clinical, radiologic, or histologic findings were found in 11 of 17 cases of hypercalcemia associated with childhood tumors. Four children had undifferentiated lymphoblastic lymphoma with extensive bone involvement, but no visceral or neurologic involvement. At diagnosis, four adolescents with rhabdomyosarcoma had numerous metastases, particularly in the breasts and bone marrow. Three infants had renal tumor without bone metastases. Histologically, their tumors differed from classical nephroblastoma and resembled the malignant rhabdoid tumors of the kidney. These findings allow individualization of three distinct groups of tumors with unusual features which may suggest the presence of hypercalcemia. These tumors appear to have a poor prognosis since all patients but one died of their malignancy.