Correlation between laminin and type IV collagen distribution in breast carcinomas, and estrogen receptors expression, lymph node and vascular involvement.

The laminin (Lam) and type IV collagen (Coll IV) and estrogen receptor (ER) immunodetections were assessed in a large series of 400 human breast carcinomas. In all the cases the patient's age, the tumor size, the histological type and grade, the presence or the absence of axillary lymph node metastasis and of vascular invasion in tumor borders, and ER tumor content were recorded. Monoclonal anti-Lam, anti-Coll IV were applied with the avidin-biotin-peroxidase complex and monoclonal anti ER with peroxidase anti-peroxidase complex, on frozen sections. A computerized system of image analysis referred to as SAMBA (TITN) with specific software for tissue sections analysis permitted a multiparametric quantitative analysis of immunostained surfaces. With this system, in each tumor, the cellularity, the percentage of Lam, Coll IV and receptor positive surfaces versus the total cell surface and versus the epithelial (keratin positive) surface, the integrated optical density IOD histograms were obtained and correlated to morphometrical and standard histological data. From this study, it was shown that: (1) Lam and Coll IV immunostained epithelial basement membranes in carcinomas were correlated to the presence of estrogen and progesterone receptor antigenic sites within the tumors, with a significant decrease of the positive staining in ER-ICA negative tumors in comparison to ER-ICA/PR-ICA positive tumors. (2) The combined densitometric and morphometric evaluation demonstrated a decrease of Lam and Coll IV immunostaining in malignant tumors, correlated to (i) the presence of peritumorous vascular invasion and (ii) keratin positive cells in bone marrow (iii) axillary lymph node involvement. It is concluded that the variations in Lam and Coll IV antigens distribution may be relevant indicators of tumor metastatic potential in breast carcinomas and that computerized image analysis enables the standardization of the evaluation antigens distribution.

Inflammatory and non-inflammatory inclusion body myositis. Characterization of the mononuclear cells and expression of the immunoreactive class I major histocompatibility complex product.

In ten patients with inclusion body myositis (IBM) five muscular biopsies showed profuse inflammatory exudates and three showed a few scattered inflammatory cells with partial invasion in some muscle fibers. No inflammatory cells were seen in two cases. In all patients, histopathological, histomorphometric and immunocytochemical studies were performed. Immunocytochemistry for the class I and class II major histocompatibility complex gene product (MHC) was performed in all cases and in ten control muscles including: normal muscles [3], dermatomyositis [3], polymyositis [3], scleroderma [1]. In the five cases of IBM with inflammatory exudates, subsets of lymphocytes were analyzed with a panel of monoclonal antibodies against B cells, T4 cells, T8 cells, K and natural killer cells and macrophages. Some muscle fibers expressed class I MHC antigens in the inflammatory cases of IBM. These fibers were near the inflammatory exudates and occasionally showed a partial invasion. No expression of class I MHC was found in normal muscles and in non-inflammatory cases of IBM. The antigen which triggers the mononuclear cells in the inflammatory forms of IBM is probably not the filamentous inclusions in rimmed vacuoles. In other inflammatory myopathies, expression of class I MHC was present on all fibers in polymyositis, only in the perifascicular area in dermatomyositis and in scleroderma. It could be suggested that the term "inclusion body muscle disease" be applied to cases with rimmed vacuoles and "IBM-like" filaments without inflammatory cells.

Immunocytochemical assays in human endometrial carcinomas: a multiparametric computerized analysis and comparison with nonmalignant changes.

Immunocytochemical assay (ICAs) were performed on frozen sections from human endometrial samples (n = 89) including normal endometrium, decidua, hyperplasia with and without atypia, and carcinomas. Monoclonal antiestrogen receptor (ER), anti-laminin (Lam), anti-type IV collagen (Coll IV), and anti-Ki67 were applied with avidin-biotin-peroxidase complex or peroxidase-antiperoxidase complex. The results of the ICAs were evaluated through a computerized system of image analysis referred to as SAMBA. It was shown that this system provided for an accurate reliable and reproducible analysis of ICAs in tissue sections. It is concluded that this multiparametric and standardized method of analysis of ICAs can further be applied in correlations with clinical and biochemical data.

Immunodetection in fine-needle aspirates and multiparametric (SAMBA) image analysis. Receptors (monoclonal antiestrogen and antiprogesterone) and growth fraction (monoclonal Ki67) evaluation in breast carcinomas.

