[Is systematic radiography needed before spinal manipulation? Recommendations of the SOFMMOO]. / Faut-il faire des radiographies systématiques avant manipulation vertébrale? Recommandations de la Société française de médecine manuelle orthopédique et ostéopathique (SOFMMOO).

OBJECTIVES: The French National Health Authority recommends systematic radiography before lumbar manipulation. However, current research shows radiography to have more disadvantages than advantages. For the cervical spine, in cases of complication following cervical manipulation, the lack of prior radiography is a fault, even if such x-ray results would have advised manipulation. To establish clear recommendations on this topic, we searched in the literature for complications of spinal manipulations that could have been prevented had preliminary radiography been performed to determine whether these complications were anticipated or not by the international guidelines. MATERIAL AND METHODS: Literature search. RESULTS: For the lumbar and thoracic spine, the international guidelines are sufficient, except in cases of aneurysmal bone cyst, which can appear until the age of 25 years. For the cervical spine, despite a lack of data in the literature, prudence and medicolegal issues justify the performance of systematic radiography. CONCLUSION: The SOFMMOO recommends systematic radiography prior to cervical manipulation and in case of back or neck pain in patients of less than 25 years of age. In all other cases, the international guidelines provide sufficient recommendations.

Evidence for multiple sequences and factors involved in c-myc RNA stability during amphibian oogenesis.

To investigate the molecular mechanisms regulating c-myc RNA stability during late amphibian oogenesis, a heterologous system was used in which synthetic Xenopus laevis c-myc transcripts, progressively deleted from their 3' end, were injected into the cytoplasm of two different host axolotl (Ambystoma mexicanum) cells: stage VI oocytes and progesterone-matured oocytes (unfertilized eggs; UFE). This in vivo strategy allowed the behavior of the exogenous c-myc transcripts to be followed and different regions involved in the stability of each intermediate deleted molecule to be identified. Interestingly, these specific regions differ in the two cellular contexts. In oocytes, two stabilizing regions are located in the 3' untranslated region (UTR) and two in the coding sequence (exons II and III) of the RNA. In UFE, the stabilizing regions correspond to the first part of the 3' UTR and to the first part of exon II. However, in UFE, the majority of synthetic transcripts are degraded. This degradation is a consequence of nuclear factors delivered after germinal vesicle breakdown and specifically acting on targeted regions of the RNA. To test the direct implication of these nuclear factors in c-myc RNA degradation, an in vitro system was set up using axolotl germinal vesicle extracts that mimic the in vivo results and confirm the existence of specific destabilizing factors. In vitro analysis revealed that two populations of nuclear molecules are implicated: one of 4.4-5S (50-65 kDa) and the second of 5.4-6S (90-110 kDa). These degrading nuclear factors act preferentially on the coding region of the c-myc RNA and appear to be conserved between axolotl and Xenopus. Thus, this experimental approach has allowed the identification of specific stabilizing sequences in c-myc RNA and the temporal identification of the different factors (cytoplasmic and/or nuclear) involved in post-transcriptional regulation of this RNA during oogenesis.

p185 overexpression in 220 samples of breast cancer undergoing primary surgery: comparison with c-erbB-2 gene amplification.

In breast cancer, DNA amplification of the oncogene c-erbB-2, encoding for the p185 protein, is associated with a poor prognosis. A retrospective study on a population of 220 cases of primary breast cancer permitted a quantitative measure of p185 oncoprotein overexpression by an immunoenzymetric assay and the determination of c-erbB-2 amplification by the Southern blot method. A correlation existed between the two measurements (r=0.85) using the double cut-off: DNA 2 copies and p185 400 U/mg protein, and only 2.7% of the cases were discordant. 13.2% of the tumors showed p185 overexpression. The percentage of tumors overexpressing p185 was significantly different between the groups with amplified and non-amplified c-erbB-2. We observed a significant correlation between p185 levels and tumor grade (p=0.03), and an inverse correlation with hormonal receptors (p=0.0001). The p185 assay could be an additional prognostic factor to better define patient subgroups with node negative, grade II, and positive or negative hormonal receptors.