Immunocytochemical assays (ICA) using monoclonal antiestrogen receptors (ER ICA), antiprogesterone receptors (PR ICA), and monoclonal antibody Ki67 (Ki67 ICA) were performed in 127 breast carcinomas. The immunostaining procedures were applied on frozen tissue sections, tumour imprints, and fine-needle aspirates in order to compare the variations in the distribution of the antigens detected in the three different types of preparations. Positive reactions detected with peroxidase-antiperoxidase and avidinbiotin-peroxidase, and alkaline phosphatase-antialkaline phosphatase complexes were evaluated through a computerized system of image analysis referred to as SAMBA 200 (SAMBA TITN, Grenoble, France). Application programs specifically developed for the analysis of tissue sections and of cytologic preparations were applied. This system allowed a multiparametric, accurate, reliable, reproducible and automatized evaluation of the heterogeneity of the antigenic sites in tumors. For each markers positive cell surface (PS), and integrated and mean optical densities (IOD, MOD) and IOD histograms were compared. It was shown that (1) there was no significant variation in optical densities in cell imprints and aspirates whereas PS significantly (P less than 0.01) differed in both preparations; (2) there were significant differences of the optical densities between tissue sections and cytological preparations, either imprints or aspirates, likely due to randomly cut nuclei in tissue sections; and (3) there was a significant difference between the PS of tissue sections and aspirates but no significant difference between tissue sections and imprints. It is concluded that fine-needle aspiration constitutes a convenient method for cell sampling, reliable for the diagnosis of malignancies. However, it may not reflect the heterogeneity of cell subpopulations in tissue.

Type IV collagen immunostaining and computerized image analysis (SAMBA) in breast and endometrial disorders.

Type IV collagen immunostaining was performed on tissue sections from a large series of non-malignant and malignant disorders of the breast and endometrium. The results were analysed by means of a computerized system of image analysis referred to as SAMBA. It was shown that this system provided an accurate, reliable, reproducible, automated and multiparameteric analysis of collagen IV immunoprecipitates. It was concluded that this standardized method of analyses can be routinely used for the measurement of collagen IV, thus enabling correlations to be sought with histopathological and clinical data.

[Growth fraction (Ki67), ploidy balance and proliferation index in tumors of the urogenital tract and breast]. / Fraction de croissance (Ki67), balance de ploidie et index de prolifération dans les tumeurs du tractus urogénital et du sein.

The practical applications of computer-assisted image analysis systems are multiple in oncology. The computerized system of image analysis referred to as SAMBA (TITN) is particularly relevant to analyse coloured images resulting from immunostaining or histochemical procedures assessed either on tissue sections of any type or cytological preparations (imprints, smears). The SAMBA analysis of positive Ki67 surfaces in tissue sections from breast, endometrial, ovarian, cervical or urinary bladder samples enables a multiparametric evaluation of the growth fraction (GF) in intraepithelial, borderline or invasive proliferations. Moreover, the SAMBA analysis after Feulgen staining procedures provides a parametric evaluation of the nuclei densitometry and morphological features and of the chromatin texture, which serve to compute the ploidy balance (BP) and the proliferation index (PI). In benign tumors, GF and PI are low and tumor cells are diploid with an overall high positive PB values. In malignant tumors, GF, PI and the percentage of aneuploid cells increase with tumor grade and stage whereas PB decreases. In borderline proliferations, FC, PI and PB intermediate values are recorded. These new criteria of prognosis should be assessed routinely in pathology departments and the results from these new investigations are likely to be soon implicated in the selection of patient therapy.

Multiparametric evaluation (SAMBA) of growth fraction (monoclonal Ki67) in breast carcinoma tissue sections.

Breast tissue samples, including normal breast, nonmalignant disorders, and breast carcinomas (n = 257), were tested with monoclonal antibody Ki67 to define the growth fraction in each tissue subgroup. Immunocytochemical assays using anti-Ki67 and avidin-biotin-peroxidase complex and/or alkaline phosphatase anti-alkaline phosphatase were applied in frozen sections. The immunoreactions were analyzed with a computerized system of image analysis referred to as SAMBA (Systeme d'Analyse Microphotometrique à Balayage Automatique). This system permitted a multiparametric and automatized analysis of colored images. The results obtained were: (a) the SAMBA analysis of Ki67-positive staining was accurate, reliable, and reproducible; (b) the anti-Ki67 immunostaining was significantly (P less than 0.01) increased in malignancies and was related to the tumors' degree of differentiation, the vascular invasion, and the presence of axillary lymph node metastases; (c) anti-Ki67 immunostaining is increased (P less than 0.01) in tumors in which estrogen receptor and progesterone receptor antigenic sites are not detected. It is concluded that the SAMBA analysis of the anti-Ki67 immunocytochemical assay provides relevant information in selecting subgroups of patients with higher risk for relapse.

Multiparametric analysis (SAMBA 200) of the progesterone receptor immunocytochemical assay in nonmalignant and malignant breast disorders.

An immunocytochemical assay using monoclonal anti-progesterone receptor (PR-ICA) was performed in nonmalignant (N = 57) and malignant (N = 200) breast disorders. The results were analyzed with a computerized system of image analysis referred to as SAMBA and correlated with binding assays (DCC), and with standard histopathologic findings. It was shown that there was a correlation between 1) the PR-ICA and the binding assays (91.5%), and 2) between the binding assays and the multiparametric computerized (SAMBA) analysis of the PR-ICA. It was also shown that SAMBA provides an accurate, reliable, and reproducible evaluation of PR-ICA that is complementary to binding assays and constitutes a standardized method of evaluating the heterogeneity of the progesterone receptor (PR) distribution in tumors. It is concluded that SAMBA analysis of both the PR-ICA and estrogen receptor (ER-ICA) should improve the prognostic evaluation and the prediction of responsiveness to endocrine therapy in breast carcinomas.

Multiparametric study (SAMBA 200) of estrogen receptor immunocytochemical assay in 400 human breast carcinomas: analysis of estrogen receptor distribution heterogeneity in tissues and correlations with dextran coated charcoal assays and morphological data.

Estrogen receptor (ER) immunocytochemical assay (ER-ICA) was assessed in 400 human breast carcinomas. In all cases, patient's age, tumor size, histological type and Scarff-Bloom-Richardson grade, and presence or absence of axillary lymph node metastases and of vessel invasion in tumor borders were recorded. In 310 cases estrogen and progesterone receptors were concomitantly evaluated (dextran coated charcoal method). In 60 of these cases the ER immunoenzymatic assay (ER-IEA) was also assessed. Monoclonal H222sp gamma and peroxidase antiperoxidase procedures (Abbott kit) were applied in frozen sections, tumor imprints, and fine-needle aspirates. A computerized system of image analysis referred to as SAMBA (TITN), permitted a multiparametric quantitative analysis of ER-positive surfaces. With this system, in each tumor, the cellularity, percentage of ER surface versus the total cell surface and versus the epithelial (keratin-positive) surface, integrated optical density, mean optical density, index of the concentration of labeled objects, and integrated optical density histograms, were obtained and correlated to histological and biochemical data. It was shown that (a) ER antigenic sites were heterogeneously distributed in ER-positive tumors, with a specific nuclear localization in epithelial cells; (b) SAMBA 200 multiparametric analysis of the ER sites distribution in tissue was appropriate, accurate, reproducible, and therefore more reliable than the semiquantitative analysis; (c) standardization and complete automation of this method of immunoprecipitates evaluation on tissue section permits daily and routine analysis of a large number of preparations; (d) there was a correlation between ER binding sites evaluation (dextran coated charcoal) and ER antigenic sites immunodetection (ER-ICA and ER-IEA); (e) there was a correlation between the SAMBA evaluation of ER-ICA and other histological prognostic factors such as small tumor size, low Scarff-Bloom-Richardson grade; (f) the preliminary SAMBA analysis of ER-ICA in tissue sections, imprints, and fine needle aspirates suggest that fine needle aspirates may not reflect accurately the tumor cell heterogeneity.

[The immune status of the central nervous system]. / Le statut immunitaire du système nerveux central.

The central nervous system (CNS) has no true lymphatic outflow and is protected by the blood-brain barrier. It shares special relationships with the immune system. Though heterologous grafts can sometimes survive inside the brain, explaining that it has been considered as an immunologically privileged site, the CNS is able to generate immunological immune reactions and can exert a regulatory role on the extracerebral ones. Inside the brain, the immunological reactions are probably due to populations of immunocompetent cells and potentially macrophagic cells which are there permanently but in an inactivated state. Nevertheless the first event triggering these reactions and responsible for the activation of immunocompetent cells remains highly hypothetical. The astrocyte-endothelial cells complex which is the anatomical support of the blood-brain barrier probably plays a vital role in this process. Permanent exchanges between the CNS and the immune system are assumed by soluble mediators: lymphokines, neurotransmitters and hormones. These allow the CNS to control to some extent the extracerebral immunological reactions by feed-back regulation mechanisms.