Quantitative determination of c-erbB-2 in human breast tumours: potential prognostic significance of low values.

The purpose of this prospective multicentric study was to quantify the c-erbB-2 protein and investigate its relationship with DNA amplification and with various prognostic parameters of breast cancer. A total of 1062 primary operable human breast tumours were collected from six French anticancer centres. The c-erbB-2 protein was measured using an enzymoimmunoassay using two monoclonal antibodies directed against the extracellular domain of the protein. The results were expressed in arbitrary units/mg membrane protein (AU) after adjustment for the anticancer centre. A significant association was found between the dosage of the protein and DNA amplification (P = 0.0001). A value of 200 AU was found to maximise sensibility and specificity and was chosen as a cut-off for over-expression. Significant associations were found between c-erbB-2 values and oestrogen receptor (ER) (P = 0.01), progesterone receptor (PgR) (P = 0.0001) and histological grading (P = 0.01). The extreme high values (above the mean plus one standard deviation, S.D.) were significantly more numerous in ER- (P = 10(-16)), PgR- (P = 10(-14)) and grade III (P = 10(-8)) tumours. The extreme low values (below the mean minus one S.D.) were significantly more numerous in ER- (P = 10(-9)) and PgR- (P = 0.02) tumours. This prospective study confirms that high c-erbB-2 protein values are linked to poor prognostic factors and shows for the first time that low values are also linked to hormone receptor negative tumours, suggesting that these low values might also have a negative prognostic significance.

Paraffin section immunocytochemistry and cytosol-based ligand-binding assays for ER and PR detection in breast cancer: the time has come for more objectivity.

The importance of the receptor level in breast cancer as an indicator of hormone response has been extensively studied for more than 20 years. Besides cytosol-based ligand-binding assays (dextran-coated charcoal assay, DCC), new methods using monoclonal antibodies raised against estrogen and progesterone receptors allow for the detection of receptors both in cytosol extracts (enzyme immunoassay, EIA) and in tissue sections (immunocytochemical assay, ICA). The biochemical assays (DCC and EIA) as well as the immunochemical detection (ICA) have specific qualities and produce original information which is useful for the therapeutic decision. While DCC gives a measure of the receptor level, whatever the real source of synthesis (normal and/or neoplastic tissue), ICA locates the positive cells and their relative proportion in the tumor. Both methods present their own advantages and disadvantages which are summarized in this study.

[Value of detection of hormone receptors by biochemical and immunochemical methods in therapeutic decision for breast cancer]. / Intérêt de la détection couplée biochimique-immunohistochimique des récepteurs hormonaux dans la décision thérapeutique des cancers du sein.

Receptor assays for both ER and PR in breast cancer specimens is used either to choose adjuvant therapy during the first treatment, or to choose hormone therapy for metastatic patients, or for the diagnosis of tumors. Whereas biochemical methods, either by radioligand or by monoclonal antibodies, have progressed over the last 20 years, other methods such as immunohistochemistry are used now for receptor detection, and bring new data useful for patient therapy. This paper summarizes the qualities and pitfalls of these different methods, and shows that the use of data from both biochemical and histochemical methods can help the physician in therapeutic decisions.

Improvement of quality control for steroid receptor measurements: analysis of distributions in more than 40000 primary breast cancers. French Study Group on Tissue and Molecular Biopathology.

All French laboratories that routinely assay estradiol (ER) and progesterone (PR) receptors participate in the European EORTC quality control program based on twice-yearly analysis of 5 cytosolic preparations. This system has considerably reduced inter-laboratory variations, but does not cover all aspects of these assays. Analysis of receptor value distributions is also crucial to ensure that receptor measurements remain stable with time, independently of the laboratory and assay method. This study involved 83907 receptor assays carried out in the last 17 years by 17 laboratories belonging to the French Study Group on Tissue and Molecular Biopathology. The assays were based on radioligand binding (RLA) or immunoenzymology (EIA). For each laboratory, the medians and positivity rates were analysed according to two totally objective criteria, the patient's age and the year of assay, and according to histological grade and histological type of the tumor in order to verify the correlations classically described. Age-related distributions varied little between laboratories, compared with data published by 7 European EORTC laboratories [1]. The results remained relatively stable with time in the RLA method for ER and PR, and in the EIA method for PR. Median ER-EIA data showed a marked increase between 1987 and 1989, mainly due to changes in the quality of Abbott reagents during this period. Otherwise, this analysis confirms previous pathophysiological observations.