[Immunohistochemical detection of estrogen receptors and image analysis (SAMBA 200) in breast carcinomas]. / Détection immunohistochimique des récepteurs oestrogéniques et analyse d'image (SAMBA 200) dans les carcinomes mammaires.

Estrogen receptor (ER) immunocytochemical assay (ER-ICA) was assessed human breast carcinomas. The patient's age, the tumor size, the histological type and SBR grade, the presence or the absence of axillary lymph node metastases and of vessel invasion in tumor borders were recorded in all the cases included in the series (n = 469) estrogen and progesterone receptors were concomitantly evaluated (DCC method) and or immunoenzymatic assays. Monoclonal H222 sp gamma and PAP procedure (Abbott kit) were applied in frozen sections, tumor imprints and fine needle aspirates. A computerized system of image analysis referred to as SAMBA (TITN), permitted to achieve a multiparametric quantitative analysis of ER positive surfaces. With this system, in each tumor, the cellularity, the percentage ER surface versus the total cell surface and versus the epithelial (keratin positive) surface, integrated optical density (IOD), mean optical density, index of the concentration of labelled objects, and IOD histograms, were obtained and correlated to histological and biochemical data. It was shown that: 1) ER antigenic sites were heterogeneously distributed in ER positive tumors, with a specific nuclear localization in epithelial cells; 2) the SAMBA 200 multiparametric analysis of the ER sites distribution in tissue was appropriate, accurate, reproductible and therefore more reliable than the semi-quantitative analysis; 3) the standardization and the complete automation of this method of immunoprecipitates evaluation on tissue section permit to daily and to routinely analyse a large number of preparation; 4) there was a correlation between ER binding sites evaluation (DCC) and ER antigenic sites immunodetection (ER-ICA and ER-EIA); 5) there was a correlation between the SAMBA evaluation of ER-ICA and other histological prognostic factors such as small tumor size, low SBR grade, the absence of axillary lymph node metastasis and vessel invasion; 6) the preliminary SAMBA analysis of ER-ICA in tissue sections, imprints and fine needle aspirates suggest that fine needle aspirates may not reflect accurately the tumor cell heterogeneity.

Aluminium overload of parathyroid glands in haemodialysed patients with hyperparathyroidism: effect on bone remodelling.

In the course of chronic renal failure, aluminium may deposit and accumulate in different tissues. The aluminium content of parathyroid glands was measured in 31 haemodialysis patients at the time of a parathyroidectomy. The values were compared with those obtained from ten control patients with primary hyperparathyroidism without renal failure, and were related to bone remodelling. Of the 31 patients, 27 had a bone biopsy after double tetracycline labelling, at the time of parathyroidectomy. Twenty-one patients had severe hyperparathyroidism, three patients had hyperparathyroidism associated with osteomalacia, three patients had mild hyperparathyroidism with reduced bone formation. Seven patients had bone aluminium deposits, associated with osteomalacia in one case. The parathyroid aluminium was 62 +/- 35.7 (mumol/g glandular dry weight) in haemodialysis patients and 14.3 +/- 6.3 in control patients (P less than 0.001). A significant positive correlation existed between parathyroid aluminium and serum aluminium (P less than 0.01). The parathyroid aluminium was not different in the patients with and without bone aluminium deposits. A weak correlation was found between parathyroid aluminium and plasma parathyroid hormone. A significant negative correlation existed between parathyroid aluminium and osteoblastic surfaces (P less than 0.05), but no correlation was found with bone formation rate at tissue and bone multicellular units levels. We conclude that aluminium accumulates in parathyroid glands of dialysed patients. Severe hyperparathyroidism may coexist with aluminium overload of parathyroid glands. A marked aluminium overload, however, may cut short the course of hyperparathyroidism and may decrease parathyroid function and cellular activity in bone.

Immune complex vasculitis associated with mediterranean spotted fever.

We report a case of mediterranean spotted fever complicated by leucocytoclastic vasculitis. Rickettsia conorii, IgA, complement and fibrin deposits were found in a skin biopsy. Treatment with tetracycline was successful.

Immunohistochemical study of thyroglobulin in thyroid carcinomas with monoclonal antibodies.

The effectiveness of an immunoperoxidase technique using four monoclonal antibodies (mAb) is compared to a technique using one polyclonal antibody (pAb) to detect human thyroglobulin (Tg) in paraffin sections of 55 thyroid carcinomas. With the pAb, a positive reaction was found in 82% of the cases. With the four mAb, the presence of Tg was demonstrated in 96.5% of the cases. The mAb gave better results than the pAb on poorly differentiated and anaplastic thyroid carcinomas. Many of the thyroid carcinomas in this study, especially the poorly differentiated and anaplastic type, failed to react with all four mAb to Tg. These results confirm the notion of the heterogeneity of Tg in thyroid carcinomas and indicate that a battery of carefully selected mAb can be successfully used for routine histopathologic detection of Tg in these tumors.

[Microphotometric system of analysis with automatic scanning and immunodetection: application to tissue sections (breast, endometrium, uterine cervix, ovary]. / Système d'analyse microphotométrique à balayage automatique (SAMBA) et immunodétection: applications sur coupes de tissu (sein, endomètre, col utérin, ovaire).

In the present study the immunodetection of various markers in a large series of tissue samples have been analysed through the computerized system of image analysis referred to as SAMBA 200. The immunodetection of estrogen and progesterone receptors, Laminin and type IV collagen, Ki 67, keratin, papilloma virus have been performed in frozen sections or in imprints and fine needle aspirates from tumorous and non tumorous disorders of human breast, endometrium, cervix and ovaries. Staining procedures varied with the type of the marker studied (PAP, ABC-P, ABC-GO, APAAP). The results obtained by the multiparametric SAMBA analysis were accurate, reproducible and reliable. Therefore, the SAMBA analysis providing a standardized and automatic analysis of immunodetection performed in tissue sections can be routinely used in pathology laboratories particularly for the prognostic evaluation and for the prediction of hormonotherapy and chemotherapy response of patients with solid malignant disorders.

[Pseudo-hypertrophic pelvi-crural amyloid myopathy in lambda light-chain myeloma. Clinical, morphological and immunocytochemical study]. / Myopathie amyloide pelvicrurale pseudo-hypertrophique au cours d'un myélome a chaines légères lambda. Etude clinique, morphologique et immunocytochimique.

The case of 65 year old woman with progressive enlargement and "wooden" induration of the pelvic girdle and thigh muscles due to an amyloid infiltration is reported. Muscle changes appeared two years after a diagnosis of myeloma with free lambda light chains. The patient complained of muscle pain, lassitude and weakness. Macroglossia was present. Skeletal muscle (vastus lateralis) contained large amounts of amyloid substance and showed type 2B atrophy. There was no fiber type grouping. Some amyloid deposits abutted on the muscle fiber, destroyed the basal lamina and sarcolemma, but never infiltrated it. Besides the amyloid phagocytosis by macrophages, a relationship between amyloid filaments and fibroblasts was present. Another non-congophilic substance was revealed using the Avidin-Biotin peroxidase complex to localize lambda light chains by light microscopy and corresponded to a granular substance in electron microscopy. Clinicopathological results are discussed with a review of thirteen similar cases previously reported.

[Tomaculous neuropathy. A histopathological study and electroclinical correlates in 10 cases]. / Neuropathie tomaculaire. Etude histopathologique et corrélations électrocliniques dans 10 cas.

Among 980 sural nerve biopsies, the nerves of 10 patients showed a great number of focal sausage-shaped thickenings of the myelin sheaths and were investigated by light and electron microscopy, teasing and quantitative studies. Single teased nerve fibres revealed myelin thickening in more than 25 p. 100 of internodes. This condition defined the tomaculous neuropathy and differed from other degenerative or toxic neuropathies which displayed a small number of internodes with myelin thickenings, in less than 5 p. 100. Segmental demyelination and remyelination were found in 12 p. 100 to 65 p. 100 of myelinated fibres. Tomaculous swellings were observed in the internodes of these fibres. Except axonal constriction within the sausage-shaped thickenings, no fibers with axonal degeneration was observed. The density of myelinated and unmyelinated fibres was normal. The loss of large myelinated fibres was interpreted as resulting from the myelinic changes. Clinical and electrophysiological data were similar in the ten cases of tomaculous neuropathies and in hereditary neuropathy with liability to pressure palsies, i.e.: autosomal dominant inheritance, higher incidence in males, recurrent nerve trunck and/or brachial plexus involvement related to compression, slowing of nerve conduction velocities in clinically affected and unaffected nerves more pronounced in anatomical narrow sites and increased F wave latencies. One patient (case 10) showed a mixed sensory motor progressive neuropathy but signs of widespread neuropathy were noted in more advanced cases. A great number of tomaculous swellings of myelin sheaths is considered as a specific but non constant change of hereditary neuropathy with liability to pressure palsies.