Detection of breast tumor progesterone receptor isoforms with the PR-enzyme immunoassay kit (Abbott). Effect of heat shock proteins on epitope recognition.

Eighty-nine human breast tumor progesterone receptors were assayed both by a radioligand assay (RLA; 3H-ORG-2058) and by the enzyme immunoassay from Abbott Laboratories (PR-EIA). The correlation obtained between the two methods was EIA = 0.83 RLA + 4.1 fmol/mg protein (r = 0.83). Great discrepancies were observed with EIA/RLA ratios varying from 0.5 to 2. After KCl 0.4 mol/l dissociation and chromatographic separation of 8 PR isoforms [12], the two PR polymeric forms (isoforms 1 and 2) which remained bound to the hsp90 heat shock protein were not or only partially detected by EIA, whereas PR-hsp70 isoforms were highly detected with EIA/RLA ratios increased up to 3.8. Free PR-A and PR-B proteins and the PR-truncated form (52 kDa) were never detected by EIA. Thus, the final result of PR assay using the Abbott method depends directly on the amount of PR-hsp70 isoforms produced through KCl dissociation during the overnight incubation of PR with the KD68 coated beads.

Biological markers and ovarian carcinomas: galactosyltransferase, CA 125, isoenzymes of amylase and alkaline phosphatase.

We present a comparative study of several biological markers (galactosyltransferase, CA 125, isoenzymes of amylase and alkaline phosphatase) with a view to ovarian carcinoma follow-up. Serum samples were obtained from a population of 75 patients under clinical observation. After a minimum 18-months period, we assessed the prognostic value of the markers. No marker permits the detection of discrete, evolving carcinomas. CA 125 is the marker that gives the best results, particularly in terms of sensitivity. Galactosyltransferase has a lower sensitivity except in the case of endometrioid carcinomas. Simultaneous analysis with CA 125 and galactosyltransferase results in no decisive improvement, other than greater precision in unfavourable prognoses. Isoenzymes of amylase and alkaline phosphatase are of no interest in the follow-up of such carcinomas.

Chromatographic separation of eight progesterone receptor isoforms in human breast tumors, and detection by radioligand and monoclonal antibodies. Association with hsp90 and hsp70 heat shock proteins.

It has been shown previously that untransformed molybdate-stabilized breast cancer progesterone receptor (PR) complexes can be dissociated by KCl (0.4 to 1M) into eight different intermediate forms, or isoforms (680-600-361-224-193-119-88-52 kDa), separated by high performance size exclusion chromatography and with the use of computer assisted smoothing and deconvolution procedures (H Cren et al [1993] J Chromatogr 615, 23-36). The purpose of this work was to study the constitution of each isoform by using different monoclonal antibodies (mabs) raised against PR-A/B (JZB39 and KD68), against PR-B (PR6 and KC 146), and against hsp90 and hsp70 heat shock proteins (9D2 and Ab72, respectively). The differential recognition of nontransformed molybdate-stabilized PR isoforms by either radioligand (RLA, 3H-Org2058) or by an enzyme immunoassay (PgR-EIA Abbott) showed the presence of two different PR isoforms in the non-dissociated PR heteropolymeric peak eluted from a TSK-3000 SW column. After PR dissociation by 0.4 M KCl and interaction of PR isoforms with the different mabs, the presence of PR-A, PR-B, hsp90 and hsp70 was studied. Results showed that hsp90 was present in isoforms 1 (680 kDa), 2 (600 kDa) and 3 (361 kDa) exclusively, whereas hsp70 remained strongly bound to isoforms 4 (224 kDa) and 5 (193 kDa). Isoforms 6 (119 kDa) reacted with PR6 antibody and represented the PR-B protein, whereas isoform 7 (88 kDa) represented PR-A protein. Isoform 8 (52 kDa) was not detected by mabs and represented a truncated form of PR. Detection of isoform 1 either by RLA or by EIA showed ratios EIA/RLA approximately 1 or 2, and these values suggested that this heteropolymeric form may contain a dimeric structure. From these observations, a model is proposed for the composition of each PR isoform obtained from dissociation of breast tumor PR.

[Intra-tissue biological markers in cancers of the breast: current assessment]. / Marqueurs biologiques intratissulaires dans les cancers du sein: bilan actuel.

Among the great many prognostic factors currently available in breast cancer, three classes of tissue biological parameters appear to be the most reliable in the establishment of a clinical decision flowchart, when they will have been technically and clinically validated: parameters of hormone dependence, tumour aggressiveness and invasion and parameters of proliferation. This article discusses the difficulties encountered in the evaluation of some of these parameters (hormone receptors by various methodological approaches, proteases, enzymes involved in cell proliferation), with emphasis on standardisation of techniques, development of quality controls, clinical validation and objective information of the medical and scientific communities.

[What can be now expected of the determination of estrogen and progesterone receptors in the treatment of breast cancers]. / Que peut-on attendre aujourd'hui du dosage des récepteurs d'oestrogènes et de progestérone dans la thérapie des cancers du sein?

Since the first published correlations around the years 1975 between estrogen and progesterone receptors and response to hormone therapy, numerous data have modified our insight on hormone dependency. For instance, it is now well established that tumors are not "receptor positive" or "receptor negative" but contain variable receptor quantities synthesized by a more or less important fraction of tumor cells. This allows to better understand events leading to partial response or to relapse. Receptor detection by classical assays gives no indications on receptor functionality, and data from molecular biology have shown that mutated receptors exist that have lost their property to induce genes, or that present new acquired properties. Nevertheless, these functional modifications are rare, and must not mask a reality: 75 to 80% of tumors with high receptor levels respond to hormone therapy, and the clinician must take this fact into account in three situations: for adjuvant therapy, for metastasis therapy, and for some special cases of difficult diagnosis.

Use of automated smoothing and deconvolution procedures for the determination of human breast cancer estrogen and progesterone receptor isoforms, after high-performance size-exclusion chromatography.

Automated smoothing and deconvolution procedures were used to analyse complex chromatographic patterns of human breast cancer estrogen and progesterone receptors, obtained by size-exclusion chromatography. By injecting, first, different known amounts of a radio-iodinated protein in a TSK G-3000 SW column, constructed complex chromatograms (twelve chromatograms) with known peak positions were obtained, and were further treated by mathematical functions to determine a smooth-deconvolution strategy, which could be used with unknown chromatographic patterns. The determination of peak areas by a "curve-fit" program showed a good correlation with the amounts of protein injected (r = 0.91). Human breast cancer estrogen receptors (56 cases) and progesterone receptors (45 cases) were chromatographed in a TSK G-3000 SW column, and further analysed with the smoothing and deconvolution procedures: eight different estrogen receptor isoforms were detected with molecular masses ranging from 530,000 [Stoke's radius (Rs) = 7.7 nm] to 23,000 (Rs = 2.6 nm), and eight progesterone receptor isoforms were observed with masses ranging from 680,000 (Rs = 8.6 nm) to 50,000 (Rs = 3.1 nm). The dissociation effect of KCl (0 to 1 M) on receptor structure yielded different proportions of receptor isoforms, but did not modify their different peak positions.

Evolution of immunoreactivity of monoclonal antibodies H222 and/or D547 used in the detection of breast cancer estrogen receptors. Varying reactivity of receptor isoforms.

From 1984 to 1990, human breast cancer estrogen receptors have been measured both by a radioligand assay (RLA[3H]estradiol) and by an enzyme immunoassay (Abbott ER-EIA kit). The ratio EIA/RLA results increased continuously from 1.04 (1984) to 1.87 (1990), and this evolution was consistent with the last trial of the E.O.R.T.C. receptor study group (Trial 1989-II, EIA/RLA = 2.5). Dilution studies of cytosols with the current ER-EIA kits showed an important parallelism defect of the standard curve, the final result of cytosols (fmol/mg protein) obtained from the upper part of the curve (between 100 and 500 fmol/ml) being 1.5 to 2 times higher than the results obtained from readings of the lower part of the standard curve (between 0 and 50 fmol/ml). Chromatographic experiments were carried out during 1986 and the measures of binding sites by RLA and of immunoreactive sites by EIA on chromatographic fractions were compared. Identical results were obtained with EIA and RLA, either on polymeric forms of the estrogen receptor, or on monomeric forms obtained after dissociation by 0.4 M KCl. The same experiments performed during 1990 showed that, in the chromatographic fractions, the concentration of immunoreactive sites was twice as large as that of ligand-binding sites, detected by tritiated estradiol. Furthermore, the detection of polymeric and monomeric receptor isoforms by monoclonal antibodies varied, and was increased by the presence of KCl (0.4 M) and/or bovine serum albumin (BSA) (1 mg/ml) in the cytosol. These findings showed that the large differences between enzyme immunoassay and ligand-binding assay results currently observed were due to differential reactivity of monoclonal antibodies for the estrogen receptor standard provided in the ER-EIA kits and for the estrogen receptor present in cytosols from human breast cancers, suggesting modifications of immunoreactivity of the monoclonal antibodies actually provided in the ER-EIA kits.

Immunoradiometric assay of pS2 protein in breast cancer cytosols.

We studied the ELSA-pS2 immunoradiometric kit (CIS Bio International) for pS2 protein assay in breast cancer cytosols according to classic validation methods. In addition, we studied correlations between pS2, steroid receptors, and cathepsin-D assays. Repeatability (CV = 1.5% to 4.8%) and reproducibility (CV = 1.6% to 4.9%) were good. The results were linearly related to pS2 concentrations between 205 and 2200 ng/L; the detection limit was 40 ng/L. The accuracy of the assay was measured by assessing recovery; analytical recoveries were near 100% throughout the standard curve. The use of different compounds for cytosol preparation (Tris 10 mmol/L or phosphate 25 mmol/L, KCl 0.4 mol/L, bovine serum albumin 1 g/L) had no effect on pS2 results. pS2 was assayed in breast tumor cytosols from 197 postmenopausal and 92 premenopausal patients. The mean value was 24 micrograms/g of protein; the median and 25th and 75th percentiles were 6, 1, and 23 micrograms/g protein, respectively. We observed a relation between concentrations of pS2 and those of estrogen and progesterone receptors, but there was no relationship between the concentrations of pS2 and cathepsin-D.

[Diagnostic value of SCC-TA4 determination in 4 localizations of epidermoid cancers. An experience of the FNCLCC subgroup of radio-analysis]. / Intéret diagnostique du dosage du SCC-TA4 dans quatre localisations de cancers épidermoïdes. Expérience du sous-groupe de radio-analyse de la FNCLCC.

A multicenter and retrospective study of the diagnosis value of SCC-TA4 in squamous cell carcinomas of 4 localisations was made with the 2 thresholds of 2 and 2.5 ng/ml. However, 3.1% of controls have a SCC value above 2.5 ng/ml. Sixteen benign gynecologic pathologies had no positive level. The benign digestive (N = 73), bronchial (N = 345) pathologies and no squamous cell carcinomas (N = 93, N = 220 respectively), had SCC-TA4 mean levels significantly lower than corresponding squamous cell carcinomas (N = 153, N = 128 respectively). Sensitivity of the test varied from 40% in the squamous cell carcinomas of the lung, to 72% in the squamous cell carcinomas of the uterine cervix. Specificity was always very high and varied from 91% in the SCC of lung, to 100% in the SCC of uterine cervix. For the SCC of uterine cervix, oesophagus and head and neck, the mean values and incidence of positive levels increased significantly with increasing tumor size and advancing disease stage. For the SCC of uterine cervix, mean SCC-TA4 levels and percentages of positive levels above 2 ng/ml were significantly higher for the patients with recurrence (22.5 +/- 4.6 ng/ml; 76%) or with metastasis appearance (23.6 +/- 5.4 ng/ml; 77%) than for the patients in remission (less than 1.5 ng/ml; 0%). In the SCC of oesophagus, we report levels before treatment that are significantly higher for the patients with metastasis at the first attempt (4.2 +/- 5.1 ng/ml; 59%), and an elevated SCC level at the diagnosis evoked a SCC of lung already disseminated (8.8 +/- 12.1 ng/ml; 50%) that will fail to respond to treatment (4.0 +/- 4.2 ng/ml; 48%).

Immunological and physiological analysis of human breast cancer progesterone receptor heterogeneity, following KCl dissociation and size exclusion chromatography.

Progesterone receptor (PR) levels were determined in 69 human breast cancer specimens by both radioligand assay (RLA) and enzyme immunoassay (EIA). These methods did not detect the same number of sites, and for each tumor there was a constant ratio between epitopes and PR-binding sites corresponding to 1/4, 2/4, ... 8/4. High performance size exclusion chromatography was performed to separate the various PR isoforms, and the ability of these isoforms to interact with the monoclonal antibodies was assessed. Determination of PR in the chromatographic fractions by EIA and RLA showed that the various isoforms isolated by chromatography presented variable quantities of steroid-binding sites and epitopes, thus confirming the differences observed in the cytosol assays. The dissociation of molybdate-stabilized PR by KCl and measurement by RLA and EIA of the isoforms obtained showed two different types of chromatographic patterns, particularly in the 8S polymeric form where the monoclonal antibodies appeared to detect mainly the heavier 8S fraction, which may correspond to the 8S-B form of the progesterone receptor. The monoclonal antibodies also detected an intermediate PR polymeric form (236 kDa) which was not always detected by the tritiated ligand. Our results suggest that breast cancer PR exhibit a marked molecular heterogeneity which may partially explain the differences in response to hormonal therapy, particularly for tumors with high receptor levels which nonetheless fail to respond to treatment.

Steroid receptors analysis in human mammary tumors by isoelectric focusing in agarose.

A high resolution and quantitative method for isoelectric focusing has been developed to separate the isoforms of estrogen and progesterone receptors in human mammary tumor cytosols stabilized by sodium molybdate. Agarose gels (0.5%) were used. Six samples can be analyzed on one gel in about 2 h, and 35-microliters samples are sufficient to determine the estrogen receptor isoform pattern. The constant yields and the reproducibility of data allow a quantitative analysis of these receptors. Four estrogen receptor isoforms have been observed (pI 4.7, 5.5, 6, and 6.5), isoforms with pI 4.7 and 6.5 being present in all tumors. After incubation at 28 degrees C in high ionic strength, the comparison of isoelectric focusing and high-performance size exclusion chromatography patterns of estrogen receptor confirms the oligomeric structure of the pI 4.7 isoform and suggests a monomeric structure for the pI 6.5 isoform. Under the same conditions of analysis, only one progesterone receptor isoform has been detected with pI 4.7.

Charge heterogeneity of human mammary tumor estrogen receptors. Relationship with a hormonal sensitivity tumor marker.

A high resolution quantitative method for estrogen receptor analysis has been elaborated using isoelectric focusing in 0.5% agarose gel, without any prior trypsin digestion. The 23 cytosols analyzed were stabilized by molybdate and prepared from human mammary tumors with progesterone receptors (PR + cytosols) or without (PR - cytosols). Progesterone receptor was used as a tumoral hormonal sensitivity marker. The estrogen receptors usually resolved as 4 isoform peaks of close isoelectric points. In PR - cytosols, the mean pI values were 4.7, 5.5, 6 and 6.5. Significant differences between the two cytosol populations were observed concerning pI 4.7 and 6.5 isoforms. In PR - cytosols, the pI 4.7 isoform occurred in greater proportions than in PR + cytosols, whereas lower proportions of pI 6.5 isoform were seen. The comparison between high performance size exclusion chromatography profiles and isoelectric focusing patterns, before and after cytosol incubation at 28 degrees C with KCl (0.4 M), confirmed an oligomer structure for the pI 4.7 isoform and suggested a monomer structure (Stokes radius 2.9 mm) for the pI 6.5 estrogen receptor isoform. The results indicated that isoelectric focusing analysis of estrogen receptors could be useful in the prediction of breast cancer hormonal sensitivity.